diff --git "a/deduped/dedup_0628.jsonl" "b/deduped/dedup_0628.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0628.jsonl" @@ -0,0 +1,44 @@ +{"text": "Cell reports the involvement of an RNA helicase in the pathway by which HIV exports partially spliced and unspliced RNA out of the nucleus. This suggests the ubiquity of RNA helicases in RNA export from the nucleus, and has novel mechanistic implications.Nucleo-cytoplasmic transport of RNA is one of many cellular pathways whose illumination has progressed hand in hand with understanding of retroviral mechanisms. A recent paper in HIV must export to the cytoplasm not only full length genomic and structural gene-encoding RNA species, but also partially and fully spliced RNAs which code for a variety of overlapping structural and accessory genes. Whereas the fully spliced HIV RNA species readily exit the nucleus and undergo translation, the partially spliced and unspliced species require the interaction of the HIV-1 Rev protein with the cis-acting, viral RRE (Rev responsive element) for expression. Simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV) solve a similar problem by encoding a cis-acting, constitutively active CTE (constitutive transporter element) in the 3'UTR of the viral genome, which uses a distinct pathway . Of lessThe Rev/RRE pathway involves the Ran-CRM-1, shuttling system: In the nucleus, Ran-GEF converts Ran-GDP to Ran-GTP. Ran-GTP bound CRM-1 binds the NES domain of Rev, which is in turn bound to the RRE, and enables CRM-1 to transport the resulting RNA/protein complex as cargo into the cytoplasm, presumably through CRM-1 interactions with nucleoporins. In the cytoplasm, Ran-GAP converts Ran-GTP to Ran-GDP, releasing the REV/RNA cargo. The asymmetric distribution of Ran-GEF and Ran-GAP between nucleus and cytoplasm ensures a constant RAN-GTP/GDP gradient to facilitate CRM-1 recycling and continued Rev/RRE export. CTE-mediated RNA export is based on binding to the Tap-NXT complex, and export through an unrelated transport pathway, which, ironically, is responsible for the bulk of mRNA transport .Cell, report the involvement of the RNA helicase, DDX3, in the Rev/RRE pathway [Enter the laboratories of Kuan-Teh Jeang and Larry Kleiman, who, in a comprehensive study in the October 29th issue of pathway . Interesin vitro (in a manner independent of its NES). Although no data are presented concerning direct in vitro binding of DDX3 to Rev, for such an interaction to exist and to be significant, DDX3 would have to bind one of the three identified functional regions in Rev. These include the NES, the coincident NLS/RBD and the Rev multimerization domain, which flanks the RBD [A number of findings not only provided evidence consistent with a role for DDX3 in the Rev export pathway, but also provided mechanistic insights. DDX3, though constitutively cytoplasmic, shuttles between nucleus and cytoplasm in a manner dependent on CRM-1, as evidenced by inhibition by leptomycin B (LMB), which inhibits CRM-1 export, but not Tap export. DDX3 also localizes to nuclear outer membranes in a speckle formation and co-immunoprecipitates with nucleoporins, in addition to having a distribution in the cytoplasm. In cell lysates, DDX3 co-immunoprecipitates with either Rev protein or CRM-1. Although the studies on cell lysates fail to distinguish between pairwise binding and ternary complex formation, ternary complex formation is a distinct possibility. DDX3 directly binds CRM-1 the RBD . No regiIn the tradition of good research, the results of Yedavalli et al. pose as On a highly speculative note, the intimate involvement of a DEAD box helicase in the Rev pathway, and the observation that DDX3 has a cytoplasmic localization, in addition to its nuclear membrane localization, recalls some old data from two independent laboratories, indicating that in some systems Rev may not act on export, but on post export events, to promote Rev/RRE dependent expression ,18. The Finally there is the possibility that specific helicases will be valid therapeutic targets for antiretroviral chemotherapy. Although targeting a cellular gene involved in a viral pathway risks inhibiting a necessary cellular function, it avoids the overwhelming problem posed by rapid viral mutation to resistance.RRE: Rev Responsive ElementMPMV: Mason-Pfizer monkey virusCTE: Constitutive Transport ElementNES: Nuclear Export SignalLMB: Leptomycin BNLS: Nuclear Localization SignalRBD: RNA Binding DomainNone. The opinions expressed are those of the author and do not necessarily express the opinion of the FDA."} +{"text": "The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction.Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive.The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv. Upon entering an uninfected host cell, the single-stranded RNA genome of Human immunodeficiency virus type 1 (HIV-1) is copied into DNA by the viral reverse transcriptase. Following integration of DNA into the host genome, transcriptional activation of the proviral DNA generates progeny virions. The post-integration events of transcription, RNA splicing, transport and translation of viral mRNAs are regulated by coordinate interaction with host proteins [Regulation of HIV-1 gene expression is controlled mainly by the two virus encoded proteins, Tat responsible for processive transcription elongation, and Rev which regulates transport of unspliced and incompletely spliced viral transcripts from the nucleus to the cytoplasm. These two regulatory proteins function by binding to structured RNA domains present in the viral transcripts, the trans-activation responsive RNA (TAR) and the Rev response element (RRE) respectively.The functional domains of Rev include an N-terminal nuclear localization signal (NLS) rich in Arg-residues, and a C-terminal nuclear export signal (NES) rich in Leu-residues. The NES of Rev interacts with host proteins that are critical for RNA export, and the NLS binds to the RRE-RNA structure, and is also involved in Rev-homodimerization -4. AfterThe Rev:RRE:Crm1 complex is translocated through the nuclear pore complex to the cytoplasm. This translocation is dependant on the RNA helicase activity of DDX3, which binds to the Rev:RRE:Crm1 complex on the nuclear side of the Nuclear Pore Complex and accompanies it through to the cytoplasmic side ,12. AfteIn murine fibroblast A9 cells, export of HIV-1 transcripts mediated by Rev is restricted due to as yet unidentified host cell factor(s) that block Rev-mediated transport. However the Rex protein, an analogue of Rev encoded by the Human T cell leukemia virus type 1 (HTLV-1) is able to mediate RNA transport in murine cells. Marques et al. reportedilf-3 gene, which by alternative splicing [4 90, MPP4 110, ILF-3, NFAR-1 and NFAR-2, which are implicated in different functions such as transcription, RNA splicing, RNA editing and export. It is unclear if any of the DRBD proteins directly influence Rev-dependent RNA transport.NF90 is encoded by the splicing ,15 givesII, the encapsidation signal \u03b5 in Hepatitis B virus RNA, and bovine viral diarrhea virus RNA [The proteins in this family are characterized by the presence of two C-terminal dsRNA binding domains of NF90ctv Fig. implicatAs outlined Fig. , two dsRAs shown in Figure 31\u201350 and anti-FLAG antibodies. Results showed that both Rev and NF90ctv proteins are expressed in HeLa cells (data not shown).The N-terminus of NF90ctv protein includes a region that is likely to be involved in protein:protein interactions . In addi31\u201350 antibodies. The results show that Rev co-immunoprecipitated with \u03b1-Flag antibodies .The C-terminal 67 amino acids (604\u2013671) of NF90ctv designated RG-, possess three SH3 motifs, that appear to be involved in protein:protein interactions . This rei.e. lysates from IPTG-induced E. coli cells (lane 1), or from uninduced (lane 2) E. coli cells or with extracts from untransfected HeLa cells (lane 3). Similar results were obtained when purified Rev expressed in E. coli replaced the HeLa cell extracts previously transfected with pRSV/Rev (lane 5). It should be noted that in all cases a double band of about 33 kDa was observed for GST/RG- but only the faster-migrating band was recognized by the \u03b1-GST/RG- or \u03b1-GST (Amersham) antibodies. The results indicate that the RG- region of NF90ctv is involved in direct interaction with Rev, in agreement with the results obtained by immunoprecipitation of full length NF90ctv of transcripts linked to RRE, we used a construct containing the ion Fig. , as did ion Fig. . As showion Fig. , Gag-GFPctv Fig. , where ictv Fig. . In presctv Fig. , Gag-GFPctv Fig. , howeverctv Fig. , suggestThe space of interactions between viral and host elements is complex, and our understanding of these dynamics is incomplete. In this study we explored whether the NF90ctv-mediated inhibition of HIV-1 Rev involved Rev-NF90ctv interaction. The results presented here show that NF90ctv inhibits the Rev mediated export of viral RNA. To refine this observation, and study its molecular mechanisms, we created a series of vectors containing segments of the NF90ctv gene to use in transfection studies. We investigated the possibility that specific regions of NF90ctv may be involved in blocking Rev-mediated RNA export activity, by expressing the NF90ctv regions in HeLa cells. Three regions of NF90ctv in particular diminished Rev function, the NES, DRBD 2 and the C-terminal RG-.The region of NF90ctv with the greatest inhibition of Rev function, the RG- domain, consists of the C-terminal 67 amino acids. This domain contains three SH3 motifs that are rich in Pro, which has been described to be involved in protein:protein interactions . SH3 motIn addition, studies have indicated that NF90 possesses several regions capable of protein:protein interactions; including PKR ,33, NF45ifl-3 gene [3LX3LXL sequence, similar to the consensus LX2\u20133LX2\u20133LXL reported to be part of the NES in Rev [The NES region of NF90ctv used in this study comprises amino acids 81\u2013190 that include a Leu-rich sequence, present in the different NF90 isoforms encoded by the l-3 gene . This reS in Rev . The NESS in Rev . Both ReS in Rev and NF90S in Rev known toHowever, since NF90ctv contains two dsRNA binding motifs, a direct interaction between NF90ctv and the HIV-1 RRE RNA structure may be equally important in blocking Rev function. These two DRBDs bind to the small and highly structured human Adenovirus transcript designated Adeno-associated virus RNA , to the Rev protein has been reported to reside mainly in the nucleus , and is Overall, the results are consistent with a mechanism of inhibition of Rev based on the interaction of Rev protein with specific domains of NF90ctv. The Rev-NF90ctv interaction may prevent Rev homodimer formation, an essential step in interaction of Rev with RRE. For the export of unspliced or incompletely spliced transcripts to be efficient, Rev must be imported back into the nucleus where it must reach a specific concentration to inducThe results described here suggest a novel means of inhibition of HIV-1 Rev function based on direct interaction between NF90ctv and Rev, interfering with the export activity of Rev Fig. . To our NcoI digestion and subcloned into the pUC18 vector upstream of and in-frame with the Flag-coding sequence. The Flag/NF90 coding sequence was then excised by NsiI and MscI digestion and ligated into the pOZ vector kindly provided by Dr. B. Howard (NIH) and previously cleaved by XhoI and NsiI, yielding the pOZ-Flag/NF90.The pEFX90 vector harboring the entire human NF90 coding sequence was kindly provided by P. N. Kao . To produce Flag/NF90, the NF90 coding sequence was recovered from pEFX90 by NheI/XbaI and XbaI/NotI digestion respectively and cloned into pCI-neo vector cleaved with the same enzymes, producing pCI-neo/N-ter NF90ctv and pCI-neo/C-ter NF90ctv respectively. To express other regions of NF90ctv proteins . The reporter plasmid pCMV128 carries the second intron of HIV-1 with the RRE structure downstream of the CAT coding sequence Fig. and therGGATCCGCGGCAGACCATGGCTAGCA and the 3'primer GCGGCCGCTTAGGCGCCGGTGGAGTG. The 5'primer presents a BamHI site (underlined) and a Kozak sequence, and the 3'primer a NotI site (underlined). On the other hand, pEGF-N1, which expresses the GFP, was cleaved with BamHI and NotI to delete the EGFP cassette, and replaced it by the mRFP PCR product to obtain the pmRFP construct used to transform Escherichia coli DH5\u03b1. Rev was amplified from pRSV/Rev using the 5'primer GAATTCTGCCGCCACCATGGCAGGAAGAAGCGGA (EcoRI underlined) and the 3'primer CCCGGGTTCTTTAGCTCCTGACTCCAA (SmaI underlined). The PCR product was digested with EcoRI/SmaI and ligated into pmRFP previously digested with the same enzymes to obtain the pRev/mRFP construct.To obtain the pRev/mRFP construct that expresses the monomeric red fluorescent protein (mRFP), the following strategy was used. The mRFP cassette was amplified from pcDNA3 using the 5'primer EcoRI/SmaI and cloned into pGEX-4T-3, yielding pGST/RG- that codes for the GST/RG- protein.To introduce the RG- region of NF90ctv Fig. into pGE5 cells per 35 mm-diameter dish 24 h before transfection with 2 \u03bcg each of the appropriate plasmid DNA using Superfect (Qiagen) following the manufacturer's instructions. Cells were harvested 24 or 48 h after transfection and lysed as described [A human osteosarcoma cell line stably transduced with CD4 and CXCR4, a T-tropic HIV-1 target cell line (GHOST/CD4+/CXCR4+), was maintained as described except tescribed . The pro31\u201350 or \u03b1-Rev71\u201390 antibodies , or \u03b1-Flag antibodies (Sigma).To evaluate expression of Rev and Flag/NF90 following co-transfection with pRSV/Rev and pOZ-Flag/NF90, cell extracts were analyzed by SDS-PAGE, the proteins transferred to a polyvinylidene-difluoride membrane , and immunodetected with \u03b1-RevHeLa cells were co-transfected with the plasmid constructs outlined in Table 5 HeLa cells were grown to about 80% confluence and cotransfected with 1.5 \u03bcg each of pRSV/Rev and pOZ-Flag/NF90, and after 24 h extracts were prepared as described above. Cell extracts (35 \u03bcl) were preincubated overnight at 4\u00b0C with 5 \u03bcl of \u03b1-Flag antibodies with gentle shaking. Protein A agarose was then added and incubation continued for 3 h. The precipitate was recovered by centrifugation, washed 3 times in PBS 1\u00d7 and the resin suspended in 20 \u03bcl of 4 \u00d7 Laemmli buffer. The protein suspension was analyzed by SDS-PAGE, transferred to a PVDF membrane and probed using \u03b1-Rev31\u201350 antibodies. The same amount of HeLa cell extract prior to immunoprecipitation was used as input control in western blot assays.For the immunoprecipitation assays, 4 \u00d7 10E. coli XL1 Blue cells transformed with pGST/RG- were induced or not overnight with 0.5 mM IPTG and lysed using lysis buffer . The recombinant protein was purified using a glutathione-Sepharose 4B column following the manufacturer's instructions. The recombinant protein was verified by SDS-PAGE and by immunoblotting with \u03b1-GST (Amersham) or \u03b1-GST/RG- polyclonal antibodies.6 HeLa cells transfected with pRSV/Rev, or 15 \u03bcg of purified Rev protein [After binding the GST/RG- protein to the glutathione-Sepharose 4B column, extracts from 10 protein [obtaine4) were co-transfected either with 1 \u03bc pRev/mRFP alone, with 1 \u03bc pRev/mRFP + 1 \u03bc pGFP/NF90 or with 1 \u03bc pGFP/NF90 alone; 1 \u03bc pmRFP was used as negative control. After 24 h, the cells were fixed for 20 min with 2% paraformaldehyde at room temperature. Following three washes with 1 \u00d7 PBS, the nuclei were stained with 4-6-diamidino-2-phenylindol (DAPI), the cells were mounted with Citifluor and the GFP or mRFP was detected using a Leica SP2 AOBS confocal microscope with a 63\u00d7, 1.32NA PlanApo lens using an Argon laser (488 nm) or a Krypton laser (568 nm), respectively. The images were assembled using Adobe PhotoShop.To determine if NF90ctv influences the cellular localization of Rev, HeLa cells on the subcellular localization of Rev and NF90ctv, HeLa cells were co-transfected as described above, and 24 h later, 50 ng/ml of LMB were added and incubation continued for 2 h at 37\u00b0C in 5% COTo examine if NF90 is able to bind the RRE RNA, the HeLa cells were transfected with either 1 \u03bc pRev/mRFP alone or with 0.4 \u03bc pSGT-5(SDM/RRE/CM-GFP) or with 1 \u03bc pNF90/mRFP with or without 0.4 \u03bc pSGT-5(SDM/RRE/CM-GFP). As negative control, we used the HeLa cells trasnfected only with pSGT-5(SDM/RRE/CM-GFP). After 24 h, the GFP or mRFP was detected by confocal microscopy, as described above.The author(s) declare that they have no competing interests.MEC carried out the studies, participated in the design and conception of the project. DH-V participated in the design and conception of the project and helped draft the manuscript. AK and GSL participated in the design and conception of the project and helped draft the manuscript. SU-I carried out the studies, participated in the design and conception of the project and drafted the manuscript. All authors read and approved the final manuscript."} +{"text": "Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP) as well as long-term nonprogressors (LTNP), longitudinal analysis of nef/long-terminal repeat (LTR) sequences demonstrated convergent nef/LTR sequence evolution in SBBC SP and LTNP. Thus, the in vivo pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than nef/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 rev alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36.The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with NL4-3, C18 or C98. D36 Rev also had a 50\u201360% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function was mapped to an unusual 13 amino acid extension at the Rev C-terminus.The ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE) in RNA binding assays was reduced by approximately 90% compared to Rev derived from HIV-1rev alleles may contribute to viral attenuation and long-term survival of HIV-1 infection in a subset of SBBC members.These findings provide new genetic and mechanistic insights important for Rev function, and suggest that Rev function, not Rev/RRE binding may be rate limiting for HIV-1 replication. In addition, attenuated Viranv genes -13. Hostnv genes -17.HIV-1 Rev is a 116 amino acid (aa), ~18 kD regulatory protein whose primary function is to mediate the nucleocytoplasmic transport, and therefore expression, of unspliced and singly spliced HIV-1 mRNA transcripts encoding viral structural proteins, via binding to the Rev response element (RRE) which is a complex RNA stem-loop structure present in these transcripts -labelled RNA transcripts bearing the RRE, as described previously [32P]-labelled RNA and increasing concentrations of His-tagged Rev protein in 10 \u03bcl binding buffer . Reactions were incubated on ice for 10 min, then applied to 5% (wt/vol) nondenaturing polyacrylamide gels containing 100 mM Tris borate (pH 8.3), 1 mM EDTA, and 3% (vol/vol) glycerol and run at 4\u00b0C followed by autoradiography and phosphorimager analysis.His-tagged Rev proteins derived from SBBC eviously . Brieflyrev alleles were subcloned into the pcDNA3.1 expression vector . Rev function in mammalian cells was quantified using the Rev-dependent reporter plasmid pDM128, which expresses the CAT gene from an intron bearing the RRE in the presence of HIV-1 Rev [To facilitate Rev protein expression in mammalian cells, SBBC IV-1 Rev . BrieflyIV-1 Rev .Lysates were prepared from CEM cells that were transfected as described above, separated in 12% (wt/vol) SDS-polyacrylamide gels, and transferred to nitrocellulose membranes, as described previously . Blots wrev nucleotide sequences reported here have been assigned GenBank accession numbers EF634153 to EF634156.The The author(s) declare that they have no competing interests.MJC and PRG designed the study, MJC and LC performed the experiments, MJC and PRG analyzed the data and wrote the paper, SLW contributed to the experimental design and data analysis.NL4-3. Dots indicate residues identical to HIV-1NL4-3 Rev, and dashes indicate gaps. Note the persistence of a dominant rev allele in each subject over the time course studied.Consensus Rev amino acid sequences from sequential SBBC blood samples. Each sequence represents the consensus of 10 independent Rev clones from each time point. Amino acid alignments are compared to Rev from HIV-1Click here for file"} +{"text": "Social scientists have suggested that cultural diversity in a nation leads to societal instability. However, societal instability may be affected not only by within-nation or \u03b1 diversity, but also diversity between a nation and its neighbours or \u03b2 diversity. It is also necessary to distinguish different domains of diversity, namely linguistic, ethnic and religious, and to distinguish between the direct effects of diversity on societal instability, and effects that are mediated by economic conditions.We assembled a large cross-national dataset with information on \u03b1 and \u03b2 cultural diversity, economic conditions, and indices of societal instability. Structural equation modeling was used to evaluate the direct and indirect effects of cultural diversity on economics and societal stability. Results show that different types and domains of diversity have interacting effects. As previously documented, linguistic \u03b1 diversity has a negative effect on economic performance, and we show that it is largely through this economic mechanism that it affects societal instability. For \u03b2 diversity, the higher the linguistic diversity among nations in a region, the less stable the nation. But, religious \u03b2 diversity has the opposite effect, reducing instability, particularly in the presence of high linguistic diversity.Within-nation linguistic diversity is associated with reduced economic performance, which, in turn, increases societal instability. Nations which differ linguistically from their neighbors are also less stable. However, religious diversity between neighboring nations has the opposite effect, decreasing societal instability. Ethnic divisions are often invoked to explain civil strife and conflict, but what evidence implicates cultural diversity as a causal factor in such strife? Social scientists have often argued that diversity within a nation might have negative effects on societal outcomes. Ethnic cleavages within a nation can create barriers to communication and exchange, factions and rivalries, and internal conflict There have been surprisingly few direct empirical studies of how cultural diversity affects social instability. The focus has instead been on the relationship between cultural diversity and economic performance across nations. Generally, the relationship is weakly negative, whether economic performance is measured as national wealth to examine different causal possibilities. SEM is a multiequational modeling system suitable for asking complex questions about the responses of systems to interconnected sets of explanatory factors To summarize, there are four groups of variables which are of interest: cultural \u03b1 diversities, cultural \u03b2 diversities, economic measures, and proxies of societal instability. We wish to examine the direct and indirect (through economic conditions) effects of \u03b1 and \u03b2 cultural diversity on societal instability. The conceptual model underlying our analysis is thus that shown in ini, the coefficient of income inequality. Some previous studies have used the rate of GDP growth over a specified period rather than GDP itself as a measure of economic performance (e.g. nstab), and an index of the occurrence of revolutions and coups d'\u00e9tats (RevCoup) drawn from A cross-national data set was assembled for 212 nations from three sources nce e.g. , but we nce e.g. . DiversiWe separately consider diversity indices for language, ethnicity, or religion and examine their interrelationships as part of the analysis. Data on religious and ethnic diversity are from D, which takes into account not only the number of groups in the assemblage but also their relative abundance. Simpson's D is calculated by first determining the proportion ip of the total number of individuals in the assemblage represented by each species i. These values are next squared and summed over all species, S to obtain D is simply the probability that two individuals chosen at random from the assemblage represent the same group. As diversity increases, D decreases, so the index is usually presented as 1-DH. Shannon's H correlates with Simpson's D at around r\u200a=\u200a0.99, so we consider it no further here.For the purposes of this study, \u03b1 diversity is the cultural diversity within a nation. We use a common index of species diversity from the ecology literature, Simpson's PS is the percentage similarity between assemblages, j is the number of species shared by the assemblages, a is the number of species in the first assemblage and b is the number in the second assemblage PS decreases, thus we employ 1-PS in the analyses. We calculated mean Jaccard coefficients for each nation and all of its neighbours for religious, language, and ethnic groups. Note that \u03b2 diversity for islands is undefined without additional assumptions so islands had no \u03b2 diversity scores in our dataset. We also calculated an alternative index of \u03b2 diversity, Sorenson's coefficient r\u200a=\u200a0.99, we consider it no further.For \u03b2 diversity, we calculated Jaccard's coefficient of similarity, defined as Structural Equation Modeling (SEM) software package Amos 7.0 2\u03a7 statistics that measure the discrepancy between observed and model-implied covariance matrices. In addition to initial assessments of overall model fit, the significances of individual paths were assessed using single-degree-of-freedom 2\u03a7 tests. Finally, we considered whether there was any indication that political instability actually drives the variations among nations in GDP (rather than vice versa) by evaluating a model that included such a feedback.We used the The structural equation model results from our analyses are shown in Notable significant pathways in For \u03b2 diversity, there is a substantial positive effect of linguistic diversity on instability in the model. Thus, nations whose linguistic composition is very different from that of their neighbors tend to have more internal strife. However, there is an interaction with religious diversity, which has an effect in the opposite direction. Increasing religious \u03b2 diversity is associated with decreased societal instability. In other words, the more religiously different a nation is from its neighbors, the more internally stable it is. As a result of this interaction, the most stable nations are those that are religiously unique, but linguistically similar to their neighbors. This interaction is represented in We reran our analyses removing the \u03b2 diversity variables, in order to make a more direct comparison with previous studies, which considered only \u03b1 diversity measures . With \u03b2 The key findings of our structural equation models are the following. First, there is an effect of within-country diversity on societal instability, with more diversity being associated with more instability. This is a novel result, as previous studies have taken economic performance as their outcome variables, rather than examining societal instability as we do here.The effect of within-country diversity on societal instability is largely mediated (and moderated) by economic conditions. We observe a negative relationship between within-country diversity and economic performance. This aspect of our findings replicates previous studies Second, our results indicate that the different types of diversity are not perfectly correlated and have different effects. Ethnic and linguistic diversities are moderately correlated, and it is linguistic rather than ethnic diversities that have the strongest relationships to other variables. We suspect that this may be due to the superior quality of the linguistic data, since languages are better-studied and easier to define than ethnic groups. Religious \u03b1 diversity does not have the same negative effects on economic performance as linguistic \u03b1 diversity. Alesina and coworkers Our most novel finding concerns the previously-unstudied effects of \u03b2 diversity on societal instability. The more linguistically different from its neighbours, the more unstable a nation becomes, whilst religious \u03b2 diversity produces an opposing effect. The more religiously distinct a nation is from its neighbours, the less instability is created by linguistic diversity. We note that these effects, unlike the effects of \u03b1 diversity on societal instability, are not mediated by economic performance. We therefore suspect they operate at a more cultural or psychological level.We are curious why religious, but not linguistic, differentiation from neighbours should produce a stabilizing effect. Alexander There are some limitations to the study which should be acknowledged. First, our model shows the direction of the causal influence between economic performance and societal instability as flowing from the former to the latter. This is likely an oversimplification, as a feedback from instability to economic performance would be expected. This result only means that effects of economics on instability are predominant, not exclusive. Finer-grained data, and in particular time series, would allow more detailed investigation of the causal nexus between economics and societal strife.A second limitation is that we do not consider the role of the institutional environment. Previous research suggests that national institutional quality can have a major impact on outcomes, and in particular moderates the relationship between within-country diversity and economic performance"} +{"text": "The HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNA through interaction with the Rev response element (RRE) by means of an arginine rich motif that is similar to the one found in Tat. Since Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its arginine rich motif, we investigated whether the Rev protein could act as a substrate for this enzyme.in vivo. Further analysis demonstrated that the presence of increasing amounts of wild-type PRMT6, as well as a methylation-inactive mutant PRMT6, dramatically down-regulated Rev protein levels in concentration-dependent fashion, which was not dependent on the methyltransferase activity of PRMT6. Quantification of Rev mRNA revealed that attenuation of Rev protein levels was due to a posttranslational event, carried out by a not yet defined activity of PRMT6. However, no relevant protein attenuation was observed in subsequent chloramphenicol acetyltransferase (CAT) expression experiments that screened for RNA export and interaction with the RRE. Binding of the Rev arginine rich motif to the RRE was reduced in the presence of wild-type PRMT6, whereas mutant PRMT6 did not exert this negative effect. In addition, diminished interactions between viral RNA and mutant Rev proteins were observed, due to the introduction of single arginine to lysine substitutions in the Rev arginine rich motif. More importantly, wild-type PRMT6, but not mutant methyltransferase, significantly decreased Rev-mediated viral RNA export from the nucleus to the cytoplasm in a dose-dependent manner.Here, we report the methylation of Rev due to a single arginine dimethylation in the N-terminal portion of its arginine rich motif and the association of Rev with PRMT6 These findings indicate that PRMT6 severely impairs the function of HIV-1 Rev. As a positive control, we used recombinant histidine-tagged Tat86, and BSA served as a negative control. The proteins were separated by SDS-PAGE, stained with Coomassie blue . Measurements by LC/MS resulted in two assigned masses that were 27.8 Da apart in the case of recombinant Rev protein that had been subjected to methylation by PRMT6; this compared well to an expected difference of 28.1 Da in the case of one arginine dimethylation. In contrast, untreated Rev that was not methylated possessed only one mass were pulse labeled with L- [methyl-3H]-methionine. The lysates were separated by SDS-PAGE for subsequent Coomassie staining and fluorography. Lysates were loaded in equal amounts and the Coomassie stain revealed very similar host protein levels when comparisons of the different lanes were enacted. However, there was a very significant difference in Rev protein amounts detected by Coomassie stain , this may explain the weak and sharp band detected at a slightly higher migratory position than the broad band produced by Rev methylation.in vivo target for PRMT6 arginine methylation.Finally, to confirm that the signals indeed originated from Rev, we carried out western blots of the lysates with anti-Rev antibody experiment to assess mRNA levels of Rev under these different transfection conditions Fig. . This asSince there is no generally accepted method for normalization of such levels -57, we cThese results show clearly that the above mentioned 7.5-fold decrease in Rev protein levels is not caused by down-regulation of Rev mRNA by PRMT6. Rather, the decrease in Rev protein is due to the posttranslational interaction of PRMT6 with the Rev protein. However, attenuation is not dependent on methyltransferase activity, but seems to be caused by a yet undefined activity of PRMT6, which may be linked to the proteasome pathway, as previously suggested .in vivo. To this end, we used the pHIV-LTR-RREIIB-CAT reporter plasmid, which is derived from the pHIV-LTR-TAR-CAT -S-adenosyl-L-methionine and TE buffer for 3 hours at 37\u00b0C in a final volume of 10 \u03bcl. Reactions were stopped by adding 10 \u03bcl of 2 \u00d7 L\u00e4mmli buffer , followed by boiling for 5 minutes and centrifugation at 16,000 g for 2 minutes. Samples were loaded on 15% polyacrylamide gels containing sodium dodecyl sulfate (SDS) and a high level of N,N,N',N'-tetramethylethylenediamine (TEMED). Gels were stained with Coomassie brilliant blue R-250 solution (Bio-Rad Laboratories) and, after destaining, soaked in Amplify for 30 minutes. Gels were dried and exposed for fluorography on Hyperfilm MP (Amersham Biosciences) for 1 to 3 days. Gels and films were quantified with GeneTools (SynGene) and the IC50 for AMI1 were calculated with Prism 4 (GraphPad Software Inc.).1\u20132 \u03bcg of recombinant histidine-tagged Rev (wild-type or mutated), Tat86, or bovine serum albumin (BSA) were incubated with 3\u20134 \u03bcg GST-tagged PRMT6 in the presence or absence of AMI1 together with 0.55 \u03bcCi of -methionine (Amersham Biosciences) for 3 hours in the presence of cycloheximide (100 \u03bcg/ml) and chloramphenicol (40 \u03bcg/ml) (both Sigma) in DMEM lacking the amino acids methionine, cysteine and glutamine to prevent protein synthesis. The cells were lysed in RIPA buffer containing complete mini EDTA-free protease inhibitor (Roche) and processed as described for the methylation assays. Rev was confirmed by western blot with anti-Rev antibody detected with an anti-Rb antibody conjugated to HRP as described above.Similar experiments were described earlier . Briefly6 HeLa cells stably transfected with siRNA against PRMT6 -chloramphenicol , 20 \u03bcl acetyl-CoA all in Tris in a final volume of 150 \u03bcl and incubated for 1 hour at 37\u00b0C. 450 \u03bcl ethyl acetate were added, mixed, centrifuged and 400 \u03bcl of the upper ethyl acetate phase were dried for 15 minutes in a Speed-vac centrifuge. Pellets were solubilized in 18 \u03bcl ethyl acetate, separated by thin layer chromatography (TLC) and exposed to Hyperfilm MP.5 \u00d7 105 HeLa cells (PRMT6- or mock siRNA) were seeded and transfected as described above, replacing pHIV-LTR-RREIIB-CAT and pSV2/TatRev by pDM128 and pT-REx-DEST30-HRev, respectively. CAT isolation, reaction and separation was carried out as described above.5 \u00d7 10in vitro assays, and was involved in revising the manuscript. MAW participated in the concept and design of the experiments, and critically revised the manuscript. All authors have read and approved the final manuscript.CFI carried out all work presented in the figures with one exception noted below. CFI also drafted the manuscript. BX cloned and isolated Tat86, PRMT6, and wild-type Tat-Rev fusion protein, and stably transfected HeLa cells with siRNA directed against PRMT6 or mock siRNA, and carried out the experiments for figure"} +{"text": "The HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear.The activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev.Rev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains. Thesees (CRS) ,8. Genetes (CRS) -11. An oes (CRS) -14. The es (CRS) . This bies (CRS) . It is, es (CRS) -19. Howein vitro -22 and i[in vivo -27. The [in vivo ,23,25-27[in vivo ,28. One [in vivo . It is n[in vivo . In the The intracellular distribution of the M4 and the M4 derived Rev mutants requires Rev in trans for expression of the p55 Gag protein [The two mutants M4-M4 and NOS-M4 also activated Gag-expression in A3.9 cells. Thus, these mutants were also active in this essentially different reporter system. The pDM138 plasmids encode mRNAs with the CAT gene flanked by the HIV-1 splice sites. These splice sites are not present within the gag mRNA expressed in the A3.9 cell line. Although the presence of a protein .trans-activity assays [The common feature of NOS-M4 and M4-M4 is the presence of two functionally similar basic domains. The dimer included two copies of the Rev basic domain while NOS-M4 contained one domain from Rev and one from Rex. The function of the HTLV-I Rex protein is similar to that of Rev and the basic domains from the two viral proteins bind to Importin \u03b2 during nuclear import ,36. It iy assays . The co-in vitro splicing of RRE containing mRNAs underscores the functional importance of this region [The rescue of activity of the mutants M4-M4 and NOS-M4 could be explained by at least two models. Both mutants comprise two basic domains with comparable functions. We found that Rex did not activate CAT expression from the Rev dependent pDM138 reporters. This indicated that the NOS-M4 mutant binds to the IIB RNA sequences by the Rev basic domain leaving the Rex domain free for other interactions. The previous observation that a peptide corresponding to the basic domain of Rev inhibited the s region ,40. ThesThe present study showed that the activity of the negative monomeric M4 mutant was rescued by addition of an extra basic domain implying that two or more basic domains must be present within the complexes that bind to target RNA. This can be important for structural reasons or for leaving free basic domains for interaction with cellular co-factors when the Rev complex remains bound to viral RNA.rev-3xNLS and pcrevM4-3xNLS encoding Rev and the M4 mutant with three C-terminal copies of the SV40 large T antigen NLS were constructed by amplifying the rev coding region from pcrev and pcrevM4 using the primer pair catgccatggcaggaaga agcggag / ccgctcgagttctttagttcctgactccaa [NcoI-and XhoI-digested pCMV/myc/nuc vector (Invitrogen). The plasmid pcrevM4-M4 encodes an artificial M4 dimer with the monomers connected by a glycine rich linker. The rev coding region from pcrevM4 was amplified using the primer pairs tcgaagctagtcgacatctcctatg / cggggtaccgcctccttctttagctcc (PCR A) and cggggtaccggaatggcaggaagaagc / ctccagttggtagagagagcag (PCR B). The reverse primer in PCR B binds 70 nucleotides downstream from an XhoI site present in pcrevM4. The PCR product A was digested with SalI and KpnI while PCR B was digested with KpnI and XhoI. The two PCR products were ligated together into the SalI and XhoI digested pcrev vector. The NOS-M4 mutant contained the overlapping NLS/NOS from the HTLV-1 Rex protein fused to the N-terminal. The plasmid was constructed by inserting the NcoI-and XhoI digested fragment from pcrevM4-3xNLS into the NcoI-and XhoI digested pCMV/myc/cyto vector (Invitrogen), and inserting an NcoI flanked oligo encoding the overlapping NLS/NOS signal from the HTLV-1 Rex protein into the NcoI digested pcrevM4-myc vector. An overview of Rev and the Rev mutants is shown in figure rex sequence was amplified from pcD-Sr\u03b1 Rex provided by Dr. Shida and cloned into the pcrev vector after excising the rev gene [env gene [The plasmids pcgactccaa ,29. The rev gene ,42. The env gene ,44. In trev plasmid DNA varied from 0.2 \u2013 2 \u03bcg in order to obtain the same amount of Rev or mutant protein expressed in the cells. The Rev protein levels were estimated by immunofluorescence. Of the control plasmids pcrex and pctat, 4 \u03bcg and 1 \u03bcg respectively were added and the expression was confirmed by immunofluorescence.The A3.9 cell line stably expressing the gag mRNA fused to the RRE sequence was provided by M.L. Hammarskj\u00f6ld and D. Rekosh. Transfection of A3.9 cells and COS-7 cells in 35 mm wells were carried out using Lipofectamine Reagent 2000 (GIBCO BRL Life Technologies) according to the manufacturer's recommendations. One coverslip was added to each 35 mm well and CAT and Rev expression by western blot and immunofluorescence respectively were analysed in parallel. Each 35 mm well was transfected with 1 \u03bcg of the pDM138 reporter plasmids. The amount of The cells were fixed or harvested 24 or 48 h post transfection for analysis by immunofluorescence and western blot respectively as previously described . The immThe Rev proteins were detected by the anti-Rev Mab 8E7 .Tat and Rex were detected using the Mabs 1D9 and 1F8 respectively (not shown) ,46. The RRE: Rev Responsive ElementHIV: Human Immunodeficiency Viruscis-acting repressor sequencesCRS: Mab: Monoclonal antibodyCAT: ChloramphenicolacetyltransferaseNOS: Nucleolar localization signalNLS: Nuclear localization signalNES: Nuclear export signalLMB: Leptomycin BThe author(s) declare that they have no competing interests.CF: Vector design and construction. Transfections and immuoassays.TA: Vector design and construction. Transfections and immuoassays.PA: Vector design and construction.JK: Experimental design, manuscript preparationAMSz: Experimental design. Transfections and immuoassays. Manuscript preparation."} +{"text": "The coral reefs of Zanzibar Island encompass a considerable proportion of the global coral-reef diversity and are representative of the western Indian Ocean region. Unfortunately, these reefs have been recently subjected to local and regional disturbances. The objectives of this study were to determine whether there are potentially non-random processes forcing the observed coral diversity patterns, and highlight where and at which spatial scales these processes might be most influential.\u03b3) into local \u03b1 diversity and among-sample \u03b2 diversity components. Individual-based null models were used to identify deviations from random distribution across the three spatial scales. We found that Chumbe and Mnemba had similar diversity components to those predicted by the null models. However, the diversity at Changuu and Bawe was lower than expected at all three spatial scales tested. Consequently, the relative contribution of the among-site diversity component was significantly greater than expected. Applying partitioning analysis for each site separately revealed that the within-transect diversity component in Changuu was significantly lower than the null expectation.A hierarchical (nested) sampling design was employed across three spatial scales, ranging from transects (\u226420 m), stations (<100 m), to sites (<1000 m), to examine coral diversity patterns. Two of the four sites, Chumbe and Mnemba, were located within Marine Protected Areas (MPAs), while the other two sites, Changuu and Bawe, were not protected. Additive partitioning of coral diversity was used to separate regional diversity and the within-transects scale as spatial boundaries within which to examine the processes that may interact and disproportionately differentiate coral diversity. In light of coral community compositions and diversity patterns we strongly recommend that Bawe be declared a MPA. Biodiversity is not homogeneously distributed across the planet. Understanding how and why diversity changes across multiple-spatial scales is still one of the most challenging tasks facing contemporary ecology in general Theoretically, the assembly of local communities can be visualized as the result of species passing through a series of \u201cfilters\u201d ; Fig. 1.Additive partitioning of species diversity is a promising approach to distinguish among spatial diversity patterns using hierarchical sampling The coral reefs of Zanzibar Island (Unguja), Tanzania, support a large proportion of regional reef-coral diversity Nevertheless, there is a paucity of data describing the composition and spatial diversity of reef-coral assemblages around Zanzibar et al.Stylophora, Pocillopora, Seriatopora and Acropora were sampled.In a noteworthy study Belmaker In this study we focused on stony-coral assemblages. Our hypothesis (to be tested) was that coral assemblages around Zanzibar vary non-randomly across spatial scales. Thus, we sought to quantitatively describe composition and diversity of stony-corals on the Zanzibar reefs and examine their distribution across a hierarchy of spatial scales. Our major objective was to identify where and at which spatial scales non-stochastic processes might interact and have direct influences on the arrival and survival of corals. This would enable us to highlight the appropriate spatial boundaries for studying these processes.Reef surveys were conducted in November 2006 on four reef sites around Zanzibar . All foucenter rules\u201d scheme of Zvuloni et al.A hierarchical (nested) sampling design was employed to assess three spatial scales of influence: (1) transects (\u226420 m), (2) stations (<100 m), and (3) sites (<1000 m). The design consisted of four sampling sites that wer\u03b3) was partitioned into local \u03b1 diversity and among-sample \u03b2 diversity . Thus, \u03b2 diversity was calculated as the total diversity minus the average local diversity (\u03b2\u200a=\u200a\u03b3\u2212\u03b1). The main advantage of additive partitioning, in characterizing the relationship between \u03b1 and \u03b2 components over the traditional multiplicative method , is that the contribution of \u03b1 and \u03b2 to the total diversity can be directly calculated and compared \u03b2 diversity) and vice versa.Additive partitioning of coral diversity was used to quantify the spatial distribution of diversity patterns \u03b1) diversity was the average number of TAUs within a transect . Since we used a nested sampling design, samples at one scale are themselves composed of samples at a smaller scale. Hence, partitioning can be applied at all spatial scales and \u03b3 diversity can be partitioned into the diversity contributed by each scale. As such, \u03b21 is regarded as the average diversity among transects, within stations; \u03b22 is the average diversity between stations, within sites; and \u03b23 is the average diversity among sites, within the region. Given our nested sampling design, these components can be calculated using the following equations:\u03b1 and \u03b2 components, as:As a first stage in this study, regional diversity was defined as the total number of TAUs found in the full collection of samples from the four sites. Alpha , and repeated this procedure 1000 times to obtain a 95% confidence interval (CI) for each diversity component.Using a null model, all the individual coral colonies within the region were shuffled through individual-based randomization. This model is more powerful than the sample-based model in detecting departures between observed and null partitions because of intra-specific aggregation of individuals. Furthermore, the individual-based model overcomes the effects of abundance and sampling effort on diversity measures and comparisons . In these four independent analyses, \u03b3 diversity was defined as the total number of TAUs found in the full collection of samples within a site. The observed components were then compared with those expected by a null model that randomized all the individual corals within the tested site.Deviations of the observed components from the null expectation indicate a non-random spatial distribution of TAUs. However, deviations of the mean diversity across the four tested sites can be the result of strong deviations within only some of the sites, whereas other sites may show diversity patterns similar to the expected overall null model. Alternatively, lack of deviations from the overall model may be the result of considerable negative deviations at some sites that are counterbalanced by high, positive deviations at other sites. Therefore, the observed diversity-component for each hierarchical level was calculated separately for each site and compared with those expected by the overall model that takes into account all the individual corals within the four sites. We then tested the relative contribution of each site to the observed regional diversity. In addition, the total diversity within each site was partitioned into the diversity contributed by each component within the site . Diversity across the three tested spatial scales was highest at Site 3, followed by Site 4, Site 2 and Site 1 was significantly lower than expected. By applying the partitioning approach and a null model separately for each site was significantly higher than expected by the null model. Thus, the local \u03b1 diversity component was significantly lower than expected. Such deviations from the null expectations suggest that forces included in neutral theory This study shows that the mean diversity observed at the three hierarchical scales was consistently lower than expected by chance, under the assumption of a random distribution of TAUs around Zanzibar . In addiet al.Stylophora, Pocillopora, Seriatopora and Acropora) in three regions across the Indo-Pacific - Red Sea, Tanzania, and the Great Barrier Reef. Their coral-sampling design consisted of two hierarchical levels, both transects and sites, within each region. They found deviations at similar spatial scales in their three studied regions. However, in contrast with previous studies that partitioned only the mean diversity components within a region, the present study also separately tested the input of each site to the deviation from the overall null model. Indeed, using such examinations we found that deviations from the null expectations were the result of strong negative deviations only within Sites 1 and 2 , but also on the within transect-scale in Site 1. Over the larger scale, these mechanisms can range from dispersal-limitation to differential survival among the sites. However, in Site 1, there are mechanisms that prevent the success of some species in specific locations and reduce local P. rus was found to be relatively abundant at Sites 1 and 2 , and was shown to be among the most important taxa contributing to the differences among sites , including number of individuals observed in the survey and species included within each TAU.(0.07 MB DOC)Click here for additional data file."} +{"text": "The increase of human life span will have profound implications in Public Health in decades to come. By 2030, there will be an estimated 1.2 billion women in post-menopause. Hormone Replacement Therapy with synthetic hormones is still full of risks and according to the latest developments, should be used for the shortest time possible. Searching for alternative drugs is inevitable in this scenario and science must provide physicians with other substances that can be used to treat the same symptoms with less side effects. Systematic research carried out on this field of study is focusing now on isoflavones but the randomised controlled trials and reviews of meta-analysis concerning post-menopause therapy, that could have an important impact on human health, are very controversial. The aim of the present work was to establish a theoretical calculation suitable for use as a way to estimate the \u201cTheoretical Efficacy (TE)\u201d of a mixture with different bioactive compounds as a way to obtain a \u201cTheoretical Efficacy Related to Estradiol (TERE)\u201d. The theoretical calculation that we propose in this paper integrates different knowledge about this subject and sets methodological boundaries that can be used to analyse already published data. The outcome should set some consensus for new clinical trials using isoflavones (isolated or included in mixtures) that will be evaluated to assess their therapeutically activity. This theoretical method for evaluation of a possible efficacy could probably also be applied to other herbal drug extracts when a synergistic or contradictory bio-effect is not verified. In this way, it we may contribute to enlighten and to the development of new therapeutic approaches. Since then, many researchers, all over the world turned their attention to isoflavones and initiated studies to explore its effects. Today, one of the major concerns about isoflavones lays in the definition of a suitable dose-dependent effect, thereby creating new therapeutic options.Since in 1984 Setchel www.menopause.org/bioidentical.html).Nowadays, over the counter (OTC) tablet preparations and nutraceuticals with isoflavones extracted from soy and other plants are widespread around the world. These OTC medicines are often used for postmenopausal treatment, like Hormone Replacement Therapy (HRT) with oestrogen and/or progesterone is most evident in genistein and daidzein and their derivatives. The characteristic way that isoflavones bind to oestrogen receptors (ERs), mainly to beta-receptors (ERs-\u03b2), makes them promising molecules in replacing hormones for therapeutic purposes.Very early, in the beginning of the research in this field of study, it became evident that isoflavones were less potent than endogenous and synthetic hormones but it also became obvious that they had fewer side effects. Despite the huge amount of results from many epidemiological trials, it is not possible to make a good correlation between isoflavones and soy extracts being sold today \u20135.Epidemiological data prove that both genders of Asian population are less affected by hormone-dependent tumours. This fact as been directly related to the cultural diet rich in soy, but other factors could be also involved, such as the low amount of saturated fatty acids ingested, different ways to deal with stress and possible low levels of cortisol, among others, since Indians did not eat much soy and are also protect against these tumours.Since the most active isoflavone, which is also the main isoflavone found in soy seeds, is genistein and its derivatives, we decided to compare OTC tablets containing isoflavones extracted from soy, available in the Portuguese market and the isoflavones extracted from soy seeds supplied by the United States Department of Agriculture (USDA) Germplasm bank. These formulations where all indicated for the treatment of hot-flushes and post-menopausal related problems where the content in isoflavones it was critical for the referred bioactivity. The major conclusion of this comparative study was that important differences existed between the OTC formulations and the content of genistein and other isoflavones in raw soy seeds extracts. The quantification of the isoflavones present in OTC formulations was sometimes lower than the expected levels \u20138. TheseIf we consider that it is not yet clear how much of a dose of isoflavones is necessary to observe a physiological effect and if the soy protein has any synergic contribution, we should be concerned about what is really contained in the OTC Tablet preparations of soy isoflavones and what kind of quality control should be established \u20139. The rDespite the number of clinical trials performed with isolated isoflavones or with isoflavones included in soy or red clover food products, results did not clarify its efficacy or whether they could be used safely as a treatment as it is claimed. On the other hand, the U.S. Food and Drug Administration (FDA) back in 1999 made a statement that soy protein can be used with beneficial results in the prevention of cardiovascular diseases, and recommended that its extracts should be standardised, however the amount of isoflavones that can be on the product was not defined.The OTC tablet preparations of isoflavones extracted from soy that we analysed showed as major bioactive compounds the glycosylated isoflavones genistin, daidzin and glycitin and minor amounts of the malonyl and/or methylated derivatives of genistin and daidzin. The aglycones only appeared in very low amounts. The seeds, supplied from the USDA Germplasm Bank, have a mixture of most of these compounds (conjugated or not) but also have low amounts, some times only traces, of the aglycones \u20139.The evaluation of the efficacy of several extracts and the different results of clinical trials carried out with soy extracts or isolated isoflavones turns its usage into a very controversial subject. Scientists are now aware of the phenomena. Many are now studying the effects of formulations available to post-menopausal women. Given the difficulty of prescribing such formulations, the aim of the present work was to establish a theoretical calculation suitable to be employed as a way to estimate the \u201cTheoretical Efficacy (TE)\u201d of a mixture of different bioactive compounds in a way to obtain a \u201cTheoretical Efficacy Related to Estradiol (TERE)\u201d. Ultimately, different extracts could be compared, even when the relative amounts present in the extracts are very different.2.hot-flush, menopause, isoflavones, genist-, daidz-, glycit-, clinical trials. We also used the selected papers by a panel of Phytohealth members that had reviewed studies and classified them according to the grading criteria proposed by Harbour and Miller [phytoestrogens, isoflavones, genistein, daidzein, equol, soy (a); cross-referenced with the keywords: post-menopausal, hot-flushes, osteoporosis, bone mineral density, bone metabolism, cardiovascular, endothelial function, vascular reactivity, blood pressure, lipid profile, breast, colon, cognition. In order to make cross comparisons between studies that used a range of sources of isoflavones the doses of isoflavones were calculated as aglycone equivalents.Data from literature and reviews of selected clinical trials: a literature survey was made, using the following search engines: Elsevier-Science Direct, PubMed, SpringerLink, Taylor & Francis, Web of Science (ISI) and B-on. The search included the following keywords: d Miller and the d Miller presenteIn this paper we use from the clinical data two important keys to the process; on one hand the relative proportions of the different isoflavones intake and on the other, the efficacy of the treatment in reducing hot-flushes that is one of the parameters more cited in the most of the essays. To do the theoretical calculation these am3.3.1.The isoflavone content (daidzin and genistin and their derivatives) clearly differs between seeds and OTC tablet preparations extracted from soy. In these OTCs the main compounds are the glycosidic forms, glycitin, daidzin and genistin, but the relative proportions change from one sample to another. From the clinical trials used in the selected reviews, the levels of active compounds used to perform the assays differed significantly and the results are very difficult to compare.We found in literature that there is a consensus that glycosylated and methylated isoflavones have an entero-hepatic absorption where they undergo hydrolysis and afterwards are converted to the sulphonated or glucuronated forms. The aglycones undergo the same metabolization.Daidzein and genistein do not have the same binding affinity to alpha- and/or beta-ERs . Also knHowever, it is also accepted that beta-ERs are mainly located in bones, brain, thymus, bladder, cardiovascular system and its activation by estrogenic compounds, or compounds with the ability to mimic estrogenic molecules, such as phytoestrogens, can improve and prevent conditions like osteoporosis and cardiovascular diseases ,18 and tIn global terms the total efficacy of the TERE will be determined adding these two values. However the amount linked to the alpha-receptors will be consider as a possible risk limitation that needs to be evaluated when the increased dose will induce an improved TERE but the risk assessment for the toxicity can be a handicap.In order to allow an assessment between results obtained from clinical trials and different substrates submitted for therapeutic evaluation, the following calculation is proposed.et al., ERs affinity binding for estradiol for some isoflavones but the some theoretical calculations can be done with other affinities published. Utilising these TERE calculations will be possible to compare results of the bioactivity between various clinical trials or other kind of bio-evaluations.To demonstrate the calculation method, let us consider for example two different clinical trials, both using test formulas containing 100 mg of total isoflavones. In one of them the patients are taking pills with 25 and 75 mg of daidzein and genistein equivalents, respectively, but in the other trial patients ingest 75 mg of daidzein and 25 mg of genistein, which is exactly the inverse of the previous formulation. Looking at Keeping the affinity differences in mind, the ER binding values for daidzein and genistein are a starting point to make the theoretical calculation of the efficacy of both mixtures see . The oveThis is currently the case of the clinical trials conducted with botanical extracts if they are not well characterized and/or not comparable in relation to the relatively amounts of the bioactive compounds. It is very common to see reported in literature that in a given trial was used an extract with bio-compounds without saying exactly which molecules were in the extract.Since isoflavones and other molecules are able to mimic estrogens and they all have a different affinity for the receptors involved in the therapeutic activity, the results cannot be compared. For example, opium has two main alkaloids, morphine and codeine, both with analgesic activity but the first is much more active that the methylated derivative (codeine). The therapeutic efficacy of different mixtures of an opium extract would be therefore dependent on the relative amounts of each compound in them even when calculated with these TE calculations.Applying the same methodology as in i.e., molecules that have a preferential binding to alpha-ERs.For all of them, nevertheless, the risk-benefit relationship is determined as roughly 2% and 98% respectively. The increase of dosage can signify an increase of the amount that can be related to the risk, Once the maximum binding rate of alpha-ERs is determined, the risk of possible tumour cell proliferation by the estrogenic activity of these compounds will be lower and the safety of these OTC tablet preparations of isoflavones extracted from soy, red clover, etc, will be more predictable. This is an example of the application for the TERE.For example, in the first calculations the deteHighly purified isoflavones, soy foods and soy supplements may stimulate the growth of pre-existing oestrogen-dependent breast cancer tumours. Using an animal model, Ju and colleagues comparedin vivo potential of equol and flavonoids, such as genistein, equol, apigenin and kaempferol was investigated by Breinholt and colleagues [The lleagues , in immain vivo\u201d estrogenicity of dietary flavonoids.In kaempferol- and equol-exposed mice the cytosolic alpha-ERs concentration was significantly increased when compared to the solvent control, which is speculated to result in an increased sensitivity of the uterus to subsequently encountered estrogens. Oral administration of equol, genistein, biochanin-A and daidzein to 6-week-old female mice revealed a large variation in their systemic bioavailability. Other researchers found similar data. Bioavailability, metabolism, the ability to alter alpha-ERs distribution in the uterus and the estrogenic potential of parent compounds and metabolites may consequently contribute to the differences in \u201cCarcinogenesis, suggests that isoflavone-containing products consumed in the US may have lost many of the biologically active components in soy and, therefore may not have the same health benefits as traditional soy foods.The essays published so far did not reveal yet the full risk assessment of these concentrated extracts. A study reported in the May 2004 online issue of the journal In fact, the profile of the isoflavones is different in soy seeds and in tet al. [This TERE can be supported by the data collected by Williamson-Hughes et al. and by Ket al. . They fo4.Much research as been published relating to the issue of \u201cphytoestrogens in human health\u201d, but few recommendations can be made because most of the results seem to be controversial . Maybe, The authors declare that they are not for or against the use of isoflavones in human health applications and that they just wish to attempt to clarify this controversial issue. For instance, the verified estrogenic bioactivity shows an important relationship between genistein dose and the reduction of hot flushes and night sweats. These unpleasant effects observed in post-menopausal women are caused by lower oestrogen levels that fail to regulate body temperature via the hypothalamus. The anti-dopaminergic effect induced by estrogens can be rIn order to prevent the risks that this therapeutic option can cause in long term use, much research still needs to be done to prove the safety of isoflavones. Fortunately, many of the issues commented here will be under investigation. Studies of the effects of soy isoflavones found in dietary supplements on various parts of the body are under way in various research groups.Some of them are investigating how different doses of isoflavones and the timing of exposure affect breast, brain and adipose tissue, and how the interaction between isoflavones and oestrogen receptors takes place. More information is needed on the biological effects of pure isoflavones and complex mixtures of the various soy isoflavones commonly found in commercial OTC tablet preparations of isoflavones extracted from soy and if the matrix induces changes in the bioability of these compounds."} +{"text": "NiCR-2H, like NiCR, can selectively oxidize guanines present in distinctive DNA structures , and notably, NiCR-2H oxidizes guanines more efficiently than NiCR. In addition, UV and 1H NMR studies revealed that NiCR is oxidized into NiCR-2H in the presence of KHSO5 at low molar ratios with respect to NiCR (\u22644).We report here a biophysical and biochemical approach to determine the differences in interactions of NiCR and NiCR-2H with DNA. Our goal is to determine whether such interactions are responsible for the recently observed differences in their cytotoxicity toward MCF-7 cancer cells. Viscosity measurement and fluorescence displacement titration indicated that both NiCR and NiCR-2H bind weakly to duplex DNA in the grooves. The coordination of NiCR-2H with the N-7 of 2\u2032-deoxyguanosine 5\u2032-monophosphate (5\u2032-dGMP) is stronger than that of NiCR as determined by Natural and synthetic nickel [especially Ni (II)] complexes can oxid50: ~70\u201380\u2009\u03bcM) toward MCF-7 cancer cells in the absence of any exogenous oxidant while NiCR had no effect on cell growth -ATP was purchased from MP Biochemicals . Quantification of 5\u2032\u2009\u200932P-labeled oligonucleotides was carried out using a Storm 860 phosphorimager and ImageQuant 5.1 software . Cell medium and supplements were acquired from Invitrogen . Cell Titer 96 AQueous One Solution (MTS) was purchased from Promega . Analysis of cell viability (the MTS assay) was carried out on a Thermo Scientific Multiskan Ex plate reader and the number of cells (dye exclusion assay) was counted using a hemocytometer under a microscope. DNA labeling was performed by incubating [\u03b3-32P]-ATP (30\u2009\u03bcCi) and T4 polynucleotide kinase (20 units) in the presence of an oligonucleotide (10\u2009pmol) at 37\u00b0C for 30\u2009min. Unreacted [\u03b3-32P]-ATP was removed using a MicroSpin G-25 column . Cells were maintained in advanced DMEM/F12 medium supplemented with 5% fetal bovine serum, L-glutamine and penicillin (50\u2009IU/mL), streptomycin (50\u2009\u03bcg/mL) and amphotericin B at 37\u00b0C in a humid atmosphere containing 5%\u2009\u2009CO2 and air.Oligonucleotides were purchased from Fisheroligos . NiCR and NiCR-2H were synthesized based on the previously published procedures , 21. Unl\u22121\u2009cm\u22121\u2009bp\u22121). Small aliquots of a concentrated stock solution of ethidium bromide (EB), NiCR, or NiCR-2H were added into a 2\u2009mL of DNA solution (1\u2009mM) in an Ostwald-type viscometer that was immersed in a thermostated water bath at 25\u00b0C to obtain the desired ligand/DNA ratios. After each addition, the solution was mixed by bubbling with N2. The time for the level of the liquid to pass between two marks on the viscometer was recorded using a stopwatch. The relative viscosities were calculated based on the published equations [Calf thymus (CT) DNA was dissolved in a mixture of sodium phosphate buffer and NaCl (100\u2009mM). The concentration of the DNA was determined by UV spectroscopy, using a molar extinction coefficient at 260\u2009nm was added to an aqueous solution (400\u2009\u03bcL) of NiCR (10\u2009mg) and incubated for 5\u2009min. The reaction mixture was then evaporated to dryness and the residue was suspended in acetonitrile (500\u2009\u03bcL). After filtration, the acetonitrile solution was concentrated under vacuum. The residue was dissolved in CF3COOD and subjected to 1H NMR measurements. The spectra of NiCR and NiCR-2H in CF3COOD were also recorded.KHSO\u03bcM) and ethidium bromide (5\u2009\u03bcM) were premixed in a 2\u2009mL of Tris-HCl buffer (10\u2009mM) and NaCl (100\u2009mM) and allowed to stand for 30\u2009min at 25\u00b0C. Small aliquots of a stock solution of the ligand were added into the DNA-EB complex solution until the 1\u2009:\u20091 ligand/DNA ratio was reached. After each addition, the mixture was incubated for 15\u2009min at 25\u00b0C prior to the fluorescence analysis (Ex: 546\u2009nm and Em: 605\u2009nm). The fluorescence spectra of the ligands in the absence of CT DNA and EB were measured and used as blanks.CT DNA (5\u20092O followed by lyophilization to dryness twice and then redissolving in D2O. A NiCR-2H stock solution (36.37\u2009mM) was also prepared in D2O. Each individual sample (600\u2009\u03bcL) was prepared by mixing the 5\u2032-dGMP stock solution with the NiCR-2H stock to make a final concentration of 50\u2009mM 5\u2032-dGMP and a desired ligand/5\u2032-dGMP ratio ranging from 0 to 0.33. The samples were then subjected to 1H NMR measurements.The 5\u2032-dGMP stock solution (100\u2009mM) was prepared by dissolving 5\u2032-dGMP in D5\u2219KHSO4\u2219K2SO4), the concentration of KHSO5 was two fold higher than that of oxone. NiCR (1\u2009mM) in water was mixed with a KHSO5 stock to obtain a desired ligand/KHSO5 ratio ranging from 0 to 10. The absorbance of each individual mixture was recorded from 350\u2013800\u2009nm.The oxone solution was freshly prepared in water prior to the reactions. Based on the molecular formula of oxone mixed with a small amount of 5\u2032\u2009\u200932P-labeled 1 in phosphate buffer and NaCl (100\u2009mM) was prepared by heating at 90\u00b0C for 5\u2009min and then slowly cooling down to 25\u00b0C, and incubated at 4\u00b0C overnight. 5\u2032\u2009\u200932P-labeled 1 was added such that the radiation of DNA solution was approximately 20,000\u2009cpm/\u03bcL. The DNA (1\u2009\u03bcM) was incubated in a mixture (10\u2009\u03bcL) of phosphate buffer , NaCl (100\u2009mM), NiCR or NiCR-2H (30\u2009\u03bcM), and KHSO5 (ranging from 0\u20131\u2009mM) at 25\u00b0C for 30\u2009min. After quenching the reaction by addition of NaHSO3 (1\u2009\u03bcL of 200\u2009mM stock), the DNA products were precipitated from NaOAc (0.3\u2009M) and EtOH at \u221280\u00b0C. After centrifugation, removal of the supernatant, and drying, the residue was treated with 10\u2009\u03bcL of piperidine (1\u2009M) at 90\u00b0C for 20\u2009min, concentrated, and resuspended in formamide loading buffer (5\u2009\u03bcL). Analytical oligonucleotide separations were carried out using 20% polyacrylamide denaturing gel ).All experiments were carried out in triplicate. A 15-mer oligodeoxynucleotide duplex (\u03bcL) containing ~3 \u00d7 103 cells was added into a 96-well microtiter plate and incubated in 5% CO2 incubator for 24\u2009h. This would allow cells to reach ~90% confluence. The nickel complex solution (50\u2009\u03bcL) was then added into the microtiter plate to reach a desired concentration and incubated for 72\u2009h at 37\u00b0C. After incubation, a Cell Titer 96 AQueous One Solution (20\u2009\u03bcL) was added to each individual well. Quantification of viable cells was done by measuring the absorbance at 492\u2009nm using a plate reader. Untreated cells and media with no cells were used as controls.All the experiments were carried out in triplicate. The nickel complex (NiCR or NiCR-2H) was predissolved in the medium and filtered using 0.2 micron sterile filter. The medium solution , the following equation was used.Atreated: The absorbance of the solution containing treated cells, Amedia: The absorbance of the media, Auntreated: The absorbance of the solution containing untreated cells.t-test was performed to show statistically significant (P < .05) and insignificant (P > .05) data.Minitab 15.0 software was used to determine the statistical significance. Two-sample Student's 50: 20\u2009\u03bcM) toward MCF-7 cells than NiCR in a statistically significant manner, and NiCR barely has any effect on inhibition of the growth of MCF-7 cells. These observations are consistent with the previous report [50 value for NiCR-2H is less than previously reported. The IC50 values of NiCR and NiCR-2H for HeLa and A549 cells could not be determined within the concentration range used for the two nickel complexes (\u03bcM) and NiCR-2H (200\u2009\u03bcM) were 25% and 35%, respectively. Surprisingly, dye exclusion staining resulted in 90\u2013100% cell viability for all cells at all NiCR and NiCR-2H concentrations. The disagreement between cytotoxicity of known drugs and dye exclusion results has been previously reported in [Our initial attempts were to find a general trend of cytotoxicity of NiCR-2H toward different cancer cells. Three cancer cell lines, HeLa , A549 (human lung cancer), and MCF-7 (human breast adenocarcinoma) were chosen for study. Dye exclusion staining and the MTS assay were used to determine the cytotoxicity. In a dye exclusion test, dead cells are blue because they cannot exclude the dye molecule (trypan blue) in the media. In a MTS assay, the absorbance of a reduction product (formazan) from a tetrazolium salt (MTS) is determined spectroscopically. Only live cells are able to release active reductases that catalyze the reduction reaction; therefore, the absorbance of formazan is proportional to the number of live cells in culture. The results from the MTS assay are shown in s report . It is nomplexes . Both Niorted in \u201325. Dye orted in . Dead ceThe pioneering work by Burrows and coworkers revealed a minimum binding of NiCR to duplex DNA . Later, Because molecular interactions of NiCR and NiCR-2H with duplex DNA had not been fully investigated until the present work, we have been able to characterize these interactions using simple and reliable procedures of viscosity measurement , 28. Non\u03bcM) was titrated with 9-aminoacridine , a decrease in fluorescence (Ex: 546\u2009nm and Em: 605\u2009nm) of the solution was clearly observed. A dose-dependent reduction in fluorescence was found with up to 97% reduction at 100\u2009\u03bcM 9-aminoacridine as compared to the control (\u03bcM) only gave rise to a subtle decrease in fluorescence (\u03bcM) or NiCR-2H (100\u2009\u03bcM) after subtracting the background intensity is 9-aminoacridine (~19\u2009\u03bcM) > NiCR-2H (~278\u2009\u03bcM) > NiCR (~327\u2009\u03bcM). The C50 values for NiCR-2H and NiCR were obtained by extending the titration curves to reach the theoretical 50% reduction. A quantitative analysis [50 values in conjunction with the previously published binding constant of EB (107\u2009M\u22121) and the EB concentration (5\u2009\u03bcM) gives the apparent binding constant of 2.6 \u00d7 106\u2009M\u22121, 1.8 \u00d7 105\u2009M\u22121, and 1.5 \u00d7 105\u2009M\u22121 for 9-aminoacridine, NiCR-2H, and NiCR, respectively. The binding constant of 9-aminoacridine to CT DNA derived from the fluorescence titration experiments can prevent positively charged species (NiCR and NiCR-2H in our case) from binding to DNA due to the electrostatic repulsion. Hence, the binding of NiCR or NiCR-2H with DNA at 100\u2009mM NaCl is expected to be weaker than that in 10\u2009mM NaCl. Based on the results of viscosity and fluorescence titration, we conclude that NiCR and NiCR-2H bind weakly to duplex DNA in the grooves under physiological conditions. The interactions between small molecules and DNA can also be determined spectroscopically , 31. Whe control . In contrescence . An apprntensity , suggest > NiCR ~7\u2009\u03bcM. Theeriments is compacridine ~\u2009\u03bcM > NiCFurther evidence for the weak binding comes from the UV denaturation experiments. The melting temperatures of a 22-mer (AT tracts) or a 16-mer (mixed base) DNA oligonucleotide duplex are independent of the concentration of NiCR or NiCR-2H , suggesting that both complexes cannot stabilize duplex DNA probably due to the minimal binding. Collectively, the little quantitative differences in the binding of NiCR and NiCR-2H with DNA lead us to conclude that the binding of the two with DNA should not be responsible for the differences in cytotoxicity. 2O and guanine) changes its geometry from square planar to octahedral (2O containing 5\u2032-dGMP (50\u2009mM) and NiCR-2H varied over the range of 0\u201316.5\u2009mM was individually prepared to guarantee an accurate 5\u2032-dGMP/NiCR-2H molar ratio, and the 1H NMR spectra of these solutions were recorded. The resulting spectra between 3\u201310\u2009ppm are shown in Metal complexes are known to coordinate with guanine because the N-7 position of guanine is the most nucleophilic site . The cootahedral . The cootahedral was used32P-labeled DNA oligonucleotide duplex containing guanines in a bulge region . KHSO5 is proved to be a necessity to produce detectable amounts of DNA damage products under the same conditions. Like NiCR, NiCR-2H in the presence of KHSO5 could not directly produce strand breaks in DNA. However, it undoubtedly damaged DNA because strand breaks (faster moving DNA cleavage products) were detected by gel electrophoresis after treatment of reacted 1 with hot piperidine. The overall cleavage patterns of 1 produced by NiCR-2H are similar to those by NiCR , the amounts of cleavage products at G2 and G3 by NiCR were (22.3 \u00b1 1.3)% and (11.4 \u00b1 0.9)% and the amounts of cleavage products at G2 and G3 by NiCR-2H were (29.4 \u00b1 0.9)% and (16.5 \u00b1 1.2)%, respectively. NiCR-2H in general provides 5%\u20139% more of damaged guanine products than NiCR , the sums of the cleavage products at G1 and A1 for NiCR and NiCR-2H were (57.4 \u00b1 4.8)% and (68.4 \u00b1 1.6)%, respectively. We believe the changes in damage sites result from the destabilization of 1 by the high concentration of KHSO5. This destabilization effect was confirmed by circular dichroism . The destabilization of 1 was only observed when NiCR and KHSO5 were both present in the solutions, and KHSO5 alone had no effect on the stability of 1 . When 1 dissociates into random coils, the bulge region no longer exists. In the random coils, G1 and A1 located at the end of the DNA are less well protected than G2 and G3 in the middle of the DNA; therefore, the nickel complexes mainly hit on the less-protected nucleobases. The oxidation of adenine (A1) observed in our experiments has not previously been reported; however, A1 could simply be overoxidized by the large excess of KHSO5. Our results for the first time directly compare the efficiency of NiCR and NiCR-2H to oxidize DNA. Both complexes mainly oxidize guanines present in the bulge of 1 in the presence of KHSO5, and NiCR-2H more readily oxidizes guanines than NiCR. The oxidation potentials of NiCR and NiCR-2H should not be responsible for their difference in guanine oxidation because both complexes have similar oxidation potential values as previously determined in [1H NMR. NiCR can selectively oxidize guanines present in distinctive DNA structures such as bulges and loops in the presence of an oxidizing agent . Informaegion 1, for the cterized . In our by NiCR . The Max by NiCR shows thhan NiCR , suggestof KHSO5 0\u2009\u03bcM, the5 at the physiological temperature with a prolonged incubation time. DNA 1 was incubated with either NiCR or NiCR-2H at various concentrations in the absence of KHSO5 at 37\u00b0C for 18\u2009h followed by hot piperidine treatment. The DNA cleavage products obtained under this reaction condition was only 1%\u20135%, which is much less as compared to those obtained in the presence of KHSO5. The Maxam-Gilbert method confirmed that both complexes still mainly oxidized guanines in 1 but with no preference to G2 and G3 in the bulge region. . NiCR-2H produced ~2\u20133.5% more cleavage products than NiCR. The minimal DNA oxidation by NiCR and NiCR-2H in the absence of KHSO5 seems not to be responsible for the observed differences in cytotoxicity to MCF-7 cells. However, this conclusion is drawn without taking endogenous oxidants into consideration. Endogenous oxidants such as reactive oxygen species (ROSs) are known to promote the DNA damage induced by metal complexes [Because the cytotoxicity was determined by incubating NiCR or NiCR-2H with cultured cells in the absence of any exogenous oxidant, we then investigated the DNA damage by NiCR or NiCR-2H in the absence of KHSOomplexes , 39. NiC5 with different molar ratios to NiCR was first investigated using UV absorption spectroscopy. The spectrum of NiCR showed a maximum absorption at 399\u2009nm and a weak absorption at 720\u2009nm (\u03bbmax\u2009) increased dramatically as a function of the concentration of KHSO5 ranging from 0 to 4\u2009mM (KHSO5/NiCR \u2264 4). A blue shift of \u03bbmax from 399\u2009nm to 394\u2009nm was observed (\u03bbmax at 394\u2009nm and no absorption at 720\u2009nm as well. When the concentration of KHSO5 was over 4\u2009mM (KHSO5/NiCR > 4), the absorption at 394\u2009nm decreased accompanying a red shift of 14\u2009nm to \u03bbmax at 408\u2009nm, suggesting that a secondary oxidation occurred was gleaned from 1H NMR. The diamagnetic NMR spectra of NiCR and NiCR-2H were obtained using CF3COOD as a solvent, suggesting that CF3COOD is a weak ligand that cannot form a paramagnetic complex with NiCR or NiCR-2H. The reactions of NiCR with 3 or 10 equivalents of KHSO5 were carried out, and the resulting products were measured by 1H NMR. The spectra of NiCR, NiCR-2H, and the oxidation products are shown in 5 is very similar to that of NiCR-2H intermediate.Additional support for the oxidation of NiCR into NiCR-2H at low KHSO NiCR-2H . The sig NiCR-2H because NiCR-2H . The res\u03bcM) and both NiCR and NiCR-2H have neglectable effect on HeLa and A549. In order to understand the differences in cytotoxicity, we in this paper have investigated the interactions of NiCR and NiCR-2H with DNA. We conclude that the differences in cytotoxicity should not result from the differences in the binding of NiCR and NiCR-2H with DNA because both complexes bind weakly in the grooves of DNA with no quantitative differences. Both NiCR and NiCR-2H damage DNA with a similar sequence preference, and NiCR-2H more readily oxidizes guanine than NiCR in the presence of KHSO5 probably due to its stronger coordination with guanine. The differences in oxidation of guanine between NiCR and NiCR-2H could be a key to the differences in cytotoxicity. However, this is not conclusive because the role of exogenous oxidants is unknown. We have also obtained the direct evidences for oxidation of NiCR into NiCR-2H at low molar ratios of KHSO5/NiCR, suggesting NiCR-2H could act as an important precursor for the previously proposed Ni (III) intermediate. The investigation of molecular interactions of NiCR and NiCR-2H with DNA is the first step toward understanding the differences in cytotoxicity. The ultimate explanation on this matter must be more complicated and requires understanding of the biological responses of NiCR and NiCR-2H in vivo such as cellular uptake and cellular metabolism.Amongst three cancer cell lines, NiCR-2H is only cytotoxic to MCF-7 cells (IC50: 20\u2009Details of experimental procedures and spectra for UV denaturation of DNAoligonucleotide duplexes with NiCR and NiCR-2H, circular dichroism titration of 1with oxone, and DNA cleavage studies under various conditions, and dye exclusionstudies. This material is available online on doi: 10.1155/2010/619436.Click here for additional data file."} +{"text": "Exposure of the cells to aluminium fluoride also reduced the affinity for BK. Bradykinin-induced release of ARA proved pertussis toxin (PTX) sensitive, with a maximum sensitivity at 10 ug/ml PTX. GTP\u03c4S at 100 \u03bcM increased the release of arachidonate. The effect of GTP\u03c4S and BK was additive at suboptimal doses of BK up to 0.5 nM but never exceeded the levels of maximal BK stimulation at 50 nM. PTX also inhibited the release of ARA induced by the calcium ionophore, A23187. Phorbol 12-myristate 13-acetate or more commonly known as tetradecanoyl phorbol acetate (TPA) itself had little effect on release by the intact cells. However, at 100 nM it augmented the BK activated release. This was downregulated by overnight exposure to TPA and correlated with down-regulation of protein kinase C (PKC) activity. The down-regulation only affected the augmentation of ARA release by TPA but not the original BK activated release. TPA displayed a similar, but more potent amplification of PAF synthesis in response to both BK or the calcium ionophore A23187. These results taken together point to the participation of G-protein in the binding of BK to BPAEC and its activation of ARA release. Possibly two types of G-protein are involved, one associated with the receptor, the other activated by Ca2+ and perhaps associated with phospholipase A2 (PLA2). Our results further suggest that a separate route of activation, probably also PLA2 related, takes place through a PKC catalysed phosphorylation.Regulatory mechanisms in bradykinin (BK) activated release of arachidonate (ARA) and synthesis of prostaglandin (PG) and platelet activating factor (PAF) were studied in bovine pulmonary artery endothelial cells (BPAEC). A role for GTP binding protein (G-protein) in the binding of BK to the cells was determined. Guanosine 5-O- (thiotriphosphate), (GTP\u03c4S), lowered the binding affinity for BK and increased the Kd for the binding from 0.45 to 1.99 nM. The Bmax remained unaltered at 2.25 \u00d7 10"} +{"text": "P=0.015), which surveillance or detection bias may not fully explain.Using data from the Geneva Cancer Registry, we found that in 2002\u20132004, breast cancer incidence in women aged 25\u201339 years increased by 46.7% per year (95% CI: 7.1\u201374.0, Health professionals and patient support groups in Geneva, Switzerland, have recently expressed concern about an apparently increasing number of very young breast cancer patients. Owing to the average 2-year delay in cancer registration, we have only now been able to investigate this observation using incidence data up to the year 2004 among women residents in Geneva.The Geneva cancer registry, functional since 1970 and covering the whole population of the canton (approximately 435\u2009000 inhabitants), is considered comprehensive with a low percentage (<2%) of cases recorded from death certificates only . Trainedn=3608), with the population at risk considered as the resident population at the middle of each relevant year, obtained from the Cantonal Population Office. We calculated annual incidence rates for five age groups: 25\u201339, 40\u201349, 50\u201369, 70\u201379, and \u2a7e80 years. Trends in age-specific annual incidence rates were calculated by log-linear Poisson regression implemented in the generalised linear interactive modelling statistical package , histological type , differentiation ), oestrogen and progesterone receptor status (positive (if \u2a7e10% of cells expressed receptors), negative, and unknown), mean pathological tumour size (in mm), and stage ). We used the pathologic pTNM classification system or, when absent, the clinical cTNM classification. Tumour stage was considered as stage I (T1 and N0), stage II , stage III , stage IV (M1) and unknown. We also examined the proportion of tumours with clinical T0 N0 M0.Variables of interest included family history of breast or ovarian cancer , method of detection , modalities of diagnostic assessment : yes P=0.003) over the whole period. The entire increase occurred in the last 3-year period (2002\u20132004) with a mean annual increase of 46.7% . Cancers at ages 25\u201339 represented 3.4% of all breast cancers in 1995 and 7.2% in 2004 (P=0.032). Also, since 1970 (when cancer registration started in Geneva) no such increase has been observed (data not shown). For the other age groups, incidence remained fairly stable, except for women aged 50\u201369 years among whom it increased at an average of 2.6% per year, from 1998 to 2002, when it stabilised . Screen-detected cancers increased non-significantly from 9 to 19%. The proportion of stage I cancers slightly decreased from 40 to 33%, whereas the proportion of stage II cancers remained relatively constant around 45%. Only three women had non-clinical T0 N0 tumours, one in the first and two in the second period. The mean tumour size remained unchanged over the whole study period, at 21\u2009mm in 1995\u20132001 and 20\u2009mm in 2002\u20132004 (P=0.817). We observed no increase in the proportion of young patients with a positive familial history of breast cancer.P=0.006).In Geneva, breast cancer incidence in women aged <40 years has recently doubled. This increase may be partly explained by a higher screening frequency of younger women and better surveillance and recognition of familial risk factors. Improved tumour detection through advances in imaging techniques may also be involved, since in 1995\u20132001 less than 30% of breast cancer diagnoses in young women involved MRI compared with nearly 50% in 2002\u20132004 starting early in the study period, but this is already a well-documented phenomenon in Switzerland and Cancer Incidence in Five Continents (CIF) . Howeverin utero and early in life, would be indicated.In conclusion, we observed a significant increase in young breast cancer patients. At present, we cannot definitively rule out an increased surveillance and detection bias and we cannot confidently conclude a sustained increase. Careful surveillance of recent trends of breast cancer incidence is required for young women. If other population-based cancer registries confirm this trend, further research on breast cancer risk factors, including any acting"} +{"text": "Community-acquired pneumonia is one of the most common causes of sepsis and ICU admissions. Patients with CAP who demand critical care had mortality rates of 25 to 50%. Thereby, the assessment of the severity is essential to guide the treatment. There are several severity scores for CAP and some of the most acknowledged are CURB-65 and CRB-65. The objective of this study was to evaluate the accuracy of CURB-65 and CRB-65 as predictors of death in patients with community-acquired pneumonia.A prospective study during 6 months was conducted with patients diagnosed with CAP admitted to the ICU of the Hospital Santa Luzia, Bras\u00edlia, DF, Brazil. Patients were stratified according to CURB-65 (0 to 5) and CRB-65 (0 to 4) and their risk categorized as: low (CURB-65: 0 to 1 and CRB-65: 0), moderate (CURB-65: 2 and CRB-65: 1 to 2) and high (CURB-65: 3 to 5 and CRB-65: 3 to 4). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), likelihood ratio positive (LR+), and likelihood ratio negative (LR-) were calculated. Validity and reliability were assessed with the Spearman correlation coefficient. Patients with chronic kidney failure and those submitted to mechanical ventilation at the time of admission were excluded.P = 0.00). See Figure A total of 62 patients were included. Twenty-seven with low risk, 24 with moderate risk and 11 with high risk according to CURB65 and their mortality rates were 7.4%, 8.3% and 54.5%, respectively. According to CRB-65, 11 were low risk, 44 moderate risk and seven had high risk. The mortality on CRB-65 stratification was 0%, 15.9% and 42.9% for low, moderate and high risks, respectively. When we gathered moderate and high risks, CRB-65 was more sensitive (1.00 vs. 0.80) and had better LR- (0.00 vs. 0.41), and NPV (1.00 vs. 0.92). CURB-65 had better specificity (0.48 vs. 0.21), LR+ (1.54 vs. 1.26), and PPV (0.23 vs. 0.20). The receiver operating characteristic curves of CURB-65 and CRB65 had areas of 0.758 and 0.686, respectively. The Spearman correlation coefficient was 0.612 (CURB-65 and CRB-65 had a high correlation. CRB-65 was more sensitive as a predictor of death as well as a guidance for hospitalization. Moreover, CRB-65 is a more practical score since it does not use laboratorial variables."} +{"text": "Aim. The aim of this study was to characterize the genetic profile of patients with chronic hepatitis C virus (HCV) infection relative to polymorphisms rs12979860 and rs8099917 in gene IL28B and the association of those polymorphisms with the response to treatment with pegylated interferon and ribavirin, performed at a reference center in Brazilian Amazonia. Methods. A total of 75 individuals with chronic hepatitis C and 98 healthy individuals from both genders over 18 years old were assessed. DNA samples were collected from leukocytes and subjected to real-time polymerase chain reaction to genotype polymorphisms rs12979860 and rs8099917. Results. Analysis of the allelic and genotypic frequencies of the investigated polymorphisms showed that both groups were in Hardy-Weinberg equilibrium; polymorphism rs12979860 exhibited no significant difference between the groups. For polymorphism rs8099917, allele T was significantly less frequent (P = 0.0195) among the patients (63.3%) than the controls (75.5%), and the patients were 1.7 times as likely to exhibit allele G. No difference in response to treatment was associated with SNP patterns. Conclusion. The results suggest a possible association of SNP rs8099917 with higher odds of chronic HCV infection but do not indicate a putative influence of the investigated SNPs on the sustained virologic response. Hepatitis C is considered to be an insidious disease, and its natural history has not yet been fully elucidated. According to some studies, 55 to 85% of the individuals affected by the acute form of disease remain infected for over six months and become chronic carriers . In thes\u03bb1), IL-28A (IFN-\u03bb2), and IL-28B (IFN-\u03bb3). These cytokines exhibit significant antiviral, antiproliferative, and antitumor activity and are expressed by mononuclear cells, monocyte derivatives, and dendritic cells when viral infection occurs TaqMan Universal PCR Master Mix [2X], [1X] TaqMan Assay [20X], and 20\u2009ng of DNA in a total volume of 10\u2009The viral load and genotyping tests were performed at Central Laboratory using the qualitative and quantitative PCR-HCV-RNA method (Roche-COBAS AMPLICOR Hepatitis C Virus (HCV) Test, version 2.0\u2009v). For the purpose of analysis, the viral load values were dichotomized as low or high . The histopathological assessment followed the French Metavir classification .t-test were used for quantitative data, and the nonparametric chi-square test (with the Yates correction), Fisher's exact test, and G-test (with Williams' correction) were used for qualitative nominal or ordinal data. The genotypic and allelic frequencies found were assessed using the Hardy-Weinberg equilibrium.Descriptive and inferential analyses were performed as a function of the normality of the data. Parametric ANOVA and Student's The degree of association between genotypes and viral infection was measured using the odds ratio (OR) and corresponding 95% confidence intervals (CI). The analyses were performed using the software Epi Info 3.5.3 and BioEClinical and laboratory assessment of the stage of hepatitis showed that 76% (57/75) of the participants did not exhibit liver cirrhosis, while 24% (18/75) did.The viral genotype was established in 70 participants, with genotype 1 in 77.1% (54/70) of the cases and genotype 3 in 22.9% (16/70). Among the participants subjected to treatment, 80.5% (33/41) exhibited genotype 1, and 19.5% (8/41) exhibited genotype 3.The viral load was measured in 61 participants and was less than 600,00\u2009IU/mL in 63.9% (39/61) of the cases.Liver biopsy was performed in 59 participants; histopathological analysis showed that 61% (36/59) of the biopsies exhibited activity periportal or periseptal scores of zero to one, and 59.3% exhibited fibrosis scores of two to four.P = 0.0813) to a higher frequency of allele T was found in the group of patients compared to the controls, but no significant difference in the genotypic frequency (P = 0.1865) was found between the two groups.Analysis of the SNP rs12979860 allelic and genotypic frequencies showed that both groups (patients and controls) were in Hardy-Weinberg equilibrium . A tendeP = 0.0195). In addition, a tendency (P = 0.0600) to a higher proportion of genotype TT (P = 0.0413) was found in the group of controls (58.2%) compared to the patients (41.3%), whereas the frequency of genotype GG was twice as high among the patients (14.7%) compared to the controls (7.1%).Analysis of the polymorphism rs8099917 allelic frequency showed tAmong all patients treated with PEG-IFN + RBV, 61% (25/41) were undergoing their first treatment for hepatitis C, and 39% (16/41) were being retreated due to the failure of treatment with conventional IFN.Intention-to-treat analysis resulted in 51.21% of ERT (21/41) and 39% of SVR (16/41). Treatment discontinuance due to NR or non-ERT occurred in 41.4% of the cases (17/41), and intolerance to side effects occurred in 9.7% (4/41) . NeitherIn this study, no significant difference was found in the genotype and allele proportions of SNP rs12979860, possibly due to the small size of the sample of individuals with hepatitis C. Nevertheless, genotype CT predominated among the patients 49.3%), which agrees with the results reported by Ge et al. for Nort%, which The allelic distribution of polymorphism rs12979860 in the group of patients was similar to the distribution described by Venegas et al. relativeP = 0.06) to a higher frequency of genotype TT among the controls and GT among the patients. The higher frequency of the heterozygous genotype GT and homozygous genotype GG among the patients agrees with the results reported by Venegas et al. [Analysis of the SNP rs8099917 genotypes revealed a tendency and SVR in individuals with genotype 1 , 11, 27,"} +{"text": "Therefore, we investigated these correlations.Atelectasis can provoke pulmonary and non-pulmonary complications after general anaesthesia. Unfortunately, there is no instrument to estimate atelectasis and prompt changes of mechanical ventilation during general anaesthesia. Although arterial partial pressure of oxygen . Atelectasis was calculated by computed tomography relative to total lung mass . We logarithmically transformed PaO2 (lnPaO2) to linearize its relationships with shunt and atelectasis. Data are given as median (interquartile range).Shunt, PaOtotal was 768 (715\u2013884) g in sheep and 543 (503\u2013583) g in pigs. Atelectasis was 26 (16\u201347) % in sheep and 18 (13\u201323) % in pigs. PaO2 (FiO2 = 1.0) was 242 (106\u2013414) mmHg in sheep and 480 (437\u2013514) mmHg in pigs. Shunt was 39 (29\u201351) % in sheep and 15 (11\u201320) % in pigs. Atelectasis correlated closely with lnPaO2 (R2 = 0.78) and shunt (R2 = 0.79) in sheep . The correlation of atelectasis with lnPaO2 (R2 = 0.63) and shunt (R2 = 0.34) was weaker in pigs, but R2 increased to 0.71 for lnPaO2 and 0.72 for shunt 12 hours after induction of ARDS. In both, sheep and pigs, changes in atelectasis correlated strongly with corresponding changes in lnPaO2 and shunt.M2 and shunt, when blood gases are measured during ventilation with pure oxygen. In lung-healthy pigs, these correlations were significantly weaker, likely because pigs have stronger hypoxic pulmonary vasoconstriction (HPV) than sheep and humans. Nevertheless, correlations improved also in pigs after blunting of HPV during ARDS. In humans, the observed relationships may aid in assessing anaesthesia-related atelectasis.In lung-healthy sheep, atelectasis correlates closely with lnPaO In in pigs . This ag2 or lnPaO2. This confirms a previous report for homogeneous, anaesthesia-related absorption atelectasis [Others suggested that the activity of HPV can be assessed by the ratio between atelectasis and shunt ,44. Theslectasis , while ilectasis .2 and/or shunt measurements during anaesthesia ventilation [Obviously, validation of our results in human patients appears necessary. However, we could not yet identify a population of lung-healthy patients who undergo CT of the lung during general anaesthesia and have an arterial catheter at the same time. Besides validation, such results would aid in providing a look-up table for approximation of the amount of atelectasis from lnPaOtilation .2) may alter the perfusion of atelectatic lung regions. Hypocapnia, which reduces or even abolishes HPV, was avoided [2, however, would have generated unrealistic results with limited applicability in the clinical anaesthesia setting. Although animals in the present study showed neither clinical nor radiological signs of pneumonia, an infection potentially interacting with HPV cannot be excluded. Furthermore, anaesthetic drugs may alter HPV. However, the drugs used in our study are frequently used in the clinical setting and seem to have a limited effect on HPV [Results from the atelectasis model used in our study may apply to patients with atelectasis who are ventilated during anaesthesia but may not be directly transferable to patients with oedematous and/or inflamed lungs. Moreover, sheep have reduced collateral ventilation, which may influence the potential for developing absorption atelectasis and also the matching of aeration and gas exchange . Changes avoided . Furthert on HPV ,48.Using pure oxygen temporarily during measurement periods might be discussed controversially, since even short periods of pure oxygen ventilation can promote atelectasis and increase shunt, particularly during and after induction of anaesthesia ,49,50. NThe protocols of our experiments in pigs and sheep, from which the data for the current study was obtained, differ in certain points . These earlier experiments investigated different research questions and there is no overlap or duplicate publication of results.In sheep with absorption atelectasis breathing pure oxygen, we could show strong linear relationships between atelectasis and both, oxygenation and intrapulmonary shunt. After lung recruitment, reductions in atelectasis correlated with increments in oxygenation and decreases in shunt. Our results in sheep, whose HPV physiology has similarities to the human one, suggest that oxygenation and shunt could be used to estimate atelectasis. These finding extend our previous findings in ARDS patients to anaesthesia-related atelectasis. Pigs, which have a much more intense HPV than humans, did not show such strong correlations for atelectasis in healthy lungs. However, after induction of ARDS, these correlations were similarly strong for pigs and sheep.2 are frequently available in the perioperative setting, especially in critically ill patients or patients undergoing major surgical procedures, estimation of atelectasis based on the PaO2 could be an instrument to individualize lung protective ventilation and minimize atelectasis during general anaesthesia, provided that adequate measures are taken to blunt hypoxic pulmonary vasoconstriction.Since arterial catheters and thus the PaOS1 ProtocolSee (PDF)Click here for additional data file.S1 ARRIVE ChecklistSee (PDF)Click here for additional data file.S1 TableMeasurements obtained under ventilation with pure oxygen and at individual FIO2 .(CSV)Click here for additional data file.S2 TableVentilation with pure oxygen. Measurements at baseline and after recruitment with PEEP of 10cmH2O or 20cmH2O.(CSV)Click here for additional data file.S3 TableMeasurements obtained under ventilation with pure oxygen.(CSV)Click here for additional data file.S4 TableMeasurements obtained under ventilation with pure oxygen and at individual FIO2 . Pigs 23 and 14 were excluded due to malfunction of computer tomography. Pig 22 was excluded due to hyperkalaemia and renal failure. Pig 6 was excluded due to a pneumothorax. Due to mentioned reasons, measurements could only be obtained for 19 of 23 pigs studied with atelectasis in otherwise normal lungs before induction of ARDS.(CSV)Click here for additional data file."} +{"text": "CDC6 gene and cell proliferation, knockdown of DP1 and DP2 expression did not affect ectopic E2F1- or E1a-induced expression of the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p53, activation of p53 or apoptosis. These observations suggest that growth related and pro-apoptotic E2F targets are regulated by distinct molecular mechanisms and contradict the threshold model, which postulates that E2F activation of pro-apoptotic genes requires a higher total activity of activator E2Fs, above that necessary for E2F-dependent activation of growth-related genes.The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression by activating growth-related genes and pro-apoptotic tumor suppressor genes, respectively. It is generally accepted that E2F binds to target sequences with its heterodimeric partner DP. Here we show that, while knockdown of DP1 expression inhibited ectopic E2F1- or adenovirus E1a-induced expression of the E2F activity is regulated by retinoblastoma (RB) family proteins during the cell cycle. RB family proteins directly bind to and inactivate E2F in the resting state. Normal growth signals, such as serum stimulation of fibroblasts, induce phosphorylation of RB family proteins by cyclin-dependent kinases (CDKs), resulting in an increase in E2F transcriptional activity, activation of the growth-related target genes, and subsequent cell cycle progression3. Since the DP subunit is an obligate partner for efficient DNA binding of E2F to canonical E2F response elements (TTTC/GG/CCGC) in growth-related target promoters, it is also essential for promotion of cell cycle progression5.E2F family proteins (E2F1-E2F5) form heterodimeric complexes with DP family proteins (DP1 and DP2), generating E2F transcriptional activity that regulates expression of growth-related target genes3. Ectopic expression of E2F1 or deregulated endogenous E2F activity resulting from expression of adenovirus E1a, which binds to and inactivates the RB family proteins, induces apoptosis in serum-starved fibroblasts8. Accordingly, ectopic expression of E2F1 or E1a activates pro-apoptotic E2F target genes, including the tumor suppressor ARF gene, an upstream activator of the tumor suppressor p5310, which results in activation of p53 and induction of apoptosis.In addition to their role in cell cycle progression, E2F proteins, especially E2F1, also play a key role as mediators of apoptosisRB1 gene, which codes for pRB, is inactivated or mutated in many forms of human cancer, resulting in enhanced, or deregulated E2F activity, due to loss of pRB function12. Hereafter, we refer E2F activity induced by loss of pRB function or ectopic expression of E2F as deregulated E2F activity, and E2F activity induced by growth stimulation as physiological E2F activity. Upon loss of pRB function, the ARF gene is activated by deregulated E2F, resulting in activation of p539. Mice heterozygous for RB1 develop pituitary tumors13 and loss of ARF accelerates tumorigenesis14. In addition, tissue specific inactivation of p53, when combined with pRB inactivation, promotes the development of lung and ovarian tumors in mice16. These observations suggest that E2F regulation of the ARF-p53 pathway is crucial for prevention of tumorigenesis resulting from dysfunction of pRB.pRB plays a crucial role in tumor suppression mainly through restraining E2F activity. The ARF gene is distinct from that of growth-related target genes in that, unlike growth-related E2F targets, which are activated by both physiological and deregulated E2F activity, the ARF gene specifically responds to deregulated E2F activity17. In human normal fibroblasts, the ARF promoter responds to deregulated E2F activity induced by ectopic expression of E2F1 or inactivation of pRB by adenovirus E1a or short hairpin RNA (shRNA), but not to physiological E2F activity induced by serum stimulation. Growth-related target promoters, on the other hand, are activated by all of these stimuli. These observations suggest that the ability of the ARF promoter to discriminate between physiological and deregulated E2F activity serves as the basis for the ARF gene to function as a tumor suppressor gene in response to dysfunction of pRB17. To gain insight into the mechanisms, by which the ARF promoter is selectively activated by deregulated E2F activity, we identified and characterized the E2F-responsive element of ARF promoter (EREA). In contrast to typical E2F binding sequences (TTTC/GG/CCGC) in growth-related target promoters, EREA lacks the T nucleotides and is composed solely of GC repeats. ChIP assay showed that E2F1 bound to EREA upon over-expression of E2F1 or expression of E1a but not upon serum stimulation in normal human fibroblasts. These results suggest that EREA specifically binds deregulated E2F1.We previously showed that E2F regulation of the 2. In this model, a total pool of free activator E2Fs contributes to E2F transcriptional activity, which, upon reaching a critical threshold, induces growth-related genes that are involved in cell proliferation. Once E2F activity exceeds a second higher threshold, pro-apoptotic genes are activated, triggering cell death. However, given that the sequence of the atypical E2F binding site in the ARF promoter (EREA) is distinct from that of canonical E2F binding sites in growth-related target promoters, we postulated that E2F recognition of and interaction with EREA may be distinct from that of typical E2F binding sites, such that E2F binds to EREA by itself or together with a binding partner other than DP. We thus hypothesized that E2F regulation of the ARF gene may not conform to the threshold model or requirement for the heterodimeric partner DP.The molecular basis of how E2F discriminates target genes involved in the opposite cell fates of cell proliferation and apoptosis is not known. To explain E2F regulation of both cell proliferation and apoptosis, the threshold model has been proposedARF gene expression and apoptosis, in human normal fibroblasts, using shRNAs against DP1 and DP2 mRNA. The threshold model predicts that ARF gene expression and apoptosis would require higher E2F activity and therefore be more dependent on DP than E2F-dependent growth-related gene expression and cell cycle progression. Surprisingly, our results show that, while DP is essential for deregulated E2F induction of growth-related CDC6 gene expression and cell cycle progression, activation of ARF gene expression and apoptosis is independent of DP. These results contradict the threshold model and suggest that E2F regulation of pro-apoptotic tumor suppressor genes is not simply a function of elevated total E2F activity in concert with DP family members.In contrast to the established functions of DP in growth-related E2F-dependent gene expression and cell cycle progression, the role of DP in deregulated E2F-mediated pro-apoptotic gene expression and apoptosis has not been elucidated. Thus, in this study, we examined the requirement of deregulated E2F for DP in induction of ARF gene expression and apoptosis by deregulated E2F, we established shRNA-mediated knockdown of DP1 expression in human normal fibroblasts. We focused on DP1 as it is the predominantly expressed DP family member in human normal fibroblasts in Drosophila, is sufficient to inhibit binding of E2F to typical E2F binding sequences, leading to a reduction in growth-related target gene expression and cell cycle progression5. These observations indicate that DP1 is the primary functional DP family protein in human normal fibroblasts, and thus a logical target to investigate the role of DP in E2F regulation of transcription, cell cycle progression and apoptosis.To determine the role of DP proteins in the induction of sts Fig.\u00a05, over Dels Fig.\u00a0 and DP3 DP1 mRNA and found two of them (Ad-shDP1-2 and Ad-shDP1-3) were effective. As shown in Fig.\u00a0DP1 mRNA and protein in WI-38 cells ectopically expressing E2F1 or E1a 22. In contrast, the E2F-responsive element of the ARF promoter (EREA), necessary for activation of the ARF gene by deregulated E2F in human fibroblasts, is an atypical site composed of only GC repeats17. We therefore examined the effects of DP1 knockdown on the DNA binding activity of deregulated E2F1 to these distinct E2F response elements. We first performed gel mobility shift assays using typical E2F sites from DHFR promoter and EREA probes. Consistent with previous reports5, DP1 knockdown in WI-38 cells dramatically reduced the level of ectopically expressed E2F1 bound to the canonical DHFR E2F sites . Similarly, the E2F responsive element of the BIM promoter, which is also specifically activated by deregulated E2F activity, is also composed of GC repeats25. Moreover, in ChIP assays, binding of E2F1 to the GC repeat was only detected upon ectopic expression of E2F1 or E1a and not detected under normal growth conditions25. Thus, these pro-apoptotic tumor suppressor genes are only activated when deregulated E2F1 binds to the distinct response elements. In addition, while binding of physiologically induced E2F to a canonical response element requires DP1, binding of deregulated E2F1 to this atypical sequence is independent of DP proteins. Taken together, these observations imply that deregulated E2F is functionally distinct from its physiologically activated counterpart.The E2F responsive sequence of ARF promoter (GC repeat) is distinct from that of proliferation-related genes usually within 100\u2009bp from the transcriptional start sites3. In contrast, ARF gene contains atypical E2F binding sequence (GC repeat)17. It is interesting to note that, in the genome-wide analysis of over-expressed E2F1 binding sites in vivo26, only 12% of the identified sites are typical E2F binding sites (TTTC/GG/CCGC), with the remainder contain CGCGC motifs similar to the atypical E2F binding sequence of the ARF and BIM genes. The genes identified in that study contain not only growth-related and pro-apoptotic genes but also genes involved in DNA damage response, DNA repair, ubiquitin-like conjugation, mRNA processing, mRNA splicing and other cellular processes. We have also identified genes, which are regulated by E2F in the same manner as the ARF gene, such as p27Kip1, TAp73, BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP728. Among these, p27Kip1, TAp73 and ABTB1 contain typical E2F binding sites, but located more than 100\u2009bp upstream from the transcriptional start sites, and BIM, JMY, MOAP1, RBBP4 and RBBP7 contain CGCGC. Although all of the genes identified by over-expression of E2F1 may not be bona fide targets of endogenous deregulated E2F, we speculate that many of the genes containing CGCGC motifs may be regulated in the same manner as the ARF gene.In this study, we analyzed the ARF gene expression and apoptosis is independent of DP family members in contrast to CDC6 gene expression and cell cycle progression. Moreover, DP knockdown suppressed induction of CDC6 gene expression and cell cycle progression but not induction of ARF gene expression or apoptosis, which contradicts expectation from the threshold model. The differential regulation of growth-related genes and pro-apoptotic genes by E2F is not sufficiently explained by the total amount of free E2Fs, suggesting an additional layer of regulatory mechanism such as qualitative difference of E2F, which awaits to be elucidated.In summary, our results show that deregulated E2F induction of Normal human fibroblast WI-38 cells (RIKEN Bioresource Center Cell Bank) and 293A cells (Invitrogen) were cultured in Dulbecco\u2019s modified Eagle medium containing 10% FCS. Cells were trypsinized and counted at indicated time points after serum stimulation. Cell counting was performed three times and values are shown as means\u2009\u00b1\u2009SD.31. E2F1 cDNA from pDCE2F and DP1 cDNA from pCMV-DP123 were cloned into pENTR-CMV, which was generated by subcloning a CMV promoter-driven expression cassette from pcDNA3 (Invitrogen) into pENTR-D-TOPO (Invitrogen), creating expression entry vectors pENTR-E2F1 and pENTR-DP1, respectively. The shRNA expression entry vectors were constructed by inserting double-stranded oligonucleotides including the target sequence into pENTR-U6, which was generated by subcloning the human U6 promoter from pSilencer 2.0-U6 (Ambion) into pENTR-D-TOPO. Nineteen-bp DNA sequences for RNA interference against human DP1 mRNA (nucleotides 396\u2013414 (for shDP1-2) and 679\u2013697 (for shDP1-3) of human DP1 cDNA; GenBank accession number: NM_007111), against human DP2 mRNA and against human ARF mRNA were selected based on a published report32. Recombinant adenoviruses were produced using the entry vectors and ViraPower Adenoviral Expression System (Invitrogen) according to the supplier\u2019s protocol.pARF-Luc(-736), pCDC6-Luc, pCMV-\u03b2-gal, the DP2 expression vector pCMV-DP2, the GST-E2F1 expression vector pGEX-2TK-RBAP-1, the GST-DP1 expression vector pGST-DP1, the Flag-tagged DP3 expression vector pCMV3-FLAG-DP3, the E2F1 expressing adenovirus Ad-E2F1, and the E1a expressing adenovirus Ad-12SE1A have been previously described17. For FACS analysis, cells were harvested with any floating cells. To measure the percentage of cells with a sub-G1 DNA content, harvested cells were fixed with lysing solution (BD Biosciences), stained with propidium iodide, treated with RNase, and analyzed by FACSCalibur (BD Biosciences). To detect a cell surface change in the apoptotic process, annexin V staining was performed using TACSTM Annexin V-FITC (R & D Systems) according to the manufacturer\u2019s protocol and the percentage of cells stained with annexin V was analyzed by FACSCalibur. To measure the percentage of cells incorporating BrdU, 10\u2009\u03bcM BrdU was added to the cell culture at 20\u2009h before harvest. Harvested cells were then fixed, permeabilized, and incubated with a BrdU antibody . The cells were then incubated with FITC labeled antibody to mouse immunoglobulin and analyzed by FACSCalibur. All assays were performed at least three times and values are shown as means\u2009\u00b1\u2009SE. Statistical significance of the difference was determined with student\u2019s t-test.Infection with recombinant adenoviruses proceeded as previously described17. Reporter activities were normalized to \u03b2-galactosidase activities as an internal control. All assays were repeated at least three times in duplicate and values are shown as means\u2009\u00b1\u2009SD. Statistical significance of the difference between the mean was determined with student\u2019s t-test.Luciferase assays proceeded as previously described17. Specific primer sets were: ARF promoter, 5\u2032-ggcggtaggcgggagggagaggaa-3\u2032 and 5\u2032-cgtgagccgcgggatgtgaacca-3\u2032, human \u03b2-actin promoter, 5\u2032-ctccctcctcctcttcctcaatct-3\u2032 and 5\u2032-cgtcgcgccgctgggttttat-3\u2032, and CDC6 promoter as described33. Antibodies for immunoprecipitating protein-DNA complexes were anti-E2F1 (KH95), anti-E2F4 (C-108), and anti-p50 (NLS) .ChIP assay was performed as previously described17. Antibodies used were anti-DP1 , anti-DP2 , anti-E1a , anti-E2F1 , anti-FLAG , anti-p14ARF , anti-p53 , and anti-\u03b1-tubulin .Immunoblotting was carried out as previously described17. Specific primer sets for PCR were: DP1, 5\u2032-ttagtcccgggaaaggcgtggtgt-3\u2032 and 5\u2032-cgccgtcttatgtttttctggtca-3\u2032, DP2, 5\u2032-ataaccatttggctgctgatt-3\u2032 and 5\u2032-ctgggcccgcttctgctttat-3\u2032, DP3, 5\u2032-ctcactgaagctaacgaagaactc-3\u2032 and 5\u2032-tcatggaaagacggcacag-3\u2032, Bax, 5\u2032-ggttgtcgcccttttctact-3\u2032 and 5\u2032-tgagcactcccgccacaa-3\u2032, those for CDC6, ARF, and GAPDH were as described34. The PCR products were resolved by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining.Isolation of mRNA and RT-PCR were performed as previously described17. A DHFR promoter fragment containing typical E2F sites and EREA were used as probes. The adenovirus E2 enhancer and its mutants were used as competitors.Gel mobility shift assay was carried out as previously describedSupplementary Figures"} +{"text": "Some epidemiological studies have implied a pathogenetic association between varicella zoster virus (VZV) and multiple sclerosis (MS); this, however, remains controversial. The present report describes a case involving an immunocompetent 10-year-old girl who developed relapsing-remitting MS following the prolonged reactivation of VZV inside the first branch of the trigeminal nerve, exhibiting herpes zoster ophthalmicus with severe optic neuritis. Symptoms related to herpes zoster ophthalmicus and MS appeared consecutively in the 10-week period after the appearance of vesicles. This suggests that the onset of MS was triggered by some mechanism involving VZV reactivation in the first branch of the trigeminal nerve. To the best of our knowledge, this report is the first to describe a relationship between the onset of MS and herpes zoster ophthalmicus. Early diagnosis and aggressive antiviral therapy are important in cases of herpes zoster ophthalmicus to prevent the possible development of MS as well as visual impairment as sequela. Varicella zoster virus (VZV) is a strongly neurotropic virus that has been hypothetically associated with multiple sclerosis (MS). A recent large-cohort epidemiological study identified a higher risk for MS development in patients who experienced a herpes zoster (HZ) attack within the previous year than in control subjects . Some stA previously healthy 10-year-old girl with a history of chicken pox at 4 months of age presented with HZ over the right side of the nasal sidewall Figures and 2, aHZO is defined as the reactivation of VZV inside the ophthalmic branch of the trigeminal nerve , commonlIn the current case, it is possible that optic neuritis developed as a symptom of MS before the brain demyelinating lesions. However, sequential appearance of HZ, conjunctivitis, and optic neuritis in a 10-week period and the ipsilateral severity of symptoms suggested that optic neuritis developed pathologically associated with HZO. Furthermore, optic neuritis was steroid resistant, contrary to the brain lesions, and completely remitted in response to plasmapheresis. This is indicative of a mechanism different from that for brain lesions, possibly including more involvement of circulating substances in serum such as autoantibodies, immune complexes, and cytokines.It is noteworthy that the delay in administration of antiviral therapy may have also contributed to the exacerbation of optic neuritis. Additionally, we highlight the possibility that persistent HZO with optic neuritis triggered the onset of RRMS, although it is undetermined whether VZV had invaded into the central nervous system or not prior to the demyelination, because the analysis of DNA in CSF and peripheral blood was performed 4 or more weeks later than the appearance of the focal neurological symptoms, which is somewhat late for evaluation of the presence or absence of infection. It has been recently reported that pain-mediated neural signals could induce an immunological reaction that triggered relapse in a mouse model of MS, which is termed the \u201cpain gateway reflex\u201d . In the To the best of our knowledge, this is the first case report of RRMS onset directly following HZO, with severe optic neuritis. The consecutive and immediate appearance of symptoms related to HZO and MS in a 10-week period after the onset of skin lesions strongly suggests that VZV reactivation in the first branch of the trigeminal nerve immunologically triggered the onset of MS.Ocular complications, including optic neuritis, are rare in patients with pediatric herpes zoster, and their symptoms cannot be easily noticed in children. However, early diagnosis and aggressive antiviral therapy are important in cases of HZO to prevent the possible development of MS as well as visual impairment as sequela. Further studies are needed to determine the exact role of antecedent and concomitant infection with VZV in the onset of MS."} +{"text": "Brain derived neurotrophic factor (BDNF) seems to serve as an important regulatory mechanism in the growth and development of neurons in many areas of the brain.Insulin-like growth factor 1 (IGF-1) is related to neurogenesis and regulation of the BDNF gene and is involved in the growth and differentiation of neurons.Cortisol is released in response to stimuli such as psychological oppression, anxiety, and fear. Stress also induces changes in BDNF. The purpose of this study was thus to examine the effects of varying intensities of aerobic exercise on resting serum BDNF, IGF-1 concentrations, cortisol, and memory of adolescents.Forty male students with no history of physical illness from the middle school by participated in this study. Participants were randomly assigned to low, moderate, or high intensity treadmill exercise group, or a stretching (control) group. Exercise was performed 4 times per week for 12\u00a0weeks. Body composition, brain derived neurotrophic factor levels, insulin-like growth factor 1 levels, cortisol levels, and working memory were assessed.The high intensity exercise group showed a significant increase in brain derived neurotrophic factor at rest, concentration level of insulin-like growth factor 1, cortisol, and working memory. For resting brain derived neurotrophic factor, the high intensity exercise group showed a more significant increase compared to the low intensity aerobic and stretching groups. The change in the working memory significantly increased for the high intensity exercise group compared to the low intensity aerobic group, moderate intensity exercise group, and stretching group.In adolescents, whose brains are still developing, aerobic exercise of moderate to high intensity levels seems to have a positive effect on levels of serum brain derived neurotrophic factor at rest and on cognitive functioning.EHPM-D-16-00107R2. ICMJE. 12 July 2016. Brain derived neurotrophic factor (BDNF) seems to serve as an important regulatory mechanism in the growth and development of neurons in many areas of the brain. It has also been reported to play a part in improving the survival of neurons by heightening the resistance to nerve damage . ExercisSince being discovered in human blood in 1995 \u20139. SeverRecent studies have reported that temporal exercise-induced increases in levels of BDNF can further escalate in response to chronic aerobic exercise bouts . ChronicInsulin-like growth factor 1 (IGF-1) is related to neurogenesis and regulation of the BDNF gene and is iCortisol is released in response to stimuli such as psychological oppression, anxiety, and fear. Stress also induces changes in BDNF; an increase in stress levels influences BDNF mRNA, which ultimately decreases BDNF expression , and sigAccording to Baddeley, working memory is the place of mental effort that demands conscious attention . GathercAlthough there is clear evidence of aerobic exercise-induced increases in BDNF, the effect of chronic resting serum BDNF levels, which are influenced by the intensity and duration of aerobic exercise, on cognitive functioning remains unclear. Also the subjects of most of these previous experimental studies were adults. The purpose of this study was thus to examine the effects of varying intensities of aerobic exercise on resting serum BDNF, IGF-1 concentrations, cortisol, and memory of adolescents.Forty male middle school students with no history of physical illness volunteered to participate in the study. Students who took part in any sports activities in addition to the usual physical education in the school curriculum were excluded. The experimental groups were as follows: low intensity exercise group (LIEG), moderate intensity exercise group (MIEG), high intensity exercise group (HIEG), and a stretching group . Homogeneity among the groups was verified. The characteristics of the subjects are shown in Table\u00a0Before participation in the exercise intervention, all subjects underwent graded exercise (GXT) testing in order to assess maximum oxygen intake (VO2max). After GXT, the subjects resumed normal daily activity, but were instructed them to avoid any physical or exercise exertion. The subjects then fasted for 12\u00a0h before blood samples were taken from the median cubital vein. BDNF, IGF-1, and cortisol concentration levels were then measured. At 9\u00a0am the next day, the subjects underwent a working memory test.The aerobic exercises were designed to burn 200\u00a0kcal, and the exercise time for each group was calculated for the consumption of 200\u00a0kcal. The exercises were applied to each individual for a duration of 12\u00a0weeks .Post-intervention blood samples were taken after one day of rest from the termination of the exercise intervention. One day following blood sampling, at 9\u00a0am, the subjects underwent a working memory test, identical to the pre-test protocol Table\u00a0.Table 2MIn order to measure VO2max, graded exercise tests were performed at a laboratory in D university located in Yongin at Korea using a treadmill , respiratory gas analyzer , and ECG monitor according to a modified Bruce protocol . During One animal study has previously reported there to be a positive correlation between concentration levels of BDNF in the cortex and concentration levels of BDNF serum .Whole blood was centrifuged at 3000\u00a0rpm for 15\u00a0min in order to separate it into serum and plasma. The blood was stored at \u221280\u00a0\u00b0C until analysis. The serum BDNF was analyzed using the ELISA (sandwich enzyme-linked immunosorbant assay) Kit . Toshiba YBA-200 FRNEO (Japan) was used for analysis of IGF-1. IGF-1 was measured using the radio-immunoassay method, and IGF-I-D-RIA-CT was taken as a reagent. Packard \u0263-Counter (USA) was used for analysis of cortisol. Cortisol was measured using the radio-immunoassay method, and SIEMENS Coat-A-count cortisol (USA) was taken as reagent.The Korean version of the Wechsler intelligence scale for children-below K-WISC-III was utilized to assess working memory. The K-WISC-III not only includes scores that represent comprehensive intelligent ability such as IQ, but also includes subtests that measure a subject\u2019s ability in particular areas of intelligence. In this study, the number subtest was applied, because it is one of the core subtests that measure memory. For this, there are two tests: repeating presented numbers in the right order and in reversed order. The first test consists of 8 questions. There are two numbers in the first question that would enable the minimum order. Then, one number is added to each question that follows until the participants reach the 8th question (a string of 9 numbers). For each task in this first test, the examiner clearly articulated all numbers at one-second intervals and the subject was then asked to repeat the numbers in the right order, one second after hearing the last number of the question. After a two-minute interval, one more test with 8 questions of different number combinations was completed. After completing these first tests, the reverse order tests were performed. These had the same number combination sets, except that subjects were asked to repeat the numbers in the reverse order. There were a total of 32 questions in the entire assessment, and one mark was scored for each correct response and zero for each incorrect response, resulting in a maximum score of 32 points.The exercise sessions were completed at D university in Yongin, and were conducted 4 times a week for 12\u00a0weeks. Low, moderate, and high intensity exercises were performed using treadmills, and exercise intensity was set at 40% VO2R, 55% VO2R, and 70% VO2R for each group, respectively, based on the scale recommended by American College of Sport Medicine (ACSM). In order to standardize the amount of work, each subject was to burn 200\u00a0kcal in each exercise session, and exercise times were calculated for each individual in order to match this energy consumption for their protocol. The SG performed whole-body stretching for 30\u00a0min at the same time, frequency, and location as the aerobic exercise groups.During the experiments, all subjects were asked not to undertake any exercises other than the ones designed for the experiments, with cooperation from their schools.Table\u00a0t-tests were used to verify the difference between pre- and post-intervention variables within each group, and a two-way repeated analysis of variance (ANOVA) was used to test the within-group effect of time and between-group differences. If sphericity was satisfied, a univariate analysis was performed. Bonferroni\u2019s analysis was employed for all post-hoc tests. A value of p\u2009<\u20090.05 was considered to be statistically significant for all analyses.All data for were analyzed using SPSS version 18.0 statistical package. Descriptive statistics by measurement items of each group were performed and Levene-F verification was adopted for the verification of the homogeneity of each variable among groups. Paired Within-group changes in the dependent variables before and after the 12-week aerobic exercises are shown in Table\u00a0p\u2009<\u20090.05) and HIEG group (p\u2009<\u20090.01) showed a significant increase in BDNF when at rest compared to pre-intervention levels. No significant changes were apparent in the LIEG or SG groups. For the effect of analysis of variance between groups and time factors of BDNF, there was a significant main effect of time (p\u2009<\u20090.001), a significant main effect of group (p\u2009<\u20090.05), and a significant interaction of group by time (p\u2009<\u20090.01). Post-hoc tests revealed that the HIEG group showed increases in BDNF with more significant difference than the LIEG and the SG groups. The MIEG group showed increase with more significant difference than the SG group.After 12\u00a0weeks of aerobic exercises, the MIEG group (p\u2009<\u20090.05). No significant differences were observed in the LIEG or the MIEG groups. For the effect of analysis of variance between group and time factors of IGF-1, time showed no significant association and also no significant difference was observed in terms of the interactive effect between group and time as well as among the groups.After 12\u00a0weeks of aerobic exercise, the HIEG and the SG groups showed a significant increase in IGF-1 when at rest compared to pre-intervention levels (p\u2009<\u20090.05). No significant differences were observed in the LIEG or MIEG groups. For the effect of analysis of variance between group and time factors of cortisol, time showed no significant association and also no significant difference was observed in terms of the interactive effect between group and time as well as among the groups.After 12\u00a0weeks of aerobic exercise, the HIEG group showed a significant decrease in cortisol when at rest compared to pre-intervention levels (p\u2009<\u20090.01). No significant differences were observed in the LIEG, MIEG, or the SG groups. For the effect of analysis of variance between group and time factors of working memory, time showed significant difference (p\u2009<\u20090.01) and the interactive effect between groups and time also showed significant difference (p\u2009<\u20090.01) and significant difference was also observed among the groups (p\u2009<\u20090.001). Post-hoc tests revealed that the HIEG group showed an increase with more significant difference than the LIEG, MIEG, and SG.After 12\u00a0weeks of aerobic exercise, the HIEG group showed a significant increase in working memory compared to pre-intervention levels . The fact that the moderate intensity exercise group showed no significant difference while its BDNF significantly increased at rest could indicate that the change in the working memory and serum BDNF will be more significant when high intensity aerobic exercise is performed. To conclude, our results support trends found for BDNF and cognitive function to be enhanced by aerobic exercise.Despite the use of different cognitive functional tests, most previous studies have reported an enhancement of cognitive functioning after acute exercise , 24 and The intensity of the exercise appeared in most of the previous studies, which supports the view that increases in BDNF can be elicited by exercise of at least 50% VO2max. However, the intensity of the LIEG group in the present study, set at 40% V02R, is thought to be too low to have any impact on the serum BDNF at rest.In this study, IGF-1 showed a significant increase in the HIEG and SG groups only, and no significant difference was observed in terms of the interactive effect between groups and time as well as among the groups. Schiffer et al. reportedCortisol is a stress hormone secreted from the adrenal cortex of the kidneys in response to stress, and, in order to allow the body to produce maximum energy, it acts to increase blood pressure and glucose levels. A study on the relationship between exercise and cortisol showed that short-term medium to low strength exercises did not change plasma cortisol levels or may even have slightly reduced those levels, whereas high-intensity exercise can elicit increases in blood plasma cortisol levels . In the During childhood and adolescence, vigorous generation of brain cells takes place along with physical growth. Brain function also completes its growth during this period, and research on the generation and activation of the brain cells at this age is therefore important . A stronWe conclude that adolescents whose brains are still developing, long-term aerobic exercise of moderate to high intensity levels may have a positive effect on concentration levels of serum BDNF at rest and on cognitive functioning. Through well-designed studies, the effect of the exercise on neural plasticity and cognitive development could be better understood, and experiments with larger sample sizes could identify the relevance between changes in the brain function and its relationship to exercise and BDNF."} +{"text": "Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These \u2018soft tissues\u2019 have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil \u2018soft tissues\u2019 differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms. The chances of establishing a real-world Jurassic Park are slim. During the fossilization process, biological tissues degrade over millions of years, with some types of molecules breaking down faster than others. However, traces of biological material have been found inside some fossils. While some researchers believe these could be the remains of ancient proteins, blood vessels, and cells, traditionally thought to be among the least stable components of bone, others think that they have more recent sources. One hypothesis is that they are in fact biofilms formed by bacteria.Centrosaurus. The bones were carefully excavated in a manner to reduce contamination, and the sediment the bones had been embedded in was also tested for comparison. Saitta et al. found no evidence of ancient dinosaur proteins. However, the fossils contained more organic carbon, DNA, and certain amino acids than the sediment surrounding them. Most of these appeared to have a very recent source.To investigate the source of the biological material in fossil bone, Saitta et al. performed a range of analyses on the fossilized bones of a horned dinosaur called Sequencing the genetic material revealed that the fossil had become a habitat for an unusual community of microbes that is not found in the surrounding sediment or above ground. These buried microbes may have evolved unique ways to thrive inside fossils. Future work could investigate how these unusual organisms live and whether the communities vary in different parts of the world. Fossils have traditionally been thought to retain little original organic material after undergoing decay and diagenesis. However, recent discoveries of relatively intact macromolecular organic material in fossils and sub-fossils challenge this view. These include ancient DNA and peptHowever, it remains unclear how long different types of organic molecules and organic structures can survive and under which conditions. DNA, which is relatively unstable, is thought to persist no longer than a million years under optimal conditions . In compDinosaur bone has previously been reported to contain endogenous organic remains such as DNA, collagen, osteocytes, erythrocytes, and blood vessels . These rSub-fossil and fossil vertebrate remains are primarily composed of bone, dentine, and/or enamel. These represent calcified tissues with both a mineral component, primarily calcium phosphate, and a protein component that is dominated by collagen. As such collagen is a common target in the analysis of ancient organic remains. Collagen is also non-labile relative to many other vertebrate proteins because of its decay resistance, partly due to its triple helical quaternary structure and high concentration of thermally stable amino acids , and it Others have criticized reports of ancient collagen based on mass spectrometric results, suggesting that they represent laboratory or environmental contamination or statiReports of dinosaur protein and complex organic structure preservation are problematic for several reasons. Firstly, it remains unclear how such organics would be preserved for tens of millions of years. If endogenous, putative dinosaur soft tissues should contain diagenetically unstable proteins and phospholipids , vulneraFurthermore, a long-term trend of protein loss and increasing contamination in ancient organismal remains, such as bone, has been shown . Fossil 14C abundance, which would suggest that the fossils are not isolated from surface processes).Since there are theoretical and empirical reasons to believe that dinosaur organics are unlikely to persist for tens of millions of years, and given the potential for contamination, we argue that the null hypothesis is that complex biomolecules (e.g. nucleic acids or proteins) recovered from dinosaur bones are not original material, more likely representing recent contamination. This hypothesis makes a series of testable predictions: (1) organic material recovered from fossil dinosaur bone will differ in composition (both chemistry and structure) from modern vertebrate proteins and tissues, beyond differences expected through normal diagenesis;\u00a0(2) fossils will show evidence for microbial presence ; (3) fossil bone organic material will show signatures of recent biological activity , energy dispersive X-ray spectroscopy (EDS), light microscopy, attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FTIR), pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), high-performance liquid chromatography (HPLC), radiocarbon accelerator mass spectrometry (AMS), Qubit fluorometer, epifluorescence microscopy (propidium iodide (PI) and SYTO 9 staining), and 16S rRNA gene amplicon sequencing.In addition to finding little evidence for the preservation of original proteinaceous compounds, our findings suggest that bones not only act as open systems just after death and exhumation, but also act as favorable habitats as fossils in the subsurface. Microbial communities appear to be localized inside the dinosaur bones collected here.For details on the analytical methods of ATR FTIR, light microscopy, VPSEM, EDS, Py-GC-MS, HPLC amino acid analysis, radiocarbon AMS, DNA extraction, 16S rRNA amplicon sequencing, and epifluoresence microscopy see Appendix 1.Centrosaurus apertus , located 3 m above the contact with the underlying Oldman Formation . The mudstone-hosted bone-bearing horizon is an aggregation of disarticulated but densely packed bones, with a vertical relief of 15\u201320 cm. Similar to other ceratopsid bonebeds from the same stratigraphic interval from Alberta Tourism, Parks and Recreation, as well as a Permit to Excavate Palaeontological Resources (No. 16\u2013026) from Alberta Culture and Tourism and the Royal Tyrrell Museum of Palaeontology, both issued to CM Brown.Samples of Late Cretaceous fossil dinosaur bone, along with associated sediment and soil controls were obtained from the Dinosaur Park Formation (Late Campanian) in Dinosaur Provincial Park, Alberta, Canada . The Dininterval , the recCentrosaurus bones were first discovered and exposed to the air under typical paleontological excavation techniques to allow for rapid detection of bones.Sandstone and mudstone overburden was removed with pick axe and shovel (~1 m into the hill and\u00a0~1 m deep) to expose a previously unexcavated region of the bonebed, stopping within\u00a0~10 cm of the known bone-bearing horizon. A few hours after commencement of overburden removal, excavation of the mudstone to the bone-bearing horizon was conducted using awl and scalpel. Subterranean At this point, aseptic techniques were then implemented to expose more of the bone in order to determine its size and orientation. It is necessary to qualify the usage of the term \u2018aseptic\u2019 in this study. Paleontological field techniques have changed little over the last century, and it is practically impossible to excavate fossils in a truly sterile manner . Considering this, the term \u2018aseptic\u2019 is used here to acknowledge the inability to provide completely sterile sampling conditions, while still indicating that efforts were taken to minimize contamination of the samples. Our success at reasonably reducing contamination is supported by the fact that our samples yielded consistent and interpretable results.Centrosaurus bone, were sampled without first removing the surrounding matrix, although fractures in the mudstone did appear during sampling so that the samples cannot be said to have been unexposed to the air, especially prevalent in the small rib sample sent to Princeton University for analysis. Also sampled were the aseptically excavated but completely exposed portions of the subterranean bone immediately next to the matrix-surrounded region, designated herein as uncovered subterranean Centrosaurus bone. In other words, these were the regions of the bone fully exposed using aseptic techniques after initial discovery of the bone in order to determine size and orientation. All samples were collected in autoclaved foil without applying consolidants, placed in an ice cooler kept in the shade, and brought back to the field camp freezer that evening.During aseptic excavation and sampling, nitrile gloves washed in 70% ethanol and a facemask were worn. All tools were sterilized with 10% bleach, followed by 70% ethanol, and then heat-treated with a propane blowtorch at the site. Bone samples several centimeters long were obtained using a diamond-coated Dremel saw or utilizing natural fractures in the bone. Certain segments of the bones, designated herein as matrix-surrounded subterranean Additionally, surface-eroded bone from BB180 and on the same ridge above BB180, mudstone excavated from the overburden-removed area of BB180 and several cm below the weathered surface of the same ridge above BB180, and topsoil on the same ridge above BB180 were similarly aseptically acquired and stored . In total, eight bone samples, eight sediment samples, and two soil samples were collected.Samples were transported to the Royal Tyrrell Museum of Palaeontology in a cooler. Following accession at the museum, similar sets of samples were either mailed to Princeton on ice or transported via plane to Bristol without refrigeration with a maximum time unrefrigerated under 24 hr . Note that warming after cold storage could lead to condensation, altering the behavior of any potential microbiome. Upon arrival, samples were stored at 4\u00b0C in Bristol or \u221280\u00b0C in Princeton as required for analysis.Gallus gallus domesticus) bone was obtained frozen from a Sainsbury\u2019s grocery store in Bristol, UK and was kept refrigerated (4\u00b0C). Other controls included amino acid composition data from a reference bone and radiocarbon data from an 82\u201371 ka radiocarbon-dead bovine right femur used as a standard from the literature (Carcharias taurus) eroded from Pleistocene-Holocene sediments were non-aseptically collected from the surface of the sand on a beach in Ponte Vedra Beach, Florida, USA without applied consolidants and stored at room temperature. It should be noted that Florida experiences a high-temperature climate relative to many samples typically studied for palaeoproteomics. Teeth samples represent a mix of dentine and enamel as opposed to normal bone tissue, with relative concentrations depending on how easily the different tissues fragmented during powdering. The decision to include subfossil shark teeth was made based on their ready availability (i.e. they are incredibly common fossils and are easy to collect from the surface of the sand), the minimal loss to science when destructively analyzed due to their ubiquity, and that the protein composition of the tooth dentine would be dominated by collagen, as in bone. Technical grade humic acid was also purchased from Sigma Aldrich as an additional control.The aseptically collected Dinosaur Provincial Park fossil bone, mudstone, and soil samples were compared to younger fossils and modern bone. Chicken were whiDemineralization products differed from those of chicken bone and PleiThese results show that the dinosaur bone yielded different structures when the bone apatite was removed compared to the more recent bone (i.e. primarily vessels as opposed to large fibrous masses). Furthermore, the dinosaur vessels are relatively inorganic in composition compared to the more recent bone, consistent with a mineralized biofilm .Centrosaurus bone revealed somewhat poorly resolved, broad organic peaks , and these can also be detected in the surrounding mudstone matrix than bone proteins. Homologous series of n-alkane/n-alkene doublets may signify the presence of a kerogen-like substance which could potentially be an ancient lipid-derived geopolymer in the dinosaur bone.Centrosaurus bone had a total hydrolysable amino acid (THAA) compositional profile that did not match collagen , but also that exposure to the surface changes the amino acid profile within these Cretaceous fossils.Interpretation of amino acid data is restricted here to only those samples that were prepared to counter peak suppression than did the modern chicken bone . All these groups plotted separately in the PCA from modern collagen collagen .Total organic carbon (TOC) content was higher in the subterranean and surface-eroded topsoil . Howevercollagen . TOC in The fossil dinosaur bone therefore yielded a TOC content similar to relatively rich environmental carbon sources, such as topsoil, but not as high as more recent bone proteins. Although, some of the C in the fossil dinosaur bone is potentially ancient, there is still a sizable contribution of recent C from the immediate environment, consistent with the presence of a microbiome.Centrosaurus bone than in adjacent mudstone matrix , with greater similarity between the mudstone and bone surface scrapings than between either of these and the interior bone core samples or microbial communities that these fossils experienced. Therefore, examining fossils from different localities, climates, lithologies, and taphonomic histories is vital to understanding variation in how biofilms in fossil bone might be mineralized.The Si dominance of the HCl demineralization products from the dinosaur bone likely suggest that they are at least partly silicified. HCl demineralization may favor mineralized biofilm retrieval , explaining why all the observed demineralization products of the dinosaur bone have high-Si content under EDS . If this is the case then it would indicate that any original organics are significantly more susceptible to acid (i.e. of different composition) than the organic masses in the identically treated younger bone samples, which survive well. It seems likely that mineral infilling in the Centrosaurus bone may come from sources other than proteins.Humic acids are common in soils and contain low molecular weight, aromatic components , and then-alkane/n-alkene doublets appears to have been detected in the Centrosaurus bone, but the doublets were very weak. Variation in the visibility of the doublets between the matrix-surrounded and uncovered subterranean Centrosaurus bone samples is likely representative of intra-bone variation in kerogen content rather than contamination since a strong kerogen signature is not likely to result from exposure to air or the sterilized excavating equipment. Future analyses should examine these samples by mass spectrometry under selected ion monitoring (SIM) scanning mode with comparison to authentic standards of n-alkane/n-alkenes or modify extraction methods prior to analysis in order to more clearly observe these potential doublets. Kerogen forming from in situ polymerization of endogenous labile lipids such as cell membranes might not be expected to preserve the tubular or hollow shape of \u2018soft tissues\u2019 such as vessels or cells in bone with high fidelity since initial hydrolytic cleavage from a hydrophilic group will eliminate the amphiphatic nature of these molecules and make them incapable of retaining their bilayer configuration in aqueous solution , suggesting the potential for different microenvironments inside the bone and different carrying capacities for a microbial community. One surface-eroded sample came from an active paleontological quarry (BB180) and was likely exposed to higher levels of contamination as a result. The high THAA concentrations observed in one of the surface-eroded Late Cretaceous bones compared to the subterranean bone further suggests that bone can be colonized by exogenous microbes, since surface exposure would be expected to result in adverse conditions for any surviving endogenous proteins. However, such comparisons should be done cautiously given the small sample size of this study.Centrosaurus bone sample had a THAA composition more closely matching surface-eroded bone than the matrix-surrounded subterranean bone, and also had an elevated THAA concentration compared to the matrix-surrounded subterranean bone, suggesting that even relatively brief aerial exposure might lead to rapid contamination of the subterranean microbial community by surface microbes. The high THAA concentration in the adjacent mudstone matrix of the subterranean bone compared to the other mudstone samples may indicate that bone provides a nutrient source that encourages microbial proliferation.The most surprising result might be that the uncovered subterranean 14C in the dinosaur bone compared to the mudstone or topsoil suggests some biologically inaccessible, old, and possibly endogenous C within the fossils. One possibility for this pattern is kerogen derived from in situ polymerization of endogenous dinosaur labile lipids, although this type of aliphatic geopolymer has only been weakly detected in the Centrosaurus bones through Py-GC-MS , and it should be kept in mind that the surrounding mudstone matrix yields a series of n-alkanes/n-alkenes after pyrolysis. Exogenous C could also become metabolically inaccessible in bone through biofilm mineralization, as suggested by the EDS data, allowing for 14C depletion. Additionally, biofilm formation and proliferation in bones could trap mobile organic C from sediment and groundwater at a rate faster than C exits the bone when not colonized by a biofilm. This would allow for a lower F14C steady state to be reached during the time it takes C outflux to increase in order to match C influx, assuming a simple 1-box model. Perhaps a combination of these three mechanisms influences F14C.As the C in the dinosaur bone is not radiocarbon dead, this suggests an influx of more modern C (i.e. not radiocarbon dead) into the fossil. However, lower FAnalyses of nucleic acids reveal a diverse, unusual microbial community within the dinosaur bone, even when compared to the immediate mudstone matrix or the exterior surface of the bone, as evidenced by a strong enrichment in DNA and differing community composition in the bone relative to the surrounding matrix. The microbial community from the EDTA demineralized bone was similar to that of the non-demineralized bone, important since EDTA can be used as the demineralizing agent to study the \u2018soft tissues\u2019 of fossil bone . Thus, bEuzebya is consistent with the shallow depth of burial, although the presence of the Deltaproteobacteria lineages may indicate that the microenvironment inside the fossil bone creates anaerobic niches to support anaerobic metabolism. Further work is required to understand the relationship of the observed mineral phases and the microbiome. The fact that Actinobacteria were the most common microbes in the dinosaur bone based on 16S rRNA amplicon sequencing is reminiscent of the results from a 38 ka Neanderthal bone, where the majority of detected DNA sequences derived from non-ancient Actinobacteria . The dominance of the aerobic bacteria . The higHCl demineralization products of dinosaur fossil bone differ structurally and elementally from the Pleistocene-Holocene and modern samples when examined using light microscopy and VPSEM. Low-pressure conditions of VPSEM and EDS, as well as charging during these analyses, may have affected subsequent light microscopy observation, but this is mitigated by the fact that light microscopy was done under a comparative framework between the samples.The Pleistocene-Holocene shark tooth and modern chicken bone demineralize to reveal large organic masses (i.e. rich in C and O) consistent with collagen protein as evidenced by discernable N and S peaks, unlike the much older dinosaur bone demineralization products. The relatively more pronounced S peak in the shark tooth as compared to the chicken bone might indicate sulfurization of the collagen protein or some other taphonomic incorporation of inorganic S from the environment into the tooth, the latter being consistent with pyrite. After all, the teeth are the only fossils in this study to derive from a marine depositional environment, so the potential for pyrite formation under euxinic conditions, for example, would not be surprising. The high Fe content in the shark tooth suggests some taphonomic mineral accumulation (e.g. iron oxide or pyrite) and may explain some of the dark discoloration in the teeth, potentially alongside a browning effect caused by the taphonomic formation of melanoidin-like N-heterocyclic polymers known as advanced glycoxidation/lipoxidation end products. Raman spectroscopy has not only been used to suggest that these N-heterocyclic polymers are present in ancient teeth, bone, and eggshell, but also that they lead to brown staining . HoweverSimilar, albeit higher\u00a0resolution, FTIR peaks to those detected here are used as evidence for purported dinosaur collagen , but, itCentrosaurus bone compared to the younger bone might suggest lower relative collagen content. However, it should be noted that the described pattern in phosphate peak alteration was observed using a diamond ATR, and this method can result in differences in spectra from those made using Ge ATR, as was done here, due to different refractive indices of the crystals , succinimides, or piperazines , even when buried and often in comparison to the immediate environment. Fossil bone likely provides an ideal, nutrient-rich open system microbial habitat inside vascular canals capable of moisture retention. The absence of evidence for endogenous proteins and the presence of a diverse, microbial community urge caution regarding claims of dinosaur bone \u2018soft tissues\u2019. Microbes can colonize bones while buried, likely traveling via groundwater. Therefore, it is unsurprising that the prevalence of these \u2018soft tissues\u2019 is not correlated with overburden depth above the fossil or cortical versus cancellous bone tissue . Rather,The study of fossil organics must consider potential microbial presence throughout a specimen\u2019s taphonomic history, from early to late. Microbial communities interact with fossils immediately following death and after burial, but prior to diagenesis. Microbes are known to utilize bone and tooth proteins and foss In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Diethard Tautz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Alan Cooper (Reviewer #2); Charles Lee (Reviewer #3).Thank you for submitting your article \"The microbiome inside of a late cretaceous centrosaurus dinosaur fossil bone\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.At the most fundamental level, this manuscript explores the possibility of dinosaur fossil bones containing ancient organic materials. The authors examined this hypothesis using a combination of analytical chemistry, physical, and molecular genetic approaches. As a microbial ecologist with a cursory understanding of many of the physical and chemical techniques used, I think the results collectively make a strong case that dinosaur fossil bones do not contain ancient organic material and that any organic material found is likely associated with living microorganisms that originated from the surrounding subsurface environments.Reviewer #2:This is a nice study, although the choice of samples gives the impression of it being perhaps a bit haphazard and opportunistic \u2013 e.g. Florida shark tooth samples from a beach site , vs. the bones for the other samples.The study should be commended in terms of using multiple approaches, and finding very convincing results \u2013 but I think that more effort should be put into presenting and explaining what is going on, imagining that many readers will not be familiar with the methods, or whether certain results are, or are not, likely for something >65Ma.Essential revisions:Overall, the impact of the manuscript would be a lot greater if more attention was made to:1) Signposting the meaning of tests and results, rather than waiting until the Discussion for the 'reveal'.For those readers not used to the techniques and assumptions and logic behind these types of studies, it will be difficult to follow quite what is going on, as the delivery is very passive and technical, and readers would be left wondering what result was expected vs what was observed \u2013 and the meaning of either.Reviewer #3:I will refrain from commenting on the physical and chemical analyses, but there are a number of issues with the microbiological analyses that need to be addressed:- Sequence data does not equal to \"microbial activity\" or \"thriving\".- The microbial ecology analysis is very, very superficial (pun not intended). It would seem to me that OTU-based analysis (as opposed to phylum-level composition) is more appropriate for making the case that bone fossils contain unique microbial communities?!- There is really very little insight that a microbial ecologist can gain from this manuscript in its current format, making the title \"the microbiome inside of [\u2026]\" a click-bait. Seems like the data support a much more conclusive title that disputes the existence of ancient collagen in bone fossils.- I take issues with the conclusion drawn from HPLC data . Please explain and justify.- Statistics need to be provided to justify words like \"unique\" and \"distinct\". Reviewer #2:This is a nice study, although the choice of samples gives the impression of it being perhaps a bit haphazard and opportunistic \u2013 e.g. Florida shark tooth samples from a beach site , vs. the bones for the other samples.The justification of use of shark teeth has been added as follows: \u201cThe decision to include subfossil shark teeth was made based on their ready availability , the minimal loss to science when destructively analyzed due to their ubiquity, and that the protein composition of the tooth dentine would be dominated by collagen, as in bone\u201d.The study should be commended in terms of using multiple approaches, and finding very convincing results \u2013 but I think that more effort should be put into presenting and explaining what is going on, imagining that many readers will not be familiar with the methods, or whether certain results are, or are not, likely for something >65Ma.See response to comment below.Essential revisions:Overall, the impact of the manuscript would be a lot greater if more attention was made to:1) Signposting the meaning of tests and results, rather than waiting until the Discussion for the 'reveal'.For those readers not used to the techniques and assumptions and logic behind these types of studies, it will be difficult to follow quite what is going on, as the delivery is very passive and technical, and readers would be left wondering what result was expected vs what was observed \u2013 and the meaning of either.We have added short summaries of the significance of the results at the end of each section in the Results.Reviewer #3:I will refrain from commenting on the physical and chemical analyses, but there are a number of issues with the microbiological analyses that need to be addressed:- Sequence data does not equal to \"microbial activity\" or \"thriving\".We have replaced \u201cmicrobial activity\u201d with \u201cmicrobial presence\u201d and \u201cthriving\u201d with \u201clocalized\u201d.- The microbial ecology analysis is very, very superficial (pun not intended). It would seem to me that OTU-based analysis (as opposed to phylum-level composition) is more appropriate for making the case that bone fossils contain unique microbial communities?!We have moved the phylum-level figure into the appendix and replaced it with a figure in the manuscript at the class level, which we consider to be sufficient to show the distinction of microbial community between the bone and adjacent mudstone. Furthermore, the comparison of microbial community on species or OTU-level was also provided in the appendix.- There is really very little insight that a microbial ecologist can gain from this manuscript in its current format, making the title \"the microbiome inside of [\u2026]\" a click-bait. Seems like the data support a much more conclusive title that disputes the existence of ancient collagen in bone fossils.We have changed the title of the manuscript to \u201cCretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities\u201d.- I take issues with the conclusion drawn from HPLC data . Please explain and justify.8 cells/g , consistent with the idea that the amino acids within the bone are likely to be largely cellular due to the discrepancy between DNA and amino acid stability over time\u201d.We have clarified this as follows, \u201cThis is fairly similar to the observed THAA concentration indicating ~3x10- Statistics need to be provided to justify words like \"unique\" and \"distinct\".We have added one-way PERMANOVA test results and PCA of the species-level data to the manuscript and appendix, respectively. We have also altered the sentence to, \u201cAnalyses of nucleic acids reveal a diverse, unusual microbial community within the dinosaur bone, even when compared to the immediate mudstone matrix or the exterior surface of the bone, as evidenced by a strong enrichment in DNA and differing community composition in the bone relative to the surrounding matrix\u201d."} +{"text": "The authors wish to make the following erratum to Reference :The The authors would like to apologize for any inconvenience caused to the readers by these changes.Corrected references for Reference Meas. Sci. Technol.1996, 12, 1743.Figliola, R.S.; Beasley, D.E. Theory and design for mechanical measurements. J. Chem. Educ.1962, 39, A606.Pace, E.L. Scientific foundations of vacuum technique . J. Vac. Sci. Technol. A Vac. Surf. Film.2013, 31, 061604.V\u00f6lklein, F.; Grau, M.; Meier, A.; Hemer, G.; Breuer, L.; Woias, P. 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Ondes \u00c9lastiques Dans les Solides; Masson: Paris, France, 1996; p. 321.Royer, D.; Dieulesaint, E. Vacuum2014, 109, 326\u2013332.Kalempa, D.; Sharipov, F. Numerical modelling of thermoacoustic waves in a rarefied gas confined between coaxial cylinders."} +{"text": "The pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways \u2265\u20096th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line.Small airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an\u00a0air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF.+, KRT5+). When cultured on ALI, hSABCi-NS1.1 cells\u00a0consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1+), mucous , neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells , observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that \u201ccilium\u201d and \u201cimmunity\u201d were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells\u00a0on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2\u20131, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells\u00a0induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF.The hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for >\u2009200 doubling levels and\u2009>\u200970 passages, continuing to maintain basal cell features contains supplementary material, which is available to authorized users. The small airway epithelium (SAE), comprised of basal, intermediate, club, mucous and ciliated cells, plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF) and most lung cancers \u20136. In huWhile isolation of human SAE and SAE BC by bronchoscopy and brushing permits assessment of primary BC, the procedure is invasive, time consuming and expensive, and the primary BC can be cultured only for 3 to 4 passages before becoming senescent. In this context, if it is possible to immortalize normal human SAE BC that retain the capacity to differentiate to ciliated, secretory, and other differentiated cell types in vitro on air-liquid interface (ALI) culture, it would be very useful to the investigation of SAE biology in health and disease, and the assessment of pharmacologic agents targeted to modify dysregulated BC biology relevant to the pathogenesis of human lung disease. In the current study, we have generated hSABCi-NS1.1, an immortalized human small airway basal cell line from the brushed epithelium of a healthy non-smoker. hSABCi-NS1.1 cells\u00a0can be passaged for at least 200 population doublings, have the capacity to differentiate into the major differentiated SAE club, mucous and ciliated cells, as well as rare SAE cell types, including surfactant protein positive cells and novel ionocytes, and have the capacity to recapitulate SAE disease-relevant biology when stressed with relevant signals.The selected donor was a 50\u2009yr. old African American male healthy non-smoker recruited under a protocol approved by the Weill Cornell Medical College Institutional Review Board. The subject was characterized as phenotypically normal on the basis of clinical history and physical examination, routine blood screening tests, urinalysis, chest X-ray, ECG and pulmonary function testing. The non-smoking status was confirmed by history, venous carboxyhemoglobin levels and urinalysis for levels of nicotine (<\u20092\u2009ng/ml) and its derivative cotinine (<\u20095\u2009ng/ml). Following written informed consent, bronchoscopy was used to collect the small airway epithelium (SAE) as previously described , 17, 20.https://www.atcc.org/). Additional quality control was performed on passage 46 hSABCi-NS1.1 cells which had been stored in the liquid nitrogen for 131\u2009days. As a control for comparison, a new vial of passage 2 primary parental cells was thawed and cultured under the same culture\u00a0conditions .The primary small airway BC were cultured in flasks coated with human type IV collagen \u00a0and supplied with Small Airway Epithelial Cell Growth Medium . Passage 1 small airway BC cells from the selected donor were immortalized using retrovirus-mediated expression of human telomerase reverse transcriptase (hTERT) with a puromycin resistance selection marker, as previously described . The par5 cells/100\u2009\u03bcl/well onto a Transwell insert coated with human type IV collagen in PneumaCult Ex Plus medium (Stemcell Technologies). The lower chamber contained 0.5\u2009ml PneumaCult Ex Plus medium. After 1\u2009day, media in both upper and lower chambers were replaced with fresh Ex Plus medium. Two days post seeding, the media in the lower chamber was replaced with PneumaCult-ALI maintenance media (Stemcell Technologies). The media in the upper chamber was removed to expose the apical surface to air and establish the air-liquid interface . The ALI cultures were then grown at 37\u2009\u00b0C, 5% CO2, with fresh PneumaCult-ALI media changes every 2 to 3\u2009days. The apical surface was washed with 1x PBS once a week to remove accumulated mucus. Transepithelial electrical resistance (TEER) was measured with a Millicell ERS-2 Voltohmmeter . For each time point, 3 independent wells/group were measured.The basal cell line was seeded at a density of 1.5\u2009\u00d7\u200910imagej.nih.gov).Cells were assessed in chamber slides (Thermo-Fisher) or on ALI wells, fixed in 4% paraformaldehyde diluted in PBS from a 16% aqueous stock for 20\u2009min and washed with PBS. Standard immunofluorescence staining methods were used , 23. TheKaryotype analysis of the immortalized hSABCi-NS1.1 cell at passage 50 was performed at the Molecular Cytogenetics-Core Facility at Memorial Sloan-Kettering Cancer Center using established protocols . At leascDNA was synthesized using TaqMan Reverse Transcriptase Reaction kit (Thermo-Fisher). All reactions were run on an Applied Biosystems Sequence Detection System 7500. Relative expression levels were determined using the \u25b3Ct method with 18S ribosomal RNA as the endogenous control. Premade TaqMan Gene Expression Assays (Thermo-Fisher) were used as listed in Additional\u00a0file\u00a0p\u2009<\u20090.05) were used as filter to generate heat map. For RNA-Seq [Trachea, large airway epithelium and small airway epithelium were collected from normal individuals via flexible bronchoscopy as described previously , 20, 23.ego, CA) , samples5 cells/100\u2009\u03bcl/well . The following day, the infectious medium was removed, and the ALI culturing protocol continued as described above. The infection efficiency was confirmed by monitoring GFP positive staining.The methods for lentivirus production were as previously described , 23. Lenp value less than 0.05 was deemed significant.The two-tailed Student\u2019s t-test was used to compare gene expression in both in vivo and in vitro data. In all analyses, a Based on our previous published sub-dataset , small a. The cells entered consistent growth after 200\u2009days. The population doubling levels between passage 5 and passage 55 were ~\u2009200 cells contained abnormalities of chromosome 19 (resulting in net gain of 19q and/or 19p) and 15 (65%) cells contained trisomy 20.Consistent with the source of the hSABCi-NS1.1 cell line, immunostaining showed that the parental cells prior to immortalization expressed KRT5 , club (SCGB1A1+\u00a0organelles), mucous (MUC5AC+ or MUC5B+\u00a0organelles), ionocytes (FOXI1+ nuclei), neuroendocrine (CHGA+ organelles), and less-defined alveolar-like cells (SFTPA+ or SFTPB+ organelles). ARL13B staining showed a characteristic apical collection of curvilinear structures while SCGB1A1, MUC5AC, MUC5B, SFTPA, SFTPB, and CHGA staining occurred in collections of intracellular puncta typical of secretory vesicles. The transcription factor, FOXI1, co-localized with nuclei consistent with its role in regulation of gene expression. Ciliated cell and club cell staining were non-overlapping while MUC5AC and MUC5B were partially overlapping as expected.To assess the differentiation capacity of hSABCi-NS1.1 cells on ALI, the ALI cells were stained with markers for SAE-associated lineages Fig.\u00a0a-e, inclRNAseq was used to confirm the differentiation capacities of hSABCi-NS1.1 on ALI. Compared to the undifferentiated basal cell stage, ALI-day 28 hSABCi-NS1.1 had down-regulation of basal cell genes , with up-regulation of genes typical of ciliated cells , secretory cells , alveolar-like cells , neuroendocrine cells (CHGB and SCG5), ionocyte cells and MHC-II genes with diverse differentiation capacities and regional features. This human SAE cell line can achieve high cell doubling levels and passage numbers. The hSABCi-NS1.1 cell line provided a novel tool to study SAE biology and pathophysiology in an airway region relevant manner. The availability of hSABCi-NS1.1 cells\u00a0will permit studies of diseases for which the SAE plays a central role. It also can be used to assess the effect of specific mediators on the SAE. As an example, we found transcription factor SPDEF can induce human COPD-relevant changes in hSABCi-NS1.1 cells. Also relevant to small airway biology, hSABCi-NS1.1 can differentiate to ionocytes, a recently identified airway cell type , 31, surThe hSABCi-NS1.1 cell line demonstrates regional identity. Along the proximal to distal axis, the airway epithelium has different morphological features , 24\u201327. The regional identity is likely directly involved in the pathogenesis of SAE-related diseases. For example, SAE express surfactant protein genes , genetic variation of which are associated with COPD and IPF , 34. SAEIn vitro differentiation models of airway epithelial cells have been widely used in studies for airway development, carcinogenesis, environmental insults and infection by pathogens , 42. ImpAdditional file 1:Table S1. List of primers for TaqMan assays. Table S2. List of immunity-related genes among the top 1500 up-regulated genes expressed by hSABCi-NS1.1 on air-liquid interface. Figure S1. Morphology of hSABCi-NS1.1 at late passage. Figure S2. Basal cell marker KRT5 staining in the parental cell prior to immortalization. (PDF 641 kb)"} +{"text": "Yearly, millions of children are treated globally with ivermectin mainly for neglected tropical diseases. Anatomical, physiological and biochemical differences between children and adults may result in changes in pharmacokinetics. However, paediatric pharmacokinetic data of ivermectin are lacking.Trichuris trichiura-infected pre-school-aged children and school-aged children were assigned to 100 or 200\u2009\u03bcg/kg and 200, 400 or 600\u2009\u03bcg/kg ivermectin, respectively (ISRCTN registry no. ISRCTN15871729). Capillary blood was collected on dried blood spot cards until 72\u2009h post-treatment. Ivermectin was quantified by LC-MS/MS, and pharmacokinetic parameters were evaluated by non-compartmental analysis.In the framework of a randomized controlled dose-finding trial in rural C\u00f4te d\u2019Ivoire, Cmax and AUC increased in PSAC and SAC with ascending doses and were similar in both age groups when the current standard dose (200\u2009\u03bcg/kg) was administered . PSAC with lower BMI were associated with significantly higher AUCs. AUC and Cmax were \u223c2-fold lower in children compared with parameters previously studied in adults, whereas body weight-adjusted CL/F (\u223c0.35\u2009L/h/kg) was significantly higher in children. Tmax (\u223c6 h), t1/2 (\u223c18\u2009h), mean residence time (MRTINF) (\u223c28\u2009h) and V/F (\u223c8\u2009L/kg) were similar in all paediatric treatment arms. Cmax with dose was observed in both age groups. Undernutrition might influence the AUC of ivermectin in PSAC. Ivermectin shows a lower exposure profile in children compared with adults, highlighting the need to establish dosing recommendations for different age groups.A positive association of AUC or Ascaris lumbricoides, hookworms and Trichuris trichiura. A total of 20% of the world\u2019s population are estimated to be infected with at least one of the STHs, and the parasites are endemic in most countries of Central and South America, Africa and Asia.T. trichiura.Ivermectin is an antiparasitic marketed to orally treat onchocerciasis and strongyloidiasis, and is used in the combination with albendazole against lymphatic filariasis.Onchocerca volvulus or Plasmodium falciparum malaria.Even though ivermectin has been in use since the early 1980s, it has not been systematically evaluated in human medicine . To date, pharmacokinetic (PK) studies of ivermectin have been conducted mostly in a low number of healthy adults or adults infected with T. trichiura in the framework of a phase II dose-finding study. Children were treated with ascending doses of ivermectin, namely 100 or 200\u2009\u03bcg/kg for PSAC and 200, 400 or 600\u2009\u03bcg/kg for SAC. A micro-blood sampling technique was performed to collect dried blood spot (DBS) samples over 72\u2009h. Ivermectin was extracted from the DBS samples and quantified with a previously validated LC-MS/MS method.T. trichiura and to anthropological measures. Finally, PK parameters were compared between the two age groups and with ivermectin\u2019s PK in adult volunteers, which were reported previously.For the first time, a PK trial was conducted in rural C\u00f4te d\u2019Ivoire with 120 school-aged children and 80 pre-school-aged children infected with 2 was synthesized by Toronto Research Chemicals . Ivermectin tablets (3\u2009mg) were kindly provided by ELEA . Ivermectin mini-tablets at a strength of 500\u2009\u03bcg were produced at the University of Basel.\u00ae Advantage A10, Merck, Darmstadt, Germany). LC-MS-grade solvents, acetonitrile and isopropanol, and Whatman\u00ae protein saver cards 903 were purchased from Merck KGaA . SOLA\u03bc solid phase extraction (SPE) plates HRP (hydrophilic reversed phased) were obtained from Thermo Fisher Scientific and protein low-binding 96-well plates (PCR clean) were purchased from Vaudaux-Eppendorf AG . Ivermectin , formic acid (LC-MS grade) and ammonium acetate (LC-MS grade) were purchased from Sigma\u2013Aldrich . Ivermectin-dT. trichiura infections. Additionally, the tolerability of the interventions was evaluated by clinical examinations, assessment of adverse events and blood analysis. Efficacy and safety data, as well as detailed information on inclusion and exclusion criteria, randomization procedure and diagnostic methods are published elsewhere.The PK study was embedded in a phase II randomized, single-blind trial in rural C\u00f4te d\u2019Ivoire with the primary objective of identifying the efficacy of ascending, single oral doses of ivermectin against T. trichiura infection (>60 eggs/g of stool for PSAC and >100 eggs/g of stool for SAC) were enrolled in the trial in the setting of Azagui\u00e9, C\u00f4te d\u2019Ivoire. Prior to treatment, children were examined for anthropometric measures, i.e. weight and height. PSAC were randomly assigned to two treatment arms (100 or 200\u2009\u03bcg/kg ivermectin) and SAC to three treatment groups . On the treatment day, participants received a standardized fatty breakfast (oily fish on bread) owing to ivermectin\u2019s enhanced bioavailability following fatty food intake.For the PK study, 80 PSAC (2\u20135\u2009years of age) and 120 SAC (6\u201312\u2009years of age) with 2) were extracted and analysed simultaneously. US FDA guidelines require a linearity of the calibration line of 2r >0.99 and an accuracy of 3/4 of calibration line and 2/3 of quality control samples of \u00b115% [\u00b120% for the lower limit of quantification (LLOQ)] versus the nominal value.The development, optimization and validation of ivermectin extraction from DBS samples and the analytical LC-MS/MS method are described elsewhere.Cmax) and time to reach Cmax (Tmax) were observed values. The half-life was calculated as t1/2=ln(2)/\u03bbZ. AUC was determined until the last measurement (AUC0\u201372) and until infinity (AUCINF). The area under the first-moment curve was evaluated until infinity (AUMCINF). AUCs and AUMCINF were calculated using the linear trapezoidal rule. The mean residence time (MRTINF) was determined by AUMCINF/AUCINF, and drug clearance (CL/F) was assessed by dose/AUCINF. CL/F was further adjusted to the participants\u2019 weights. The apparent volume of distribution (V/F) was evaluated by (CL/F)/\u03bbZ/kg.PK parameters were obtained by non-compartmental analysis using WinNonlin . Maximum ivermectin concentrations between treatment arms or age groups. Significance is illustrated in the figures.Statistical analysis was performed with GraphPad Prism 6.01 and Stata Statistical Software: Release 14 . Kruskal\u2013Wallis analysis followed by Dunn\u2019s post-test was performed to compare PK parameters (INF or Cmax. Four specific models were compared using the Akaike information criterion (AIC): (a) b3, b4 \u2260 0; (b) b3 \u2260 0, b4=0; (c) b3=0, b4 \u2260 0; and (d) b3, b4=0. The model with the lowest AIC was plotted for different values of weight or BMI. Cure rates represent the percentage of volunteers who were fully cured (egg negative) after treatment. Egg reduction rates are defined by the group geometric mean reduction in the number of excreted eggs from baseline (prior to treatment) to follow-up (2\u20133\u2009weeks post-treatment) diagnosis.Additionally, a dose\u2013response model of the following form was estimated:n\u205f=\u205f41), 400\u2009\u03bcg/kg (n\u205f=\u205f39) or 600\u2009\u03bcg/kg (n\u205f=\u205f40)] participated in the PK study, and a complete DBS sample set was available for the treatment day (0\u20139\u2009h) and the 24\u2009h timepoint, but two and four DBS samples at 48\u2009h and 72\u2009h, respectively, could not be collected. A total of 80 PSAC were enrolled and treated with 100\u2009\u03bcg/kg (n\u205f=\u205f39) or 200\u2009\u03bcg/kg (n\u205f=\u205f41) ivermectin. In total, nine DBS samples of PSAC were not taken on the day of treatment , one DBS sample was missed at each of the 24 and 72\u2009h timepoints and seven participants were not available for DBS sampling at 48\u2009h.Participants\u2019 characteristics are summarized in Table\u00a02r >0.994. A minimum of 3/4 of the calibration line and 2/3 of quality control samples passed accuracy with \u00b115% (\u00b120% for LLOQ) versus the nominal value. The extraction and analysis of 7% of samples were repeated and 72% (>2/3) deviated <20% from the initial analysed concentrations. The LLOQ for DBS samples is 3\u2009ng/mL. DBS samples that resulted in <3\u2009ng/mL were set to 0\u2009ng/mL.The calibration line of all experiments fulfilled requirements with Cmax increased with ascending doses, and median values of 15.5 and 24.4\u2009ng/mL were obtained for PSAC treated with 100 and 200\u2009\u03bcg/kg ivermectin, respectively, and 21.9, 40.7 and 66.1\u2009ng/mL for SAC treated with 200, 400 and 600\u2009\u03bcg/kg ivermectin, respectively. AUCs also correlated with dose, e.g. AUC0\u201372 increased from 169 to 369\u2009ng\u00d7h/mL in PSAC and from 331 to 880 to 1636\u2009ng\u00d7h/mL in SAC with ascending doses. The median Tmax (5.92\u20136.80 h), t1/2 (16.3\u201319.1 h), MRTINF (26.9\u201329.0 h), V/F (7.46\u201310.4 L/kg) and CL/F (5.68\u20138.58 L/h) were similar in the five treatment arms and thus independent of dose and age (2\u201312\u2009years).The mean concentration\u2013time profiles of ascending doses of ivermectin administered to PSAC and SAC are illustrated in Figure\u00a0Cmax was computed as a function of absolute dose and weight or BMI, and the results, grouped by weight or BMI classes, are illustrated in the JAC Online). There was a statistically significant negative association of weight with AUC, even after adjustment for dose in SAC but not in PSAC. Similar results were found for BMI in PSAC, whereas the association of BMI with AUC vanished in SAC after adjustment for dose . No significant association was evaluated for PSAC.Efficacy results are presented as cure rates and egg reduction rates Table\u00a0. WhereasT. trichiura and other parasitic diseases, ivermectin appears to be a promising drug candidate with its broad antiparasitic activity. Despite its distribution to millions of children (>5\u2009years of age), to our knowledge no paediatric PK characterization has yet been performed. For the first time, the disposition of ivermectin was evaluated in PSAC (2\u20135\u2009years) and SAC (6\u201312\u2009years) treated with ascending doses. Whereas drug exposure was similar in children of both age groups and increased with ascending dosage, AUCs were 2-fold lower in children than in adults when the same weight-dependent dose was administered. This finding can have major implications for the efficacy and safety for paediatric treatment of many diseases.Ivermectin is marketed for humans \u226515\u2009kg (>5\u2009years of age) to treat onchocerciasis, strongyloidiasis and lymphatic filariasis. Owing to the lack of an effective treatment against Tmax, t1/2, MRTINF, CL/F and V/F, were of similar value in all treatment arms, highlighting the comparability of ivermectin\u2019s PK in the age range of 2\u201312\u2009years. Cmax and AUCs were similar in PSAC and SAC when the same weight-dependent dose was administered (200\u2009\u03bcg/kg ivermectin) and weight was observed in both age groups and cure rate in SAC. To date, few PK studies have been conducted in participants infected with intestinal helminths, and it remains unknown whether AUC or Cmax or solely intestinal concentrations are responsible for anthelminthic activity. A recent study in hookworm-infected children treated with tribendimidine did not identify a relationship between drug exposure and efficacy.Despite moderate egg reduction rates, all doses administered to SAC and PSAC resulted in low cure rates against Cmax and AUCs are \u223c2-fold higher in adults than in children were similar in all treatment arms of the two paediatric populations but are only approximately half of the values observed in adults, and Tmax is lower in adults than in children , hepatic and renal function, and metabolic processes alter with age and thus can influence the PK of a drug.,Tmax in children. Moreover, intestinal motility is responsible for drug\u2013mucosa interaction. If this process is impaired, a lower amount of ivermectin will be absorbed, causing lower Cmax and AUC. This might be supported by the lower blood supply by the superior mesenteric artery to the intestine in children than in adults .Interestingly, the PK results of PSAC and SAC differ from our own findings in adults when treated with the current standard dose of 200\u2009\u03bcg/kg.n Figure\u00a0. Other P,Cmax and approximately half Tmax.It has been repeatedly expressed that children are not small adults and drug dosages cannot be simply extrapolated from adults to children by adjusting for the body weight. The WHO highlighted the need for licensed paediatric drugs as still millions of children suffer owing to untreated diseases, but barriers for PK trials in children remain high owing to ethical and technical challenges. PK modelling and simulations based on data derived from adults might aid in providing paediatric treatment recommendations. Recently simulated PK parameters of ivermectin of healthy adults resemble our PK parameters of adults.T.trichiura, and PK parameters of ascending doses of ivermectin were evaluated in micro-blood DBS samples. AUC and Cmax increased with ascending doses, and Tmax, t1/2, MRTINF, CL/F and V/F were dose and age independent. Malnutrition or undernutrition might influence the AUC of ivermectin in small children. Ivermectin shows a lower exposure profile in children than in adults, highlighting the need to study drug dosing carefully, in particular given the great interest in applying this drug for novel indications.In summary, a phase II clinical trial was performed in two paediatric populations (PSAC and SAC) infected with dkz083_Supplementary_DataClick here for additional data file."} +{"text": "The objective was to evaluate the 25(OH) vitamin D (25(OH)D) status of patients with juvenile idiopathic arthritis (JIA) and determine whether the 25(OH)D level is associated with disease activity and the course of JIA.Patients \u2264\u200916\u2009years of age with recently diagnosed JIA (<\u200912\u2009months) were enrolled in the inception cohort of patients with newly diagnosed JIA (ICON), an ongoing prospective observational, controlled multicenter study started in 2010. Clinical and laboratory parameters were ascertained quarterly during the first year and half-yearly thereafter.Of the 954 enrolled patients, 360 patients with two blood samples taken during the first 2\u2009years after inclusion and with follow up of 3\u2009years were selected. The serum 25(OH)D levels were determined and compared with those of subjects from the general population after matching for age, sex, migration status and the month of blood-drawing.Nearly half of the patients had a deficient 25(OH)D level (<\u200920\u2009ng/ml) in the first serum sample and a quarter had a deficient level in both samples. Disease activity and the risk of developing JIA-associated uveitis were inversely correlated with the 25(OH)D level .In this study, 25(OH)D deficiency was common and associated with higher disease activity and risk of developing JIA-associated uveitis. Further studies are needed to substantiate these results and determine whether correcting 25(OH)D deficiency is beneficial in JIA. Vitamin D deficiency was common, as it was found in 44% of patients with juvenile idiopathic arthritis (JIA) and even in 62% of healthy peersLow levels of 25(OH)D in patients with JIA were associated with higher disease activity and higher risk of developing JIA-associated uveitis2 vitamin D3 [2D3), clinical outcomes and the cutoff values that were used differed substantially. Currently, the measurement of 25(OH)D is considered standard because it is stable and reflects the vitamin D provision of the last weeks to months. In our study, we wanted to (1) evaluate the 25(OH)D level in patients with newly diagnosed JIA and compare it with that in individuals from the general population; (2) analyze whether disease activity is correlated with the 25(OH)D level and (3) determine whether the 25(OH)D level might predict the disease course.Over the last decade, it has become clear that vitamin D is more than the \u201cbone vitamin\u201d that regulates calcium homeostasis and bone mineralization. In addition to its pleiotropic functions in different cells and tissues, the differentiation, polarization and activity of immune cells are affected by 1,25 (OH)The inception cohort of newly diagnosed patients with JIA (ICON) has been described in detail elsewhere . BrieflySerum samples were taken as part of routine laboratory controls in ICON and were collected, frozen and stored at the ICON biobank at the University of M\u00fcnster, Germany, until they were shipped to Berlin, Germany, to perform the 25(OH)D assay for all samples at the same time at a medical laboratory. For our analysis, we selected patients for whom a pair of serum samples was available. The first sample was collected at a visit that occurred between baseline and the 9-month follow up (94% at baseline or at 3-month follow up), and the second sample was collected at a visit that occurred between the 3-month follow up and the 36-month follow up (92% until the 1-year follow up), with a mean interval of 7\u2009months (SD 5) between blood draws. The time points for serum collection in male and female subjects were equally distributed in winter (October\u2013March) and summer (April\u2013September).The LIAISON 25 OH Vitamin D TOTAL Assay was used to quantitatively determine the 25(OH)D level in the serum samples. The measurement range of this assay ranges from 4\u2009ng/ml (10\u2009nmol/l) to 150\u2009ng/ml 375\u2009nmol/l). To compare the 25(OH)D levels of patients with healthy subjects, we used data from the German National Health Interview and Examination Survey for Children and Adolescents (KIGGS), which was conducted from May 2003 to May 2006, to obtain representative data on health status and selected laboratory values of children and adolescents aged 0\u201317\u2009years across Germany and laboratory parameters , it has been proposed vitamin D deficiency as 25(OH)D <\u200920\u2009ng/ml (<\u200950\u2009nmol/l) \u201310. UsinThe disease activity of patients with JIA was measured by the clinical Juvenile Arthritis Disease Activity Score (cJADAS-10), a composite score based on the physician\u2019s global assessment of disease activity, the parents\u2019 global assessments of wellbeing, and the number of active joints up to a maximum of 10, according to McErlane et al. . For thet test, and the categories of serum 25(OH)D levels between patients and controls were compared by conditional logistic regression analyses. The association between disease activity and 25(OH)D level was assessed by linear regression analysis. The association between the likelihood of uveitis and serum 25(OH)D, including the covariates of prior methotrexate (MTX) therapy and established uveitis risk factors, such as age at JIA onset, female sex, oligoarticular JIA onset, and ANA positivity, were analyzed using a multivariable Cox proportional hazard model. Multivariable logistic regression analysis was used to investigate the proportion of oligoarthritis patients who progressed to extended oligoarthritis until the 3-year follow up, with serum 25(OH)D as a predictor. P values < 0.05 were considered statistically significant. The data were analyzed using SAS software version 9.4 .Serum 25(OH)D levels were compared by the paired The subgroup of patients whose 25(OH)D levels were analyzed did not differ from the whole ICON study population in terms of the male-to-female ratio, age at study inclusion or symptom onset and ILAR category Table\u00a0. Migratip\u2009<\u20090.001). Nearly half of the patients (44%) had a deficient 25(OH)D level in the first serum sample.The mean 25(OH)D levels and the distribution of deficient and sufficient 25(OH)D levels in the study population and control subjects are summarized in Table\u00a0n\u2009=\u200971, \u03b2\u2009=\u2009\u2212\u20090.38; 95% CI \u2212\u20090.65; \u2212\u20090.11; p\u2009=\u20090.007).We hypothesized that an effect of the vitamin D status later in the disease course might be abrogated by already established therapy. Thus, we used the 25(OH)D level from the first measured sample to analyze possible correlation with disease activity because at this time point, the patients were in an early phase of disease (median disease duration at that time 6.6 IQR 3.8\u201311.4) months) and any treatment was initiated only briefly (212 (59%) treated with disease modifying anti-rheumatic drugs (DMARDs), 195 (54%) with MTX, and 18 (5%) with biologic disease-modifying anti-rheumatic drugs (bDMARDs); median therapy duration 1.0\u2009(IQR 0.5\u20133.5) month). We identified negative correlation between disease activity and the 25(OH)D level and not receiving (n\u2009=\u2009152) conventional synthetic disease modifying anti-rheumatic drugs (csDMARDs) at the first measurement, the mean 25(OH)D level did not differ significantly , patients receiving systemic glucocorticoid therapy \u2265\u20090.2\u2009mg/kg body weight prednisolone equivalent (n\u2009=\u200919) had significantly lower mean 25(OH)D than patients (n\u2009=\u2009275) without such systemic glucocorticoid doses . Patients with 25(OH)D\u2009<\u200920\u2009ng/ml in the first measurement received systemic glucocorticoids \u2265\u20090.2\u2009mg/kg body weight prednisolone equivalent and csDMARDs a little more frequently at this time than patients with 25(OH)D\u2009>\u200930\u2009ng/ml ; these results were not statistically significant.While in patients receiving (Mean 25(OH)D of the two serum samples from each patient was insufficient (22.1\u2009ng/ml (SD 7.8)), with no differences between the sexes. Between the individual ILAR categories, mean 25(OH)D did not vary significantly, while the highest percentage of stable\u00a025(OH)D deficiency was in patients with systemic, psoriatic and rheumatoid factor (RF)-negative polyarticular JIA D level to those with a stable sufficient 25(OH)D level in terms of the development of the extended form of oligoarthritis or the occurrence of uveitis. Overall, 62 of the 173 patients (36%) with oligoarthritis developed an extended disease course until the 3-year follow up; 18 of these patients already had extended oligoarthritis at the time of enrollment. Twelve of the 29 patients (41%) with oligoarthritis and a stable deficient 25(OH)D level and 2 of the 14 patients (14%) with oligoarticular disease and a stable sufficient 25(OH)D level developed the extended form of disease in the first 3\u2009years in ICON .Altogether, 61 of the 360 patients (17%) developed uveitis by the 3-year follow up: 10 of these patients developed uveitis before enrollment and 51 patients developed uveitis during the first 3\u2009years in ICON. The majority had oligoarticular disease (54% persistent/13% extended), followed by RF-polyarthritis 21%). Of the 87 patients with a stable deficient 25(OH)D level, 17 (20%) developed uveitis, whereas only 2 of the 23 patients (9%) with a stable sufficient 25(OH)D level did so D deficiency was associated with higher JIA disease activityThe progression from oligoarthritis to an extended disease course occurred more often in patients with 25(OH)D deficiency than in those with 25(OH)D sufficiencyThe 25(OH)D level was inversely correlated with the risk of developing JIA-associated anterior uveitisIn this study, in which vitamin D level was repeatedly prospectively measured in patients observed as standard, with recent-onset JIA, the following clinically relevant findings were ascertained:Pelajo et al. also identified a high prevalence of vitamin D deficiency when they measured 25(OH)D in children and adolescents with rheumatologic diseases and in children with non-autoimmune disorders as controls . The risPossibly patients with JIA are screened more frequently for vitamin D deficiency in the course of clinical routine and might consequently receive vitamin D supplements. Pepmueller et al. assessed parameters of bone mineralization in patients with JIA and also found that mean 25(OH)D in patients (28.2\u2009ng/ml) was significantly higher than in healthy controls (22.0\u2009ng/ml) .2D3 did TNF-alpha blockade synergistically suppress inflammatory mediators in RA synovial fibroblast cocultures [We identified significant negative correlation between the 25(OH)D level from the first serum sample and JIA activity, as measured by the cJADAS-10. Pelajo et al. did not find this association in their cross-sectional study of 154 patients with JIA but noted that the included patients had established ongoing disease and that the majority had received some kind of disease-modifying treatment . In a sucultures .We did not observe significant differences in mean 25(OH)D between the JIA categories, but the proportion of 25(OH)D-deficient patients differed among the subgroups. Similar to the work by Stagi et al. , we idenAnalyzing the disease course and outcomes of JIA, we observed a higher proportion of stable\u00a025(OH)D-deficient patients with oligoarthritis who developed an extended disease course during the first 3\u2009years in ICON than of patients with oligoarthritis and a stable sufficient 25(OH)D level. To our knowledge, this association has not been described before, and it requires further investigation.2D3 was significantly lower in patients with active disease than in patients with inactive disease and in controls. Moreover, they demonstrated that the incubation of peripheral blood mononuclear cells (PBMC) with 1,25(OH)2D3 resulted in reduced proliferation of PBMC and reduced production of IFN-\u01b4 and IL-17A in the presence of anti-CD3 and anti-CD28 [One of the most relevant extra-articular manifestations of JIA is anterior uveitis, which is frequently associated with complications and can be accompanied by visual impairment. We confirmed the known risk factors for the development of anterior uveitis in patients with JIA, including ANA positivity, young age at JIA onset and no prior treatment with methotrexate . A new fnti-CD28 , suggestAs a large amount of in vitro data has shown the anti-inflammatory and antiproliferative effects of vitamin D on immune cells , a causaTo date, only a few papers have described the effects of vitamin D supplementation in children with autoimmune diseases. Reed et al. examined the effect of 25(OH)D supplementation at 1\u20132\u2009\u03bcg/kg body weight/day over 1\u2009year in 13 children with active polyarticular JIA and described a significant increase in 25(OH)D, but did not observe change in disease activity with this regimen . By contOur study revealed that vitamin D deficiency is common in patients with JIA, although the prevalence was lower than in matched subjects from the general population. In patients with JIA, vitamin D deficiency is associated with higher disease activity and a higher risk of developing uveitis and possibly the extended form of oligoarthritis. The strength of this study is the prospective controlled design with standardized data ascertainment. A limitation of this study is that the any use of vitamin D supplementation was not recorded. To further clarify if cholecalciferol substitution in order to achieve a sufficient 25(OH)D level might have the potential to prevent the development of uveitis and the extended form of oligoarthritis, these associations must be confirmed by an interventional study."} +{"text": "Most radiotracers developed to date have been chosen on the basis of strong PARP1\u20133 affinity. Herein, we propose to study AZD2461, a PARP inhibitor with lower affinity towards PARP3, and to investigate its potential for PARP targeting 18F-fluorodeboronation of a carefully designed radiolabelling precursor, we accessed the 18F-labelled isotopologue of the PARP inhibitor AZD2461. Cell uptake of [18F]AZD2461 in vitro was assessed in a range of pancreatic cell lines to assess PARP expression and in vivo in xenograft-bearing mice. Blocking experiments were performed with both olaparib and AZD2461.Using the Cu-mediated 18F]AZD2461 was efficiently radiolabelled via both manual and automated procedures (9\u00a0%\u2009\u00b1\u20093\u00a0% and 3\u00a0%\u2009\u00b1\u20091\u00a0% activity yields non-decay corrected). [18F]AZD2461 was taken up in vivo in PARP1-expressing tumours, and the highest uptake was observed for PSN-1 cells (7.34\u2009\u00b1\u20091.16\u00a0%ID/g). In vitro blocking experiments showed a lesser ability of olaparib to reduce [18F]AZD2461 binding, indicating a difference in selectivity between olaparib and AZD2461.[Taken together, we show the importance of screening the PARP selectivity profile of radiolabelled PARP inhibitors for use as PET imaging agents.The online version of this article (10.1007/s11307-020-01497-6) contains supplementary material, which is available to authorized users. PARP inhibitors, such as olaparib (Lynparza), niraparib (Zejula) and rucaparib (Rubraca) reduce catalytic activity and interfere with the ability of the enzyme to dislodge from DNA PARPi is a 18F-labelled PARP structurally related to olaparib [18F]FTT, it is one of the few labelled PARP inhibitors whose structure remains closely related to the native inhibitor that has been investigated in clinical trials [18F]BO or [18F]PARPi-FL, few radiotracers have been studied for their PARP PET imaging potential with a view to assess a different PARP isoform interaction profile olaparib . To datel trials . While o profile \u201325. A raP-gp substrate. A radiolabelled isotopologue of AZD2461 offers the ability to investigate the role of PARP3 in DDR and more specifically PARP imaging, as well as a wider range of possibilities when developing new compounds that possess different PARP specificity. In 2019, Mach and co-workers described a radiofluorinated compound that was structurally related to AZD2461 obtained through classic 18F-radiochemistry AZD2461 (50\u00a0kBq/well) for 1\u00a0h at 37\u00a0\u00b0C. After washing, cells were lysed , and cell-associated 18F was assessed using an automated gamma counter (PerkinElmer Wizard2 2480). To determine the specificity of cell uptake, an excess of cold, unlabelled olaparib or AZD2461 (100\u00a0\u03bcM) was co-incubated with the radiolabelled compound.To evaluate PARP-mediated cell uptake, aliquots of 5\u2009\u00d7\u200910nu/nu mice. A single tumour was implanted per animal. Tumour xenografts were allowed to grow to a size of 200\u2013400\u00a0mm3 before subsequent procedures.All animal procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and with local ethical committee approval. Tumour xenografts were generated by subcutaneous injection of PSN-1, PANC-1, CFPAC-1 or AsPC-1 cell suspensions in the hind flank of Balb/c 18F]AZD2461 (5\u20136\u00a0MBq) using a VECTor4CT scanner . Animals were anaesthetised by 4\u00a0% isoflurane gas (0.5\u00a0L/min O2) and maintained at 2\u00a0% and 37\u00a0\u00b0C throughout the imaging session. The temperature of the animals was maintained at 37\u00a0\u00b0C, using a custom-built mouse cradle. Image acquisition was performed in 10\u00a0min using a 1.8-mm pinhole collimator. Whole-body CT images were acquired at a tube setting of 55 kVp, 0.19\u00a0mA and 20\u00a0ms per view, for anatomical reference and attenuation correction. Reconstruction of both CT and PET images was performed with the MILabs reconstruction analysis using a \u03b3-ray energy window of 467\u2013571\u00a0keV (background weight 2.5\u00a0%), 0.6\u00a0mm3 voxel size, 128 subsets and 5 iterations using the manufacturer\u2019s SROSEM reconstruction type. PET images were each registered to CT and then attenuation corrected. Images were calibrated by imaging a phantom containing a fluorine-18 standard solution and analysed using PMod software package . Biodistribution studies, where selected tissues were harvested, were performed immediately after imaging. Some animals were also intravenously administered an excess of cold, unlabelled olaparib or AZD2461 30\u00a0min before [18F]AZD2461 injection, to evaluate the specificity of tumour uptake. The amount of [18F]AZD2461 uptake in selected tissue was determined and reported as a percentage of the injected dose per gram of tissue (%ID/g). Three mice were utilised per group.PET/CT images were acquired 1\u00a0h after an intravenous bolus administration of AZD2461 was prepared via copper-mediated 18F-fluorodeboronation of a protected boronic pinacol ester precursor with molar activities up to 17\u00a0GBq/\u03bcmol and radiochemical purity >\u200999\u00a0%. As shown in our previously reported studies [N-[2-(trimethylsilyl)ethoxymethyl] (SEM) protection\u00a0was performed as predicted by previous screening experiments [18F]olaparib were used here for 18F-fluorodeboronation of [18F]AZD2461. The copper catalyst for 18F-fluorodeboronation chosen for this study was Cu(OTf)2(impy)4 . 1,3-Dimethyl-2-imidazolidinone (DMI) was demonstrated to be compatible and suitable for 18F-radiofluorination. More importantly, an undesired side-product\u2014due to protodeboronation of the precursor\u2014could be separated from the desired radiotracer . A fully automated procedure, using an Eckert & Ziegler Modular-Lab platform, allowed isolation of [18F]AZD2461 in an activity yield of 3\u00a0%\u2009\u00b1\u20091\u00a0% with molar activities up to 237\u00a0GBq/\u03bcmol and radiochemical purity >\u200999\u00a0%. [18F]olaparib was synthesised, for comparison, as described in our previous study AZD2461. The biodistribution and excretion pathway was similar to that observed in our previously reported study with [18F]olaparib, with [18F]AZD2461 showing lower uptake in liver, while splenic uptake values are higher compared with [18F]olaparib, at the time point investigated . Uptake of [18F]AZD2461 in PARP1-expressing xenografts was 2.39\u2009\u00b1\u20090.65, 3.04\u2009\u00b1\u20091.37 and 3.50\u2009\u00b1\u20090.91\u00a0%ID/g for AsPC-1, CFPAC-1 and PANC-1, respectively, 1\u00a0h after intravenous bolus injection. The highest uptake in tumour was observed in PSN-1 xenografts (7.34\u2009\u00b1\u20091.16%ID/g) and was significantly higher than that in AsPC-1, CFPAC-1 and PANC-1 xenografts , correlating with higher PARP1 expression levels obtained by Western blot. To investigate the specificity of tumour uptake, injection of an excess of cold, unlabelled olaparib and AZD2461 were performed in mice bearing PSN-1 xenografts. While an excess of olaparib was able to efficiently block tumour uptake of [18F]AZD2461 , the same amount of cold AZD2461 only blocked [18F]AZD2461 accumulation in tumours to 3.21\u2009\u00b1\u20090.70\u00a0%ID/g . In contrast, while [18F]AZD2461 was efficiently taken up in PSN-1 cells and excess of non-radioactive AZD2461 was able to efficiently reduce uptake to 25\u00a0% of initial binding (P\u00a0=\u20090.0022), the same excess of cold olaparib was only able to reduce uptake of [18F]AZD2461 to a mere 70\u00a0% of native binding (P\u00a0=\u20090.024).AZD2461 inhibited PARP1\u20133 enzymatic activity in line with previously reported values and studied in clinical trials for their ability to compete with NAD18F]olaparib, a radio-isotopologue of the AstraZeneca compound olaparib, for PET imaging of PARP in a mouse model of human PDAC olaparib, in this study, we were able to efficiently radiolabel [18F]AZD2461 using the Cu-mediated radiofluorination method, originally developed by Tredwell et al. [Am up to 4\u00a0% and 237\u00a0GBq/\u03bcmol, respectively, full details are displayed in Supplemental Information). One of the major side-products formed during reaction course is the undesired proto-deborylated compound 2 were found suitable for following in vitro and in vivo experiments.ies (Am) , 23. Howl et al. . This prmination , 32. Witin vivo in a range of tumour xenograft models of pancreatic cancer"} +{"text": "Spinal anesthesia is optimal choice for transurethral resection of the prostate (TURP), but the sensory block should not cross the T10 level. With advancing age, the sensory blockade level increases after spinal injection in some patients with spinal canal stenosis. We optimize the dose of spinal anesthesia according to the decreased ratio of the dural sac cross-sectional area (DSCSA), the purpose of this study is to hypothesis that if DSCSA is an effective parameter to modify the dosage of spinal anesthetics to achieve a T10 blockade in geriatric patients undergoing TURP.A\u2009=\u2009\u03c0(D/2)2, (\u00a0\u03c0\u2009=\u20093.14). The modified dosage of bupivacaine was adjusted according to the decreased ratio of the DSCSA.Sixty geriatric patients schedule for TURP surgery were enrolled in this study. All subjects were randomized divided into two groups, the ultrasound (group U) and the control (group C) groups, patient receive either a dose of 2\u2009ml of 0.5% isobaric bupivacaine in group C, or a modified dose of 0.5% isobaric bupivacaine in group U. We measured the sagittal anteroposterior diameter (D) of the dural sac at the L3\u20134 level with ultrasound, and calculated the approximate DSCSA (A) according to the following formula: P\u2009<\u20090.001) in group U compared with group C . The dosage of bupivacaine was significantly decreased in group U compared with group C (P\u2009<\u20090.001). The regression times of the two segments were delay in group U compared with group C (P\u2009<\u20090.001). The maximal decrease in MAP was significantly higher in the group C than in group U after spinal injection (P\u2009<\u20090.001), without any modifications HR in either group. Eight patients in group C and two patients in group U required ephedrine (P\u2009=\u20090.038).The cephalad spread of the sensory blockade level was significantly lower .on 8, April, 2018.This study was registered in the Chinese Clinical Trial Registry (Registration number: The stretch sensation of the bladder is carried by the S2 to S4 parasympathetic fibers. Considering this innervation, the height of the regional blockade level up to T10 is sufficient for TURP operation. A higher level of blockade may mask the pain upon perforation of the prostatic capsule. Intrathecal anesthesia is optimal choice for TURP, but the height should not cross the T10 level. The factors such as concentration and volume are the major factors affecting the distribution of local anesthetics after spinal injection .. Because the functional of critical organ and compensate ability for stresses are decreased, it is harmful for geriatric patients to inject more local anesthetics [. Thus, it is important to optimize the dosage of spinal anesthetics for geriatric patients.Hypotension is the major risk after the spinal injection. The systemic vascular resistance may decrease by 25% in elderly patients, whereas it may decrease only by 15\u201318% in normovolemic healthy patients . Becausesthetics . Thus, i. It is necessary to diagnosis and effective management the TUR syndrome timely [. In a case report, the authors emphasize that it is very important to diagnosis and treatment the TURP syndrome early, the patient have not been found developed hyponatremia until decreased to 90\u2009mmol\u2009l\u2212\u20091 under general anesthesia during a TURP procedure [. The patients can clearly describe the early features of TUR syndrome when patient is conscious, so spinal anesthesia is therefore desirable to facilitate early recognition [.Spinal anesthesia can reduce the stress response relate to surgery , and rece timely . In a carocedure . The patognition ..The major challenges of spinal anesthesia for geriatric patient are the changes of anatomical and physiological. Some of anatomical irregularities and physiological changes such as reduction in the number of neurons, especially spinal canal stenosis, etc. always associated with increasing age. The blockade level increases after epidural anesthesia and spinal anesthesia , 13.. A\u2009>\u200930% reduction in the DSCSA and sagittal anteroposterior diameter has been observed in patients with lumbar spinal stenosis [. The DSCSA is a more sensitive measurement parameter to predict lumbar central canal spinal stenosis [. Thus, measuring the sagittal anteroposterior diameter of the dural sac with ultrasound can evaluate the degree of lumbar central canal spinal stenosis.Previous study shown that the depth of intrathecal spaces can accurate prediction by ultrasound imaging . A\u2009>\u200930%stenosis . The DSCstenosis . Thus, mOptimal blockade levels by intrathecal anesthesia is favorable for TURP operation for adequate blockade of the stimulation of bladder traction and less hypotension and bradycardia by too high thoracic block. For geriatric patients, sensory blockade up to T10 is favorable for adequate anesthesia with less hypotension and bradycardia. Most anesthesiologists may reduce the dosage of intrathecal anesthetics to prevent too high blockade by experience. However, as lumbar central canal spinal stenosis is more frequently found in geriatric patients, we hypothesized that local anesthetics would spread more cephalad with a limited space. With goal to achieve T10 sensory blockade in patients receiving TURP operation, we modified the dose of bupivacaine according to the decreased ratio of the DSCSA. By comparing with controlled groups receiving 10\u2009mg of 0.5% isobaric bupivacaine, we analysis the levels of sensory blockade, and the changes of mean arterial blood pressure (MAP) and heart rate (HR).The purpose of present study is to determine the hypothesis that if DSCSA is an effective parameter to modify the dosage of spinal anesthetics to achieve a T10 blockade in geriatric patients undergoing TURP.We conducted a prospective, double blinded, randomized study to measure the sagittal anteroposterior diameter of the dural sac by ultrasound for geriatric patients aged more than70 years undergoing TURP with spinal anesthesia, and then calculated the DSCSA, optimizing the dosage of local anesthetic according to the decreased ratio of the DSCSA.Sixty geriatric patients schedule for TURP surgery were enrolled in this study. The medical ethical committees of The Affiliated AnQing Hospital of Anhui Medical University approved this study on 26, December, 2017, and the study was registered in the Chinese Clinical Trial Registry (Registration number: ChiCTR1800015566). The informed consent were written by all patients.The exclusion criteria of this study as following: local infection at the puncture site, administrated with anticoagulants, intracranial hypertension, and patients who did not to accept spinal anesthesia. Relative contraindications included some neurologic diseases (e.g. multiple sclerosis), lower limbs pain, and so on.n\u2009=\u200930) and the control groups, according to the random number table generator by computer (prepared by AJS).All subjects were randomized divided into two groups, the ultrasound . The MAP and HR were monitored throughout the operation also.All patients transported to the operating room, where they were subjected to standard monitoring electrocardiography (ECG), and pulse oximetry . Epidural puncture was located at the L 3\u20134 intervertebral space, the spinal needle was inserted into the subarachnoid space after successfully epidural puncture, 2\u2009ml of 0.5% isobaric bupivacaine was injected in group C, and group U received a modified dose according to the DSCSA measured by ultrasound when cerebrospinal fluid (CSF) appeared in the needle hub. Then, the spinal needle was withdrawn.MAP and HR were measured every 2.5\u2009min during surgery in the first 30\u2009min after spinal injection and, then every 15\u2009min until the end of the study.\u2212\u20091\u2009min\u2212\u20091 was treated intravenous continuous infusion to maintain a sufficient analgesia level.The cephalad sensory level was measured via a cold alcohol cotton swab every 5\u2009min until 30\u2009min after the spinal injection and, then every 15\u2009min until regression below L4. Ten minutes after the spinal injection, if the sensory blockade level was below T10, remifentanil 0.1\u20130.2 \u03bcg kgThe motor block level was measured by modified Bromage scale every 5\u2009min until 30\u2009min after the spinal injection and, then every 15\u2009min until complete motor recovery occurred. Modified Bromage scale: 0: able to move the hip, knee, ankle, and toes; 1: able to move the knee, ankle, and toes; 2: able to move the ankle and toes; 3: only able to move the toes; and 4: unable to move the hip, knee, ankle, and toes.The local anesthetics was prepared by an anesthesia assistant (HPY), and she did not assessed all patients. Another anesthesiologist (JCD or HX) assessed the cephalad sensory level and measured the Bromage scale, who remained blinded to the local anesthetics.\u2212\u20091, intravenous 0.5\u2009mg atropine was treated.If the systolic blood pressure decrease more than 30% compare with the baseline, intravenous 5 to 10\u2009mg ephedrine was treated, and a HR of less than 45 beats minWe assessed and recorded the variables, the maximal sensory level, sensory level regression by 2 dermatomes, and complete motor block recovery.2 in the control group and 80.04\u2009\u00b1\u200935.36\u200amm2 in the lumbar central canal spinal stenosis group. Thus, we hypothesized that the dosage would be more excessive for some geriatric patients with lumbar central canal spinal stenosis, and that would be a greater cephalad spread of local anesthetics. We measured the sagittal anteroposterior diameter (D) of the dural sac at L3\u20134 with ultrasound, and calculated the approximate DSCSA (A) according to the formula: A\u2009=\u2009\u03c0(D/2)2, (\u00a0\u03c0\u2009=\u20093.14). For example, to determine the DSCSA /150\u2009=\u20090.48), thus, the modified dose of bupivacaine was decreased by 48%, so 5.2(10\u201310*0.48\u2009=\u20095.2) mg bupivacaine was spinally injected.We confirmed that the primary DSCSA was 150\u200ammWe using G*Power software to estimate the sample size. Taking into consideration the results of previous studies, we set an alpha as 0.05 and a power as 0.8, the result of software shown that at least 26 patients in each group, therefore, 30 patients in each group was a sufficient sample size.P\u2009<\u20090.05 was considered statistically significant.The various parameters were statistically analysed using the SPSS 17.0 . Continuous data were evaluated with independent samples t-test, sensory level with Mann-Whitney U test, and frequency data with Chi square test. The patients flow diagram of this study is shown in Fig.\u00a0P\u2009<\u20090.001) in group U compared with group C.Demographic characteristics , ASA classification, duration of surgery, dosage of bupivacaine and DSCSA were compared in two groups Table\u00a0. The dosP\u2009<\u20090.001) in group U compared with group C . The regression times of the two segments were delay in group U than in group C and specificity (80.8%) for predicting lumbar central canal spinal stenosis [. This optimal cut-off value is less than that of some patients without lumbar central canal spinal stenosis. Therefore, greater cephalad spread results from an excessive dose without regulation according to the DSCSA in group C.The most anatomical change in geriatric patients is lumbar central canal spinal stenosis. The most frequently applied criteria are the measurement of the anteroposterior diameter of the cross-sectional area of the dural sac and of the osseous spinal canal for lumbar central canal spinal stenosis . Thus, tstenosis . This opThere were several limitations of the current study. Unlike magnetic resonance imaging (MRI), ultrasound cannot be used to accurately discriminate the AC from PC, thus, some errors may arise in the sagittal anteroposterior diameter of the dural sac. Second, the DSCSA is not a normal circle, so, the DSCSA we calculated according to the formula is only an approximate value. Third, the research population included a small number of lumbar central canal spinal stenosis patients. The demographic characteristics, such as weight, height and degree of obesity still various.Despite these limitations, the results are important for spinal anesthesia in geriatric patients to compare the DSCSA and the dose of local anesthetics.The DSCSA is a highly effective parameter for spinal anesthesia in geriatric patients undergoing TURP, a modified dose of local anesthetic is a critical factor for controlling the sensory level."} +{"text": "Gadus morhua) is an economically and ecologically important marine species across the North Atlantic. The application of new genomic resources, including SNP arrays, allows us to detect and explore novel structure within specific cod management units. In Norwegian waters, coastal cod (i.e. those not undertaking extensive migrations) are divided into two arbitrary management units defined by ICES: one between 62\u00b0 and 70\u00b0N and one between 58\u00b0 and 62\u00b0N . Together, these capture a fishery area of\u00a0>25,000 km2 containing many spawning grounds. To assess whether these geographic units correctly represent genetic stocks, we analysed spawning cod of NCC and NCS for more than 8,000 SNPs along with samples of Russian White Sea cod, north\u2010east Arctic cod (NEAC: the largest Atlantic stock), and outgroup samples representing the Irish and Faroe Sea's. Our analyses revealed large differences in spatial patterns of genetic differentiation across the genome and revealed a complex biological structure within NCC and NCS. Haplotype maps from four chromosome sets show regional specific SNP indicating a complex genetic structure. The current management plan dividing the coastal cod into only two management units does not accurately reflect the genetic units and needs to be revised. Coastal cod in Norway, while highly heterogenous, is also genetically distinct from neighbouring stocks in the north (NEAC), west (Faroe Island) and the south. The White Sea cod are highly divergent from other cod, possibly yielding support to the earlier notion of subspecies rank.Challenging long\u2010held perceptions of fish management units can help to protect vulnerable stocks. When a fishery consisting of multiple genetic stocks is managed as a single unit, overexploitation and depletion of minor genetic units can occur. Atlantic cod ( The manPopulation genetics of coastal cod in Norwegian waters have been investigated, both in the northern . Otolith types 1 and 2 depict coastal cod, whereas 4 and 5 are assigned to NEAC, according to Rollefsen and BergAll samples were genotyped using a custom Illumina SNP array containing assays for 10,913 SNPs >0.5 criterion, that is \u201csubstantial\u201d evidence for selection according to Jeffreys (1961), was chosen to define non\u2010neutral markers. In LOSITAN, a neutral distribution of FST with 1,000,000 iterations was simulated, with forced mean FST at a significance level of 0.05 under an infinite allele model. It has been suggested that outlier tests may produce high false\u2010positive rates because of population demography and bottlenecks , and a second one containing SNPs on LG1, LG2, LG7 and LG12, but outside the regions of these chromosomes that were deemed to be under positive selection, which was called \u201cNeutrals\u2010B\u201d . A further four sets of candidate markers to positive selection were defined within the selected areas on LG1 , LG2 , LG7 and LG12 , respectively.Consensus genome scan of outlier detection revealed large portions of LG1, LG2, LG7 and LG12 to be under likely positive selection between sampling sites were computed separately for each of the six subsets of SNPs defined above using Weir and Cockerham (Pairwise genetic distances (ockerham unbiasedockerham . StatistFST) and geographic distance (defined as the shortest marine path between sites and measured using Google Earth). The analyses were conducted using PaSSaGE v.2 pattern of isolation by distance (IBD). A two\u2010tailed Mantel test was\u22121.0001. The average dispersal distance obtained for markers under positive selection was compared with the distance for neutral loci to assess whether gene flow might be influenced by any form of environmental pressure.The slopes of IBD tests allow to obtain qualitative estimates of mean dispersal distances using the theoretical model elaborated by Kinlan and Gaines and base2.6K\u00a0=\u00a04 according to STRUCTURE as suggested by Frichot et\u00a0al.\u00a0. Then, LFMM, \u201clatent factor mixed model\u201d in all of the samples revealed that LG1, LG2, LG7 and LG12 contained relatively large genomic regions deviating from neutrality, as indicated by a large number of SNPs scoring a high probability for being under positive selection Figure\u00a0andS2. Co3.2K\u00a0=\u00a02 was the most common in all northern samples between White Sea and Vest, but completely absent from the Irish samples. In the southern samples, haplotypes in green and red were present in high frequencies in the NCS sample from Oslofjord, but completely absent in NCC, NEAC and White Sea samples Figure\u00a0.LG7 displayed several genomic regions where haplotypes were found in high frequencies Table\u00a0. The geo3.4FST for neutral loci was low, albeit statistically significant: 0.007 (p\u00a0<\u00a0.0001) for Neutrals\u2010A and 0.010 (p\u00a0<\u00a0.0001) for Neutrals\u2010B. Following expectations, global FST for loci under positive selection within the different LGs ranged from 0.122 to 0.253 (p\u00a0<\u00a0.0001). Pairwise FST matrices for both sets of neutral loci can be found in Table\u00a0FST of the remaining comparisons was 0.02 (28\u2010fold difference).Global M\u00a0=\u00a00.439\u20130.662, p\u00a0=\u00a0.033\u2013.000) except the markers under selection within LG1 except for LG2 and LG7 . The dispersal distance estimated for neutral markers, when considering the full set of samples was 320\u00a0km, whereas the dispersal for loci under selection within LG was significantly smaller (<50\u00a0km).A series of two\u2010tailed Mantel tests were conducted for each set of SNPs in different subsets of sampling sites to assess the correlation between geographic and genetic distance. The total suite of twelve sites significantly followed IBD expectations for all the set of loci (range from 3.510(PO)=15, 69% of the loci in LG7 showed association with temperature in March and 84% in July. Among the rest of the linkage groups, the highest value was 6% of loci overcoming this threshold in July at LG12 conversely to the 34% of the loci in LG1 and 55% in LG2. In all cases, percentages slightly increased when testing for sea temperature in July. However, in terms of the strength of the association, LG7 singled out in both comparisons. By setting the threshold of \u2010log4This is the first study to investigate genetic structure of Norwegian coastal cod across the two management units using a population genomics SNP approach. We observed pairwise genetic differences between all six coastal cod populations, from Porsanger fjord in the far north to Oslo fjord in the south\u2010east. Although the degree of differentiation among samples depended on the set of markers being considered, population genetic differences were observed in all four genomic regions under selection , as well as in both sets of neutral markers . The present ICES management regime for coastal cod in Norway is formulated around the 62\u00b0N latitude divide, that is Norwegian coastal cod is at present managed as two stocks. Our data demonstrate that while the present management division at 62\u00b0N does capture break in gene flow to the south of this border, it does not sufficiently represent genetic structuring in the north and is in need of revision. In order to aid in defining further management boundaries for coastal cod, finer geographic sampling is needed.Norwegian coastal cod spawn in sheltered fjords and more open coastal areas , and genetic analyses using mtDNA polymorphisms have confirmed its recent divergence from Gadus morhua and south (NCS) of 62\u00b0N, is not sufficient to reflect the true biological units and needs to be revised. Although 62\u00b0N can be the natural border between north and southern populations, most likely there is an even finer structure both north and south of this border. It will be challenging to devise an optimal management strategy that adequately reflects the patterns of genetic variability within coastal cod. The apparent north\u2013south cline in large parts of the genome does not easily lend itself to interpretation in distinct units, and much more geographic fine\u2010scaled approach seems necessary to resolve genetic units. There are still issues to look further into, as the potential areas under selection, that could maybe help in changes in management plan in the future.This study has shown that coastal cod in Norway, while highly heterogenous, is also genetically distinct from neighbouring stocks in the north , west (Faroe Island) and the south (Irish samples). We further found that the White Sea cod are highly divergent from other cod, possibly yielding support the earlier notion of subspecies rank. The other two outlier samples were clearly different from the Norwegian coastal cod and NEAC but showed only subtle differences within, which could be influenced by ascertainment bias.Appendix S1Click here for additional data file."} +{"text": "Nature Communications 10.1038/s41467-020-15891-9, published online 24 April 2020.Correction to: The original version of this Article omitted the source of genetically modified mouse strains in the Acknowledgements section. The Acknowledgements section should read: \u201cMarkus Geuking, Kathy McCoy, and Jakob Zimmermann carried out germ-free derivations of all genetically modified strains used in this study, with exception of NOD1/NOD2 knockout mice that were shared by Dana Philpott, University of Toronto, and generated by Elena Verdue and the Axenic Gnotobiotic Facility at McMaster University. We thank staff and management team of the Clean Mouse Facility, DBMR University of Bern, for gnotobiotic animal maintenance and services,\u201d This has now been corrected in both the PDF and HTML versions of the Article."} +{"text": "Few studies have reported on brain functional differences between healthy individuals with auditory verbal hallucinations (Hi-AVH) with and without insight, so we designed a study to address this knowledge gap. We enrolled 12 Hi-AVH with insight, 15 Hi-AVH without insight, and 15 AVH-free controls . Global functional connectivity density (gFCD) mapping was used to estimate brain networks. We found that the most common alterations in both Hi-AVH groups were increased gFCD in superior parietal lobule and superior temporal gyrus. We also found that distinct brain functional patterns of Hi-AVH without insight comprised lower gFCD in the frontal lobe oculomotor area, dorsolateral prefrontal cortex, supramarginal gyrus, primary auditory cortex, sensorimotor cortex, ventral anterior, and posterior cingulate Our pilot findings support the hypothesis that abnormal reciprocal action in the circuits for processing perception, memory, language, and attentional control may be pathological features of auditory verbal hallucinations. According to the strictest criteria proposed by Johns (\u201cDid you at any time hear voices saying quite a few words or sentences when there was no one around that might account for it?\u201d), 0.7% of the general population have experienced auditory verbal hallucinations (AVH) have AVH satisfying the proposed criteria by Johns \u201cDid you at any time hear voices saying quite a few words or sentences when there was no one around that might account for it?\u201d; 2) no other psychotic symptoms as determined by structured clinical interviews using the DSM-IV Axis I Disorders-Patient Edition (SCID-I/P) conducted by two senior psychiatrists with more than 10\u00a0years of experience; 3) IQ >80. The exclusion criteria were as follows: 1) other psychotic or affective disorders, mental retardation, alcohol dependence, drug dependence, organic brain lesions, or physical and neurological diseases; 2) history of unconsciousness for more than 5\u00a0min caused by any reason; 3) contraindications for MRI examination; 4) claustrophobia; 5) IQ\u2009<\u200980. All subjects were right-handed. Controls were distinguished by a professional psychiatrist using the SCID non-patient version.In this study, AVH severity was assessed using the auditory hallucinations rating scale (AHRS) was performed on a 3\u00a0T GE Discovery MR750 scanner equipped with an eight-channel phased-array head coil. The participants were instructed to lie down in a supine position and to rest without falling asleep during the scan. Whole-brain resting-state fMRI data depicting blood oxygen level-dependent signals were obtained using a gradient-echo echo-planar imaging sequence with the following parameters: repetition time (TR)\u2009=\u20092000\u00a0msec; echo time (TE)\u2009=\u200945\u00a0msec; slices\u2009=\u200932; slice thickness\u2009=\u20094\u00a0mm; gap\u2009=\u20090.5\u00a0mm; field of view (FOV)\u2009=\u2009220\u2009\u00d7\u2009220; matrix size\u2009=\u200964\u2009\u00d7\u200964; and flip angle (FA)\u2009=\u200990\u00b0. All scans were acquired by parallel imaging using the sensitivity encoding (SENSE) technique with a SENSE factor of 2. Structural images were obtained with a high-resolution 3D Turbo-Fast Echo T1WI sequence with the following parameters: 188 slices, TR/TE\u2009=\u20098.2/3.2, slice thickness\u2009=\u20091\u00a0mm, no gap, FA\u2009=\u200912\u00b0, matrix size\u2009=\u2009256\u2009\u00d7\u2009256, FOV\u2009=\u2009256\u2009\u00d7\u2009256.http://www.fil.ion.ucl.ac.uk/spm). To allow for imaging unit stabilization and subject familiarization, the first 10 volumes of each scan were discarded. The remaining volumes were corrected for slice-timing and motion artifacts. Head translation movement for all participants was less than 2\u00a0mm, and rotation was less than 2\u00b0. Covariates, including head motion, white matter signal, and cerebrospinal fluid signal, were regressed out from the time series of every voxel. The Friston 24-parameter model was used to regress out head motion effects. Data were regressed out if the framewise displacement of a specific volume was >0.5. The datasets were filtered with band pass frequencies ranging from 0.01 to 0.08\u00a0Hz. Individual structural images were co-registered to the mean functional image, and the transformed structural images were co-registered to Montreal Neurological Institute (MNI) space using a linear registration. Motion-corrected functional volumes were spatially normalized to MNI space using parameters estimated during the linear co-registration. Finally, the functional images were re-sampled into 3\u00a0mm cubic voxels for further analysis.SPM8 software was used to process the resting-state fMRI scans with age, gender, and education level as covariates, followed by post hoc intergroup comparisons. The post hoc intergroup comparisons were conducted within a mask showing gFCD differences from the ANCOVA analysis, and T value was used to aid in contrast indicators or one-way ANOVA (age and education level). Differences in the social demographic information between the two Hi-AVH groups were tested using a two-sample t-test.X2\u00a0=\u20090.198, P\u00a0=\u20090.911), age , and education level . There were no significant differences in AVH severity or duration between the Hi-AVH groups with and without insight. There was, however, a significant difference in GAF score between groups (P\u00a0<\u20090.05; Table Sixteen Hi-AVH with insight, 17 Hi-AVH without insight, and 20 healthy controls were enrolled in the analysis. These three groups were matched in terms of gender (Compared to AVH-free controls, the Hi-AVH group without insight showed higher gFCD located mainly in the postcentral gyrus, superior parietal lobule, superior temporal gyrus, and temporal pole, and lower gFCD located in the superior frontal gyrus and lingual gyrus Fig.\u00a0. CompareCompared to Hi-AVH with insight, Hi-AVH without insight had lower gFCD in the frontal lobe oculomotor area, dorsolateral prefrontal cortex, supramarginal gyrus, primary auditory cortex, sensorimotor cortex, ventral anterior, and posterior cingulate Fig. . We defiWe did not observe any significant correlation between gFCD and AVH severity in either Hi-AVH group.In this pilot study, we aimed to detect the common and distinct intrinsic functional features in Hi-AVH with and without insight. We observed that gFCD alterations in the Hi-AVH group without insight were more severe than in those with insight.Brain regions that were altered in both Hi-AVH groups were ones involved in perception, memory, language, and central executive control. These findings support the \u201cResting-State Hypothesis of AVH III: Reduced Rest-external Stimulus Interaction in the Auditory Cortex\u201d (Northoff G, et al., Our findings also support the hypothesis that abnormal reciprocal action of the perception, memory, language, and central executive networks can contribute to AVH (Alderson-Day et al. We did not observe any significant relationship between ARHS severity and alterations in gFCD, although such a relationship has been reported by others (Lefort-Besnard et al. There are several limitations to this pilot study. First, the small sample size limits the generalizability of our findings. Second, since it was only a cross-sectional study, prospective longitudinal studies will be necessary to more fully describe the relationship between functional brain alterations and clinical symptoms and to explore early intervention strategies.To the best of our knowledge, this is the first study focusing on alterations in functional connectivity density in Hi-AVH with and without insight. The salient findings of this pilot study were that there are indeed common and distinct brain functional alterations related to insight. Moreover, these findings support the hypothesis that abnormal reciprocal action of perception, memory, language, and attentional-control circuits contributes to the pathological features of AVH. Although some limitations existed, our pilot study nevertheless provided important clues to guide further studies."} +{"text": "Telepsychology is increasingly being incorporated in clinical practice, being offered in many psychotherapy centers, especially afterthe impact of the pandemic. However, there seems to be a remarkable discrepancy between the offer, or interest in, and real-worlduptake of e-mental health interventions among the population. A critical precondition is clients\u2019 willingness to accept and usetelepsychology, although this issue has thus far been overlooked in research.The aim of this study was to examine people\u2019s acceptance and use of telepsychology by adopting an extended model of the unified theory of acceptance and use of technology (UTAUT) that integrates perceived telepsychology advantages and barriers, usefulness perceptions, behavioral intention, and telepsychology use.An online survey was conducted with a convenience sample of 514 participants. Structural equation models were computed totest a mediation model.Results supported the UTAUT model to explain participants\u2019 acceptance and use of telepsychology. They showed a causal chainin which perceived telepsychology advantages and barriers were related to telepsychology use through the perceived usefulness of andintention to use telepsychology.Telepsychology use may be explained according to the UTAUT model when coupled with participants\u2019 perceptions of telepsychology advantages and barriers. Mental health stakeholders could consider these factors in order to increase the acceptance and use of telepsychology. Every year, a high percentage of the population requires mental health services . HoweverInformation and communication technologies (ICT) have great potential to facilitate access to interventions. In this regard, telepsychology, which the American Psychological Association (APA) defines as \u201cthe provision of psychological services using telecommunication technologies,\u201d has appeared in recent years as an alternative to traditional face-to-face interventions, at least for a significant proportion of the population. Telepsychology involves the use of different electronic tools to deliver health care, which may range from telephones and fiber optics to interactive satellite video . This woLiterature on telepsychology use has increased exponentially in recent years . In thisFor really potentiating the use of telepsychology, a fundamental precondition, as with the implementation of any other new technology or application ,15, is tUnfortunately, research has overlooked this issue. Only 3% of studies on eHealth, in general, focus on people\u2019s acceptance, making this an understudied domain ,23. ConsTechnology acceptance is a relatively mature area of research, and there is a significant amount of literature on the matter . It presThis model has been applied and tested in multiple contexts to provide insight into the forces that motivate individuals to adopt technology. In the case of eHealth, most empirical research singles out performance expectancy as the strongest predictor of technology acceptance -37. PercThe UTAUT model underpinA relevant line of research has expanded the UTAUT model by including success factors and resistance factors that drive people to adopt and use a certain technology . Our conceptual model is displayed in Hypothesis 1 stated that perceived telepsychology barriers are negatively related with telepsychology usefulness.Hypothesis 2 stated that perceived telepsychology advantages are positively related with telepsychology usefulness.Hypothesis 3 stated that telepsychology usefulness is positively related with the intention to use telepsychology.Hypothesis 4 stated that the intention to use telepsychology is positively related to telepsychology use.Hypothesis 5 stated that telepsychology usefulness mediates the relationship between perceived telepsychology barriers (H5a) and advantages (H5b) and the intention to use telepsychology.Hypothesis 6 stated that the intention to use telepsychology mediates the relationship between telepsychology usefulness and telepsychology use.As we wanted to study the general population perspective, we recruited a convenience sample through an online advertisement published on our university\u2019s website. The ad explained the research project, explained its main objective, and asked for volunteers who might be willing to participate in our research by taking an open online survey. The ad also provided the link to the survey, which was implemented using the Qualtrics platform. In order to increase response rates, the researchers sent this link along with a brief summary of the research project to their contacts via email.All of the surveys implemented in the host institution for research purposes are implemented using the Qualtrics platform, since it guarantees data protection. Qualtrics allows downloading responses in different formats. Once the survey closed, we downloaded data in Excel format and moved it to SPSS.The survey was responsive to different devices, but we recommended that potential participants complete it using a computer since it was perceived by the research team and users who tested it in advance to be easier. The survey assessed the dimensions (presented in the order used for the survey) that are presented in the following sections. Questions had to be completed to progress in the survey and move to the next screen . There was a maximum of 10 screens (some of them did not appear if they were not applicable for the specific participant by taking into account his or her previous responses). There was not a specific number of items per screen since it depended on the type of item, but we always tried to avoid excessive scrolling.The user\u2019s IP was not registered to guarantee anonymity; however, the Qualtrics system maintains an opened survey and saves a participant\u2019s progress for a week. So, during this period, if participants stopped and restarted the survey, they were directed to the exact place they were when they left the survey (if they used the same computer and browser). At the bottom of the screen, there was a progress bar.The only inclusion criterion for participation was being older than 18 years. In the data collection process, anonymity and confidentiality were guaranteed, and participants provided their consent to participate by accessing the survey and accepting the conditions . No incentive was offered to participants. The protocol was previously approved by the university\u2019s ethics committee. The final sample was composed of 514 participants. A total of 568 persons entered the system; of these, 54 did not complete the survey and were excluded.The current literature did not offer measures for the specific variables in this study. Accordingly, a specific online survey was created following similar studies and taking into account the available literature. The survey was created and reviewed in an iterative manner by the authors. In addition, before making the survey available to participants, it was tested by 4 volunteers who suggested changes that were implemented. They could judge both the format and functionality of the online survey and the content of the items. Regarding the content of items, they could assess if they were appropriate for the targeted construct and easily understandable. The measures of this study were perceived telepsychology advantages, perceived telepsychology barriers, telepsychology usefulness, intention to use telepsychology, and telepsychology use.The perceived telepsychology advantages were assessed by computing the participants\u2019 answers to the statement: \u201cPlease indicate the different advantages that might motivate you to use telepsychology.\u201d According to the literature , the poThe perceived telepsychology barriers were measured by means of a self-developed scale. It included a general statement: \u201cPlease indicate to what extent the following elements would present a barrier to doing online psychotherapy,\u201d with 9 items that reflected the main barriers identified in the literature eg, ,41,43). ,43. 41,4Telepsychology usefulness was also measured using a self-developed, 5-point Likert scale. It included the general statement: \u201cPlease indicate to what extent you think telepsychology can be effective for the following issues,\u201d with 8 items reflecting the most common presenting problems in psychotherapy. More specifically, the items were: (1) improvement of mood disorders , (2) improvement of relational problems , (3) improvement of work-related stress problems, (4) health problems , (5) personal growth issues, (6) mild psychological problems (interfering little with daily life), (7) moderate psychological problems (interfering moderately with daily life), and (8) severe psychological problems (interfering seriously with daily life). The response options ranged from 1 to 5 (very much so).Intention to use telepsychology was assessed with a mono-item scale asking: \u201cIf you had a problem today, how likely would you be to use telepsychology?\u201d The response options ranged from 1 (very unlikely) to 5 (very likely).Telepsychology use was measured as a dummy variable with the following question: \u201cHave you ever attended any kind of online psychological therapy?\u201d Two answer options were provided: (1) no and (2) yes.The following preliminary analyses were computed: mean, SD, and correlation. In addition, given that the measures of telepsychology barriers and usefulness were self-developed, we examined their validity and reliability through confirmatory factor analysis (CFA) and Cronbach alpha. Later, structural equation models (SEM) were performed to test our hypotheses on mediation effects. Two models were computed: (1) a full model that included the direct and indirect relationships among all our variables and (2) a hypothesized UTAUT model. Mplus software was usedOf the 514 participants, 79.8% (410/514) were women, and 20.2% (104/514) were men. The mean age was 36.27 (SD 10.35) years. Only 0.4% (2/514) of the participants had not completed any level of education, while 2.7% (14/514) had studied at elementary school, 27.0% (139/514) had studied at secondary school, 43.2% (222/514) had studied at college, and 26.7% (137/514) had studied a postgraduate course. Up to 61.9% (318/514) of participants reported having undergone face-to-face psychotherapy, and 6.4% (33/514) had experienced telepsychology formats. Finally, 17.1% (88/514) had a monthly salary lower than \u20ac600 (US $708.54), 16.7% (86/514) had a salary between \u20ac600 and \u20ac999 (US $1179.71), 26.7% (137/514) earned between \u20ac1000 (US $1180.86) and \u20ac1499 (US $1770.14), 20.6% (106/514) earned between \u20ac1500 (US $1771.32) and \u20ac1,999 (US $2360.58), 10.7% (55/514) had an annual income between \u20ac2000 (US $2361.72) and \u20ac3000 (US $3542.63), 3.5% (18/514) earned more than \u20ac3000, and finally 24 participants did not disclose their salary range.r=0.50). Hypothesis 3, which suggested a positive relationship between telepsychology usefulness and the intention to use telepsychology, was supported as well, with results showing a significant positive relationship. In other words, participants that perceived telepsychology as useful tended to show a greater intention to use it.Hypothesis 4 was supported, as there was a significant positive relationship between the intention to use telepsychology and actual telepsychology use, indicating that participants with higher levels of intention to use telepsychology presented higher telepsychology use than those with low intention.P=.00) and perceived telepsychology barriers and the intention to use telepsychology.Finally, all the hypotheses about mediation effects were also supported. Regarding the mediator role of telepsychology usefulness (H5), the results showed that it mediated the relationship between perceived telepsychology advantages . In sum, perceived advantages and barriers affected participants telepsychology use through their perception of telepsychology usefulness and their intention to use telepsychology.The results presented a significant indirect effect of telepsychology usefulness on telepsychology use through the intention to use telepsychology (H6), showing that the intention to use telepsychology mediates the relationship between telepsychology usefulness and telepsychology use but also additional determinants such as perceived telepsychology advantages and barriers.Our results supported the viability of the UTAUT model in assessing telepsychology acceptance and use. It showed that telepsychology use is predicted by telepsychology usefulness and the intention to use telepsychology. These results are congruent with the extensive literature on the acceptance and use of new technology and on eHealth acceptance and use in particular ,36,38-40Perceived advantages and barriers also played a relevant role in explaining telepsychology acceptance and use. These factors determined participants\u2019 perceptions of telepsychology usefulness, which affected their intention to use it and, in turn, their actual use of it. A positive perception in the balance between telepsychology advantages and barriers seems to be critical in determining whether people will accept and use this treatment option, with barriers having the strongest effect. These results are also congruent with previous literature on perceived eHealth advantages and barriers ,36,41,42Overcoming barriers and fostering a positive perception of telepsychology has become a central issue since the outbreak of the COVID-19 pandemic. In this new context of social distancing, online psychotherapy has become more a necessity than an option. Many European and American mental health providers and policies relied on using technology to mitigate COVID-19 risks and to respond to elevated mental health demands. Our results can help stakeholders to strategically design ways of facilitating access and readiness to this treatment modality by focusing on the tested UTAUT model. Furthermore, as suggested by Pierce et al , telepsyFinally, the focus of this study was on the perception of synchronous videoconferencing, which is the most similar form of internet-delivered treatment to face-to-face psychotherapy. Other forms of telepsychology, such as internet-based treatment or self-guided, internet-based psychological interventions, could share some critical aspects with the model presented in our study. However, further research should be carried out to understand specific barriers and perceived usefulness when the intervention involves minimal or nonexistent contact with professionals.Despite the interesting insight provided by this study, some limitations must be taken into consideration. First, all the measures were self-reported by participants, making common method variance possible. Future research should consider using additional measures from other sources. Second, the research design of this study was cross-sectional. Thus, it was not possible to infer causal relationships. Further research with longitudinal designs will be necessary to appropriately examine the possible causal effects as well as the stability of the UTAUT model over time.Third, a convenience sampling method was used to collect data, which may limit the extrapolation of our results, especially to clinical settings. However, as it happens in other studies , it is uThis study represents a first step towards applying the UTAUT model to telepsychology. However, we focused on the most important factors to explain participants\u2019 acceptance and use of telepsychology, thereby overlooking other factors that are also relevant. In fact, it is congruent with the recent work by Ammenwerth , who conThis study describes the main factors that must be taken into account to promote acceptance and use of telepsychology among potential clients. Our results provide evidence of the need to foster a positive perception of telepsychology, with a focus on its advantages, and to come up with ways to overcome perceived barriers that do not otherwise hinder conventional face-to-face psychotherapy. In this respect, mental health care stakeholders have a critical role, as van Voorhees et al demonstrated , showing"} +{"text": "We sought to describe the association between serum chloride levels and mortality among unselected cardiac intensive care unit (CICU) patients.We retrospectively reviewed adult patients admitted to our CICU from 2007 to 2015. The association of dyschloremia and hospital mortality was assessed in a multiple variable model including additional confounders, and the association of dyschloremia and post-discharge mortality were assessed using Cox proportional-hazards analysis.9,426 patients with a mean age of 67\u00b115 years were included. Admission hypochloremia was present in 1,384 (15%) patients, and hyperchloremia was present in 1,606 (17%) patients. There was a U-shaped relationship between admission chloride and unadjusted hospital mortality, with increased hospital mortality among patients with hypochloremia or hyperchloremia . After multivariate adjustment, hypochloremia remained associated with higher hospital mortality . Post-discharge mortality among hospital survivors was higher among patients with admission hypochloremia .Abnormal serum chloride on admission to the CICU is associated with increased short- and long-term mortality, with hypochloremia being a strong independent predictor. Serum chloride abnormalities are common among hospitalized patients, and both hypochloremia and hyperchloremia have been associated with increased in-hospital mortality among general intensive care unit (ICU) patients \u20134. HypocSerum chloride is influenced by numerous pathophysiologic processes and plays a key role in the maintenance of osmotic pressure, acid-base disturbances, and regulation of renal function . Serum cThe modern cardiac intensive care unit (CICU) cares for a heterogeneous population of critically ill patients with concomitant cardiovascular disease, yet there are no published studies examining the significance of abnormal serum chloride levels among CICU patients. The aim of our study was to clarify whether an abnormal admission chloride level was associated with higher hospital and post-discharge mortality among CICU patients and to provide insights about the effects of associated electrolyte and acid-base disturbances.The Mayo Clinic Institutional Review Board approved this historical cohort study as a minimal risk study that was exempt from informed consent. We analyzed a database of adult Mayo Clinic CICU patients \u226518 years old whose admission fell entirely between January 1, 2007, and December 31, 2015 and consented to have their medical records used for research under Minnesota state law statute 144.295 . The MayDemographics, vital signs, laboratory results, diagnoses, procedures, therapies and length of stay (LOS) were extracted from the electronic medical record (EMR) through the Multidisciplinary Epidemiology and Translational Research in Intensive Care Data Mart , 19. AdmWe defined admission chloride as the chloride value closest to the time of CICU admission. Serum chloride levels were the default, but plasma chloride values were used if serum chloride was not available. Patients were grouped based on the normal reference range for admission chloride levels: normal admission chloride (admission chloride 98\u2013107 mEq/L), admission hypochloremia (admission chloride <98 mEq/L) and admission hyperchloremia (admission chloride \u2265108 mEq/L). The maximum and minimum chloride levels during the CICU admission were collected, and patients were grouped as follows: normal chloride (minimum chloride \u226598 mEq/L and maximum chloride <108 mEq/L), hypochloremia (minimum chloride <98 mEq/L) and hyperchloremia (maximum chloride \u2265108 mEq/L). Hyponatremia was defined as admission sodium level <135 mEq/L, and hypernatremia was defined as an admission sodium level \u2265145 mEq/L . The admHospital mortality was the primary enpoint. Secondary endpoints included CICU mortality and mortality in the initial five years after discharge among hospital survivors. Continuous variables were compared using analysis of variance (ANOVA), while categorical variables were compared using Pearson chi-square tests. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI). Multivariable analysis was performed to identify predictors of hospital mortality using a non-adaptive elastic net penalized regression model for variable selection, with candidate variables including demographics, comorbidities, illness severity, admission laboratory values, admission diagnoses, and CICU therapies and complications . Ideal tS1 Fig). The mean age of included patients was 67\u00b115 years with 3,519 (37%) females. The baseline characteristics of the final study population are listed in Table 1. The mean admission chloride value was 102.8\u00b15.5 mEq/L, with a distribution shown in S2 Fig.12,904 CICU admissions were screened and 2,900 met the initial exclusion criteria for the study cohort, as previously described , 22, 27.Table 1). Patients with admission hypochloremia had a higher prevalence of HF, worse renal function, and greater overall illness severity than patients with admission hyperchloremia or normal admission chloride. Patients with admission hypochloremia had lower admission sodium and higher admission anion gap and bicarbonate, while patients with admission hyperchloremia had higher admission sodium and lower admission bicarbonate. CICU and hospital length of stay were higher in patients with hypochloremia compared with normal chloride or hyperchloremia (p <0.001).Admission hypochloremia was present in 1,384 (15%) patients, and admission hyperchloremia was present in 1,606 (17%) patients. Patients with admission hypochloremia or hyperchloremia differed from patients with normal admission chloride in terms of baseline characteristics, including comorbidities, illness severity, admission diagnoses, admission laboratory values and procedures, and therapies , an elevated risk of unadjusted hospital mortality was observed in patients with admission hypochloremia or admission hyperchloremia, and patients with admission hypochloremia had higher hospital mortality than patients with admission hyperchloremia . There was a U-shaped relationship between admission chloride and unadjusted hospital and CICU mortality .Hospital mortality occurred in 800 (8.5%) patients, including 489 (5.2%) patients who died in the CICU. Compared to patients with a normal admission chloride (Table 2), a higher admission chloride level remained associated with lower in-hospital mortality ; the final model validation AUC was 0.937. Those with admission hypochloremia were at elevated risk of in-hospital mortality compared with patients with normal admission chloride or admission hyperchloremia . Patients with admission hyperchloremia were not at increased risk of in-hospital mortality .After multivariable adjustment . The relationship between admission chloride and sodium levels with hospital mortality is shown in Fig 3A .A positive correlation was observed between admission chloride and sodium , with abnormal serum sodium levels being common in patients with either hypochloremia or hyperchloremia (Table 1). Patients with an elevated admission anion gap >12 mEq/L had higher overall mortality than those with an anion gap \u226412 mEq/L at all levels of admission chloride .A negative correlation was seen between admission chloride level and anion gap . An elevated anion gap was present in 3022 (32.1%) patients (Table 1). The hospital mortality varied as a function of admission bicarbonate and admission chloride levels .A negative correlation was seen between admission chloride and bicarbonate levels . Metabolic acidosis was present in 2,429 (25.8%) patients, and metabolic alkalosis was present in 2,218 (23.5%) patients were seen in patients with hyperchloremia, hypochloremia or both during CICU admission, compared with patients having normal CICU chloride levels. U-shaped relationships were observed between both minimum and maximum CICU chloride levels and CICU and in-hospital mortality . The range of chloride variation (maximum minus minimum) during CICU stay was positively associated with hospital mortality . Hospital mortality was higher among patients with increasing chloride or decreasing chloride compared with patients without a change in chloride .Hypochloremia during the CICU stay was present in 1,980 (21.0%) patients, while hyperchloremia was present in 2,595 (27.5%) patients, including 149 (1.5%) patients with both hypochloremia and hyperchloremia during their CICU stay. Among patients with normal admission chloride, 8.8% developed hypochloremia, and 14.2% developed hyperchloremia, including 0.7% who developed both. Incremental increases in CICU and hospital mortality , hospital survivors with admission hypochloremia had lower unadjusted post-discharge survival than patients with either admission hyperchloremia or normal admission chloride (p <0.001 by log-rank); post-discharge survival was not different for patients with admission hyperchloremia compared with normal admission chloride levels. The admission chloride level was inversely associated with post-discharge mortality . Hospital survivors with admission hypochloremia had higher adjusted post-discharge mortality than patients with either admission hyperchloremia or normal admission chloride ; post-discharge survival was not different for patients with admission hyperchloremia compared with a normal chloride level (p = 0.46).A total of 2,862 (33.2%) out of 8,626 hospital survivors died during follow-up, with a mean survival of 3.4\u00b12.9 years; 1,200 (13.0%) patients had less than 1 year of follow-up after discharge. On Kaplan-Meier analysis . After adjustment, hospital survivors with isolated hypochloremia during the CICU stay had higher adjusted post-discharge mortality compared with patients with normal chloride or isolated hyperchloremia . The presence of hyperchloremia was not associated with mortality compared to patients with normal chloride or concomitant hypochloremia.Hospital survivors with hypochloremia during the CICU stay had lower unadjusted post-discharge survival (p <0.001 by log-rank), regardless of the presence or absence of hyperchloremia during the CICU stay; patients with isolated hyperchloremia had similar survival compared with patients having normal CICU chloride has been linked to several pathogenic mechanisms that could plausibly contribute directly to adverse outcomes in this group, including disruption of acid-base and fluid balance.Because the pathologic role of chloride is poorly understood, it is unclear why hypochloremia was an independent predictor of poor outcomes while hyperchloremia unexpectedly was not. This may relate to the interplay between serum chloride and acid-base homeostasis, whereby either anion gap metabolic acidosis or hypochloremic metabolic alkalosis can contribute simultaneously to low chloride levels and worse outcomes. However, hypochloremia was an independent predictor of mortality among unselected CICU patients independent of acid-base status, and the majority of these patients had a concomitant metabolic alkalosis. Alkalosis has been associated with poorer outcomes among critically ill populations and could have contributed to the increased mortality rates seen in our hypochloremic patients . HoweverThe correlation between hypochloremia and adverse outcomes could also relate to the lower plasma tonicity in hypochloremic patients; similarly, hyponatremia was associated with higher adjusted mortality in a prior study from this CICU cohort but hypernatremia was not . ChloridWhile our study shows that hypochloremia is a novel adverse prognostic factor among CICU patients, it is unclear whether correction of underlying chloride abnormalities would affect in-hospital or post-discharge mortality. This is particularly salient considering the numerous potential ways in which chloride levels can change in response to various therapies. An increase in serum chloride concentration within 24 hours was shown to correlate with improved survival among hypochloremic patients with severe sepsis or septic shock, suggesting the potential for therapeutic interventions affecting serum chloride to improve outcomes . HoweverAs with all historical cohort studies, this study cannot be used to determine causal relationships and unmeasured confounding variables may have mediated correlation between chloride levels and mortality. Specifically, patients with hypochloremia were substantially sicker than other patients and it is possible that the observed association between hypochloremia and adverse outcomes is due to residual confounding despite multivariable adjustment. This was a single-center study, and our CICU practice may differ from other medical facilities or regions. We could not obtain data on the use of diuretics, type of resuscitation fluids used, or overall fluid balance, which are all known to influence chloride levels; this limits our ability to infer the specific causes of dyschloremia. As such, we are unable to clearly delineate the etiology of the abnormal chloride levels to determine whether any specific cause of dyschloremia was associated with worse outcomes. The ICD-9 admission diagnoses we obtained reflect all diagnoses and cannot distinguish the primary admission diagnosis.Our study demonstrated a U-shaped relationship between admission chloride levels and unadjusted hospital and CICU mortality. After multivariate adjustment, hypochloremia was associated with in-hospital and post-discharge mortality, independent of other electrolytes, including sodium. Hypochloremia remained an important predictor of mortality in patients with concomitant abnormalities of sodium and acid-base balance. Our data highlights the importance of serum chloride abnormalities in the CICU population and suggests an important role of disorders affecting chloride homeostasis among patients with cardiovascular disease. Future studies are needed to determine the importance of chloride within different cardiovascular sub-populations, to establish the etiology of the underlying dyschloremia, and to determine whether correction of underlying chloride abnormalities will lead to improved patient outcomes.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigCICU and hospital mortality as a function of minimum (A) and maximum (B) chloride level during the CICU stay.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file."} +{"text": "It is imperative to preoperatively distinguish dural ossification (DO) and thus anticipate the risks and outcome of the surgery for patients with ossification of ligamentum flavum (OLF). However, studies have disagreed as to the efficacy of the radiographic signs or factors to predict DO and surgical outcome. In additon, the association between the cerebrospinal fluid cross-section area ratio (CCAR) and DO or clinical outcome had not been reported. The purpose of this study was to analyse CCAR and its role in prediction of DO and neurological function recovery rate in patients with OLF.Fifty-two consecutive patients with OLF, who underwent posterior thoracic decompression and fusion between September 2012 and March 2019 at a single institution, were retrospectively reviewed. Demographic data, radiographic signs of DO, CCAR, pre- and postoperative modified Japanese Orthopedic Association (mJOA) score were recorded.P\u2009=\u2009.000). According to the value of CCAR, three zones were defined as DO zone (\u226414.3%), non-DO zone (\u226544.5%), and gray zone (14.3 to 44.5%). When the value of CCAR\u226414.3%, the recovery rate was poor or fair, while it had good or excellent recovery when CCAR\u226545.2%.There were 27 patients in the DO group and 25 patients in the non-DO group, with a mean age at surgery of 57.4\u2009years and 53.9\u2009years, respectively. No significant differences were found in sex, age, segment of maximum compression and preoperative mJOA score between the two groups. The receiver operating characteristic curve showed that the value of CCAR had a relatively high value for diagnosis of DO and prediction of neurological function recovery rate (The value of CCAR had a high diagnostic value for prediction of DO and neurological function recovery rate in patients with OLF. Ossification of the ligamentum flavum (OLF) is the major cause of thoracic myelopathy and has been frequently reported in Japan and other East Asian countries. The incidence of OLF ranges from 3.8 to 64% as reported in previous literatures \u201310. SurgPreoperative identification of DO and prediction of surgical outcome are very important for surgeons to adopt an appropriate surgical strategy and manage dural tear during surgery , 15, 21.The aim of this retrospective study was to (1) measure the value of cerebrospinal fluid cross-section area ratio (CCAR) on magnetic resonance imaging (MRI) and computed tomography (CT) images; (2) investigate the role of CCAR in prediction of DO and neurological function recovery rate.The patients of OLF with thoracic myelopathy, who underwent posterior thoracic laminectomy and fusion with instrumentation between September 2012 and March 2019 at a single institution, were retrospectively reviewed. The study was conducted in accordance with the Declaration of Helsinki and informed consent was waived, which was approved by the Ethical Committee of Peking union medical college hospital. No patients participated in this study and private information was protected. The exclusion criteria were patients with diffuse idiopathic skeletal hyperostosis and concurrent thoracic ventral compressive lesions, such as thoracic disc herniation with protrusion or extrusion, thoracic kyphosis and ossification of the posterior longitudinal ligament, previous history of thoracic surgery, thoracic trauma, infection and tumor.All patients had both CT and MRI images. CT had been performed on a dual-energy CT scanner, Discovery CT1750 or Somatom Definition Flash . MRI had been performed on a 3-T scanner, Discovery MR1750 or Magnetom Skyra . Intraoperative features of DO included an ossified dura that was fused with OLF, the inability to resect the OLF from the ossified portion of the dura mater during surgery , 17 and Demographic data, Sato classification of OLF, radiographic signs of DO, including tram track sign, comma sign and bridge sign, pre- and postoperative neurological function were recorded. The modified Japanese Orthopedic Association (mJOA) scoring system for thoracic myelopathy was used to evaluate pre- and postoperative neurological status. The recovery rate (RR) was calculated by the following formula: RR\u2009=\u2009(mJOA score at last follow-up \u2013 preoperative mJOA score)/ (11 - preoperative mJOA score)\u2009\u00d7\u2009100% . A rate Image J software was used for radiographic measurement. To ensure reliability, imaging evaluation was performed before the review of medical records and two independent observers were responsible for the measurement. All images were measured twice by each observer, and the mean of the two measurements was considered for statistical analysis.It is inaccurate to measured CSA of cerebrospinal fluid directly on MRI, because the demarcation between dura mater and ossification mass on MRI is not clear. Therefore, we calculated the CSA ratio of cerebrospinal fluid to spinal canal indirectly by measuring the CSA ratio of ossification mass to spinal canal on CT and that of spinal cord to dural sac on MRI, respectively. CSA of normal spinal canal on CT image was measured at the pedicle region near the narrowest segment, where the distance between the pedicles was the widest Fig.\u00a0A. The avP value <\u20090.05 was considered statistically significant.The Statistical Package for Social Sciences software was used for data analysis. Interclass correlation coefficients (ICCs) were used to assess interobserver reliability. t-test was used for continuous variables, whereas the chi-square test was used for categorical variables. Pearson\u2019s and Spearman\u2019s correlation coefficients were used to analyze the association between CCAR and DO or RR. Receiver operating characteristic (ROC) curve was drew and area under the curve (AUC) value was used to determine the best cut-off value of the parameters for diagnosing DO and prognosticating RR. A Fifty-two consecutive patients were ultimately included in the study sample. The duration of symptoms varied from 2\u2009months to 28\u2009years (5.3\u2009\u00b1\u20096.2\u2009years) before visiting the hospital. The mean age at surgery was 55.6\u2009\u00b1\u20099.4\u2009years (30\u201374), with a mean follow-up time of 4.6\u2009\u00b1\u20092.2\u2009years (0.6\u20137.2). There were 27 patients in the DO group and 25 in the non-DO group, with a follow-up of 4.5\u2009\u00b1\u20092.1\u2009years and 4.6\u2009\u00b1\u20092.3\u2009years, respectively. The most commonly affected segment of DO was T9\u201312, with an incidence of 34.6% (18/52).No significant differences were found in sex, age, segment of maximum compression and preoperative mJOA score between the DO group and non-DO group Table\u00a0. In the P\u2009=\u20090.000) (Table P\u2009=\u20090.000), and the cut-off value was 36.4% Table . The ROC.4% Fig.\u00a0A. The seP\u2009=\u00a00.000), with a sensitivity and specificity of 89.5% and 69.7%, respectively had thoracic myelopathy, while 28 moderately compressed patients had no neurological symptoms . The reaThis study had some limitations. First, the sample size was relatively small. Nevertheless, it is difficult to adopt large number of cases in one single center due to the low incidence of thoracic OLF and strict inclusion criteria. Therefore, further multicenter studies with large numbers of patients are necessary to verify these preliminary findings. Second, there is concern of possible selection bias due to retrospective study design. However, all patients of OLF were reviewed and only patients who met our criteria were enrolled. Third, there might have errors associated with radiographic measurement on MRI or CT image due to differences in cutting angles induced by technical problems. However, radiologists were well trained to follow the same guidelines when performing CT or MRI scans and the cutting angle for the same segment was the same.This study showed that CCAR had a close relationship with DO as well as the recovery rate of neurological function. A value of CCAR\u226414.3% indicated the presence of DO and poor neurological outcome. To achieve better neurological function recovery, surgical decompression is recommended when the value of CCAR\u226545.2%."} +{"text": "Both interventions included patient therapeutic education and were delivered in three sessions per week over eight weeks. The main outcome was perceived fatigue as assessed by the Spanish version of the Functional Assessment of Chronic Illness Therapy-Fatigue subscale (FACIT-F). Other evaluated outcomes were pain measured on a visual analogue scale, and distance measured using the 6-Minute Walk Test. Data were collected at baseline, immediately post-intervention, and at three and six months after baseline. Significantly greater improvements across all variables were observed in the STE-G throughout the entire follow-up period with the exception of pain. Conclusions: A supervised therapeutic exercise program plus patient therapeutic education significantly reduce perceived fatigue and increase functional capacity in breast cancer survivors suffering from cancer-related fatigue compared to an unsupervised physical exercise program based on individual preferences with patient therapeutic education.This study aimed to determine the effectiveness of therapeutic exercise plus patient therapeutic education on perceived fatigue, functional capacity and pain in breast cancer survivors with cancer-related fatigue. A randomised, single-blind, clinical trial was conducted with a total of 80 breast cancer survivors who presented cancer-related fatigue. Women were randomised into a supervised therapeutic exercise group (STE-G) ( Cancer and its treatments are usually accompanied by symptoms that affect body function and health-related quality of life, such as fatigue, pain, depression, or insomnia ,2. SeverCancer-related fatigue (CRF) is one of the side effects associated with poorer health-related quality of life outcomes in cancer survivors ,4 and enSeveral pharmacologic and non-pharmacologic interventions have been described to treat CRF . While vHowever, the evidence supporting the efficacy of supervised versus unsupervised exercise under equal conditions of adherence is insufficient. Therefore, a randomised clinical trial was conducted to test the hypothesis that, under similar conditions of adherence to exercise, supervised TE combined with a therapeutic education program could be more effective in reducing fatigue and pain and improving functional capacity in BC survivors with CRF compared to unsupervised exercise of their preference plus an educational strategy.A single-centre, single-blind, 2-arm parallel randomised clinical trial was conducted in BC survivors with CRF at the Physiotherapy Unit for Women\u2019s Health Research of the University of Alcal\u00e1 between July of 2018 and October of 2020. The study was registered at ClinicalTrials.gov (NCT02828189) and approved by the Research Ethics Committee of the University of Alcal\u00e1 (CEI2013011). Participant consent was obtained in all cases, and all procedures were performed in accordance with the Declaration of Helsinki and the CONSORT statement.Women who were diagnosed with CRF were recruited in a consecutive order by the Physiotherapy in Women\u2019s Health Research Group at the University of Alcal\u00e1 . The inclusion criteria were: cancer-free women who had undergone unilateral BC surgery with chemotherapy or radiation therapy (RT); having completed their adjuvant RT and/or chemotherapy treatment at least 6 months before the beginning of the trial; diagnosis of CRF by their family doctor according to ICD-10 criteriaa priori estimation was based on the findings of a previous pilot study carried out ad hoc to test the methods and estimate the sample size. We estimated a sample size of 40 individuals in each arm for a power of 80%, an alpha level of 0.05, and a potential maximum dropout rate of 10%. The statistical software Granmo 7.12 was used for sample size calculation.Statistical power and sample size were estimated to detect a between-group difference of 1.4 points in perceived fatigue as scored on the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) subscale with a 5-point variance. This n = 40 in each group) into a supervised therapeutic exercise group (STE-G) or an unsupervised exercise group (UE-G) by an independent physical therapist (MTL: Pt2) blind to the group assignment. The randomisation was performed using a computer randomisation list at a ratio of 1:1 . Following baseline assessment, Pt2 disclosed the group allocation to the two physiotherapists delivering the interventions (VPG: Pt3 & IRD: Pt4) as well as to the participants via a phone call.After checking compliance with the inclusion criteria and reading the study information, the eligible women signed the informed consent form for participation. Participants were individually assessed before the intervention(V0) (MJYS: Pt1) and randomly allocated (Three follow-up visits were scheduled: immediately after completing the intervention (V1), and at three (V2) and six months (V3) after baseline. These follow-up appointments were arranged to match participant availability, who were also contacted by phone or text message one week and three days in advance to confirm or reschedule the date.A different physiotherapist specialised in women\u2019s health (Pt1), who remained blind to participant group allocation throughout the trial, performed all assessments (V0\u2013V3). The baseline assessment was conducted on the day the women agreed to participate in the study and prior to randomisation, and subjects were instructed not to reveal their allocation to Pt1 to ensure blinding success.Personal and clinical data collected at V0 included age, education level, employment status, BC surgery, postoperative complications, adjuvant therapies, pain intensity, duration of pain, and CRF symptoms. Pt2 recorded the primary and secondary outcomes at V0 and follow-up assessments.Perceived fatigue was assessed with the Spanish version of the FACIT-F scale , a self-Secondary outcomes were pain reported on a visual analogue scale (VAS) and distance in metres using the 6-Minute Walk Test (6MWT).The participating women reported their pain intensity over the last two days on a 100The 6MWT is a simple and safe test used to objectively assess functional capacity. Women were asked to walk as far as possible along a 30-m minimally-trafficked corridor for six minutes, and the traveled distance in metres was recorded as the outcome measure. The 6MWT is a valid and reliable instrument tested in cancer patients . A MCID The two interventions lasted 8 weeks, with three weekly sessions of 50\u201360 min each except for the first 6 weeks that included therapeutic education, so the duration was longer by 30 min in two of the three weekly sessions in the experimental group or by 50 additional weekly minutes in the control group. Therapeutic education was imparted at the Physiotherapy in Women\u2019s Health Research Unit of the University of Alcal\u00e1 . The patient therapeutic education for BC survivors suffering from fatigue and persistent pain contained specific content on healthy lifestyle habits and pelvic floor health (patient therapeutic education description can be found elsewhere) ,30. PatiThe exercise program for the STE-G followed ACSM\u2019s Guidelines for Exercise Testing and Prescription in the cancer population ,15,31,32n = 16, 40%), dancing , or cycling . The program consisted of: (1) 5 min of warm-up (mobility and ludic aerobic exercises); (2) 30\u201345 min of their preferred exercise at moderate intensity reported as a perceived exertion of 3\u20134 . The exercise diaries were completed by the patients from both groups and collected weekly during the first six weeks of the intervention, immediately after the intervention (week 8), and at each follow-up assessment (weeks 12 and 24).t-test for independent samples was used for the adjusted means in the comparison between groups. The results of the intergroup comparisons are represented by their adjusted mean with the relevant confidence interval at 95% (CI95%) and corresponding p-value.Descriptive statistics were used for the intergroup comparison of participant characteristics, relevant clinical variables, perceived fatigue, self-reported pain, and distance in metres during the 6MWT at baseline. Separate linear regression models were generated to estimate the average change in continuous outcomes since the baseline at subsequent assessments , adjusting for the basal value. As basal values are frequent covariates, removing the variance associated to such covariates (adjustment) avoids a potential bias resulting from imbalances in baseline values between participants. The Student\u2019s p-value < 0.001). Thus, the Greenhouse-Geisser correction was used to analyse the presence of changes throughout the different visits and whether this evolution was similar between groups. These results are presented in graphs, which include the p-values of the Greenhouse-Geisser comparisons. An \u03b1 = 0.05 was set for all tests. All statistical tests were performed using Stata Data Analysis and Statistical Software .A repeated-measures generalised linear model was employed for the assessment of all outcomes in the intragroup comparison, where the repeated measures (assessment visits) were the intrasubject factor and the intervention group was the intersubject factor. A full factor model was used and the type III sum of squares was estimated. Homogeneity between assessments was evaluated using Mauchly\u2019s sphericity test, yielding statistical significance for all measured outcomes .n = 40; UE, n = 40) with no dropouts or losses to follow-up and MCID at all post-treatment visits .Significant intergroup differences were found in the FACIT-F scores (p > 0.05) in the pain VAS were found at any of the three post-intervention assessments . In contrast, significant intergroup differences in the 6MWT travelled distance (p < 0.001) and MCID were observed at all follow-up visits .No significant intergroup differences . The women in this study showed high therapeutic adherence to the TE programmes.The present study evaluated the effectiveness of two interventions consisting of supervised or unsupervised TE in combination with patient education on perceived fatigue, functional capacity, and pain after the intervention and at three and six months of follow-up in BC survivors suffering from CRF.A greater improvement in perceived fatigue and functional capacity was observed in BC survivors who underwent STE plus patient therapeutic education versus those who performed UE combined with patient therapeutic education.The present clinical trial was designed according to the standardised method for reporting exercise programs in clinical trials . Therefo2peak) during cardiorespiratory exercises is widely considered as the gold standard for measuring cardiovascular capacity, which directly correlates with fatigue levels, functional capacity, and health-related quality of life [Perception of fatigue as measured by the FACIT-F scale was very low at baseline in both groups compared to the general population ,39. Alth of life . However of life ,42 and d of life , for whiAlthough lung function and alterations in breathing capacity were not specifically assessed in the present study, the decrease in lung function following BC treatments is considered as a possible conditioning factor for the appearance and/or perpetuation of fatigue. Thus, the STE program included an approach for the recovery of thoracic and diaphragmatic flexibility, as well as for re-educating breathing to achieve diaphragmatic ventilation. This approach aimed to mitigate some side effects of adjuvant-therapy, such as the decreased lung function in the medium and long term caused by RT. Side effects of RT such as thickening of the pleura, tissue contractions, atelectatic areas, or elevation of the ipsilateral hemidiaphragm can be found in 87% of BC women ,44. NeveIn addition, several studies have reported the superiority of supervised TE over other unsupervised TE modalities. Supervision enhances therapeutic adherence, promotes individualisation and safety of the exercise program, and reinforces trust between the health professional and patient ,13. AlthPersistent pain is a common problem among women being treated for BC, with a prevalence following treatment ranging from 24% to 47% . At the Among the main strengths of this study is the fact that the resources required for assessing the measured variables and implementing the program are easily transferable to routine clinical practice, thus facilitating the inclusion of effective, safe, and easy-to-administer TE in programs for women with BCF. Furthermore, none of the women included in this study developed lymphoedema secondary to BC, not even those in the STE-G who performed specific strength-resistance exercises. These results reinforce the evidence that supervised, individualised, and progressive TE of the upper limb does not increase the likelihood of developing or worsening lymphoedema in BC survivors suffering from, or at risk of, developing lymphoedema ,55,56,57One of the main limitations of this study was the difficulty of comparing the results to previous studies on the effectiveness of TE for CRF management due to heterogeneity in participant characteristics, stage of the oncologic process, associated comorbidities, diagnostic instruments, and characteristics of the intervention in these studies. In addition, no device or application was available to corroborate if appropriate levels of exercise intensity were reached in the medium- and long-term, nor were results of pelvic floor dysfunctions collected, despite having incorporated pelvic floor health into the program. Finally, controlling drugs intake, such as pain relievers, was not possible.A supervised TE program combined with patient therapeutic education significantly reduces perceived fatigue and increases functional capacity compared to an autonomous physical exercise program based on individual preferences combined with patient education in BC survivors suffering from CRF. However, even though pain intensity progressively decreased in all participants, no significant differences were observed between groups. Future studies are needed that include the assessment of lung function and integrate individualised respiratory physiotherapy prior to and/or concomitant with exercise interventions."} +{"text": "Salmonella (NTS) infection is critical to children\u2019s health, and the ceftriaxone is the important empirical treatment choice. With the increase resistance rate of ceftriaxone in Salmonella, the molecular epidemiology and resistance mechanism of ceftriaxone-resistant Salmonella needs to be studied. From July 2019 to July 2020, a total of 205 NTS isolates were collected, 195 of which (95.1%) were cultured from stool, but 10 isolates were isolated from an extraintestinal site. Serogroup B accounted for the vast majority (137/205) among the isolates. Fifty-three isolates were resistant to ceftriaxone, and 50 were isolated from children younger than 4years of age. The resistance rates for ceftriaxone, ciprofloxacin, and levofloxacin were significantly higher in younger children than the older children. The resistance genes in the ceftriaxone-susceptible isolates were detected by PCR, and ceftriaxone-resistant Salmonella were selected for further whole-genome sequencing. Whole-genome analysis showed that serotype Typhimurium and its monophasic variant was the most prevalent in ceftriaxone-resistant isolates (37/53), which comprised ST34 (33/53), ST19 (2/53), and ST99 (2/53), and they were close related in the phylogenetic tree. However, the other isolates were diverse, which included one Enteritidis (ST11), one Indiana (ST17), one Derby (ST40), four Kentucky (ST198), two Goldcoast , one Muenster (ST321), one Virchow (ST359), one Rissen (ST469), one Kedougou (ST1543), two Uganda (ST684), and one Kottbus (ST8839). Moreover, CTX-M-55 ESBLs production (33/53) was found to be mainly responsible for ceftriaxone resistance, followed by blaCTX-M-65 (12/53), blaCTX-M-14 (4/53), blaCTX-M-9 (2/53), blaCTX-M-64 (1/53), blaCTX-M-130 (1/53), and blaCMY-2 (1/53). ISEcp1, IS903B, IS Kpn26, IS1F, and IS26 were connected to antimicrobial resistance genes transfer. In conclusion, the dissemination of ESBL-producing Salmonella isolates resulted in an increased prevalence of ceftriaxone resistance in young children. The high rate of multidrug resistance should be given additional attention.The non-Typhi Salmonella is a genus of Gram-negative bacteria of the family Enterobacteriaceae, and Salmonella enterica subsp. enterica is the most frequent pathogen triggering human sickness , and non-Typhi Salmonella , which include the TEM, SHV, CTX-M, VEB, and GES enzymes and SS agar and cultured at 35\u00b0C for 18h. Blood samples were cultured with a Bactec FX400 System , and the other types of samples were incubated on blood agar . Then, the correct clones were picked and streaked to purity for the tests. Patients\u2019 information, including age, sex, and infection site, was obtained from the digital record system, and their names were masked. The age of children was calculated in decimal years by considering a month approximately 0.083years, and a day approximately 0.0027years. This study was approved by the Ethics Committee of Children\u2019s Hospital of Zhejiang University School of Medicine (2021-IRB-031).In Children\u2019s Hospital of Zhejiang University School of Medicine from July 2019 to July 2020, all patients for whom clinical specimen were collected and found to host All the isolates were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry systems . The serogrouping was conducted by a seroagglutination test , and serogroups A, B, C1, C2-C3, D1, and E1 were determined. These serogroups were also identified as O:2, O:4, O:7, O:8, O:9, and O:3, 10, respectively.Escherichia coli ATCC 25922 was used as the quality control. The agar dilution method was used following the guidelines of the Clinical & Laboratory Standards Institute (CLSI), and the breakpoint was interpreted according to the CLSI guidelines (M100 29th edition). The AST of colistin was determined by the micro broth dilution method, and a minimal inhibitory concentration (MIC)>2mg/L was considered resistant following European Committee on Antimicrobial Susceptibility Testing breakpoint interpretation.Antimicrobial agent susceptibility was tested for all collected isolates, including against ampicillin, ceftriaxone, chloramphenicol, ciprofloxacin, levofloxacin, meropenem, and trimethoprim/sulfamethoxazole. Salmonella were selected for whole-genome sequencing, and genomic DNA was extracted with a QIAamp DNA Mini Kit . The Illumina HiSeq X-Ten platform was chosen for whole-genome sequencing with a 150bp paired-end strategy, as previously described was classified as Salmonella Typhimurium .Detection of the multilocus sequence typing (MLST) and the Inc-type plasmid of the strain were screened and determined by using the MLST 2.0 server and Plasmid Finder 1.3 at the Center for Genomic Epidemiology,blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, and blaCMY-2 among the ceftriaxone susceptible isolates were screened by polymerase chain reaction (PCR) and Sanger sequencing, and the primers used in this study are shown in For ceftriaxone-resistant isolates, antimicrobial resistance genes (ARGs) were identified with whole genome sequences (WGSs) by ABRicate with parameter identity >90% and minimum length>60%,The contigs that contained ESBL genes were extracted and annotated by Prokka , and parAll 53 ceftriaxone-resistant isolates were imported for phylogenetic relationship analysis based on their single-nucleotide polymorphisms (SNPs). The genome alignment was established by Snippy with default parameters,p<0.05 was considered as statistically significant.The comparison of antimicrobial resistance rates was performed by Fisher\u2019s exact, and Salmonella spp. were deposited in the NCBI database under BioProject accession number PRJNA749852.The WGS of ceftriaxone-resistant Salmonella isolates were obtained. The isolates from feces accounted for the vast majority (195/205), and the remaining 10 isolates were from several types of sterile body fluids. Six out of 10 were from blood samples in the gastroenterology department, respiratory department, neonatal department, and pediatric intensive care unit (PICU). One strain (SM-201) was isolated from pleural fluid and the other (SM-290) was isolated from abdominal pus . There were two isolates from subperiosteal pus . A total of 86.8% (178/205) of Salmonella isolates were from children less than 4years of age in this study, and the median age of these children was 1.25 (IQR: 0.79 to 2.29), suggesting that younger children were the most potentially susceptible population.During the period of the current study, a total of nonduplicated 205 Salmonella serogroups and antimicrobial susceptibility in a tertiary children\u2019s hospital , 23.4% (48/205), 11.7% (24/205), 75.6% (155/205), and 37.1% (76/205), respectively. The multidrug resistance (MDR) rate in the current study was 37.6% (77/205), and there was no meropenem-resistant Salmonella. Interestingly, the resistance rates for ceftriaxone, ciprofloxacin, and levofloxacin were significantly higher in younger children (age<1) . For instance, the ciprofloxacin resistance rate was 41.5% (22/53) in the ceftriaxone-resistant group, compared with 17.1% (26/152) in the ceftriaxone-susceptible group; and the difference resistance rates were 34.0% vs. 3.9% for levofloxacin.The seroagglutination test and AST were performed to explore the distribution of hospital . Fifty-t (age<1) , and 94.Salmonella isolates, followed by serogroup D1 (21/205), serogroup C1 (19/205), serogroup E1 (12/205), and serogroup C2-C3 (10/205). There were six isolates that could not be classified into a specific serogroup by the seroagglutination testing. One of these isolates, SM-233, was resistant to ceftriaxone, and further whole genome sequencing showed that SM-233 belonged to serovar Kedougou, which is serogroup G Salmonella among these lmonella . SerogroSalmonella isolates were utilized to further clarify their molecular characteristics and phylogenetic relationship. All the ceftriaxone-resistant Salmonella isolates were from stools except one from blood. The MLST results showed that ST34 was the predominant sequence type , and the others were diverse, including one ST11, one ST17, two ST19, one ST40, two ST99, four ST198, one ST2529, one ST321, one ST358, one ST359, one ST469, one ST1543, and two ST684. Furthermore, one novel ST (ST8839) was found with the MLST profile , and it was nearest to ST808, with the point mutation C102T in aroC.Whole-genome sequencing data of ceftriaxone-resistant Among these isolates, serovar Typhimurium and its monophasic variant were predominant, and 37 serovar Typhimurium and its monophasic variant isolates corresponded to more than one sequence type, which included ST19, ST34 and ST99. ST358 and ST2529 belonged to serovar Goldcoast. In the other serovars, there was a one-to-one relationship between the serovar and the MLST, for instance, serovar Kentucky corresponded to ST198, and serovar Uganda corresponded to ST684.The phylogenetic tree showed that serovars, such as Enteritidis, Muenster, Rissen, Virchow, Indiana, Kedougou, and Kottbus, were differently positioned on the phylogenetic tree and unrelated . The resSalmonella spp. isolates, the antimicrobial resistance genes were analyzed. Almost all ceftriaxone resistant isolates were CTX-M-type ESBL producers, except one producer of CMY-2 \u03b2-lactamase, which is an AmpC cephalosporinase. In contrast, no CTX-M ESBLs or AmpC cephalosporinase genes were detected among the ceftriaxone-susceptible isolates, suggesting that those enzymes were possibly responsible for ceftriaxone resistance. Among the CTX-M-producing isolates, blaCTX-M-55 (33/53) was the most prevalent, followed by blaCTX-M-65 (12/53), blaCTX-M-14 (4/53), blaCTX-M-9 (2/53), blaCTX-M-64 (1/53), and blaCTX-M-130 (1/53). In particular, SM-55 harbored two different types of ESBLs, blaCTX-M-14 and blaCTX-M-55. TEM \u03b2-lactamases were mainly distributed (40/53) in ceftriaxone resistant isolates; however, the blaTEM-1 gene was still detected in 62.5% (95/152) of ceftriaxone susceptible isolates, showing its limited effect on ceftriaxone resistance. Among the ceftriaxone-resistant isolates, 38 isolates harbored blaTEM-1, and two isolates (SM-20 and SM-22) had blaTEM-215, which were both ST684. Four isolates were blaOXA-1 positive; eight isolates were blaOXA-10 positive, and there were three isolates contained blaLAP-2 type plasmid reached 8mg/L; however, the colistin MIC of isolate SM-66 (accession number: SAMN20422894) was just 0.5mg/L, remaining susceptible. WGS analysis showed that the deletion mutation (GTGGCGAGTGTTG) of mcr-1 in SM-66 was observed between nucleic acid positions 55 and 67. The plasmid type of these two mcr-1-harboring isolates was IncHI2, which is common plasmid groups carrying multidrug resistance determinants.Interestingly, SM-66 and SM-212 both harbored the Salmonella infection, we found other invasive NTS (iNTS) infections, such as bloodstream infections, abdominal infection, and osteomyelitis, and even ceftriaxone-resistant Salmonella bloodstream infection, which caused an increased burden on clinical treatment. Chen\u2019s report indicated that iNTS would cause prolonged hospitalization, increased medical costs, and elevated mortality , which pblaCTX-M-55 and qnrS1 on the same contig, which indicated that these genes were located on the same plasmid. Paterson indicated that ESBL gene harboring plasmids likely carry genes for resistance to many other antimicrobial agents, such as aminoglycosides, trimethoprim, sulfonamides, tetracyclines, and chloramphenicol, which may be the reason for MDR of ceftriaxone-resistant isolates also showed resistance to ciprofloxacin, and the percentage was higher than that in the ceftriaxone-susceptible group. The antimicrobial resistance genes ciprofloxacin and ceftriaxone are usually carried by plasmids, which facilitates the coexistence of these genes on the same plasmids. The results showed that strain SM-15 contained the for MDR . In thesn Brazil , Spain can be found at: in silico analysis. QS drafted the manuscript. YJ reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.YJ and YY participated in the design of the study. QS, YY, and MZ collected the isolates and clinical information. PL and YY performed the antimicrobial susceptible testing and serotyping. QS and XH prepared for whole-genome sequencing and PCR. JQ and XH performed the This work was supported by the National Natural Science Foundation of China (grant nos. 81830069 and 81861138054).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": "The COVID-19 pandemic caused many educational institutions in the world to switch to the distance education process, and this process was called \"Emergency Remote Teaching\". This urgent transition process has caused many problems in educational environments. One of the problems is the subject of measurement and evaluation. Along with the pandemic, many institutions have used various online assessment systems to make measurements and evaluations online, and researchers have conducted research on these online assessment systems. This research focus on the features of the online assessment systems and aims to examine the trends towards the prominent features of the online assessment systems in the Emergency Remote Teaching period. For this purpose, the prominent online assessment systems have been determined by systematically analyzing academic studies published in 2020, and answers have been sought to the following research questions: (1) which platforms they support, (2) which security features they have, and (3) what common features they have. Identifying trends in the characteristics of online assessment systems is expected to guide practitioners, decision-makers, researchers, and system developers in the process of selecting and/or developing an online assessment system for use in online measurement and evaluation. It is an undoubted fact that exams are extremely important to achieve teaching goals supported platforms, (2) security features used, and (3) common features of the systems. For this purpose, answers were sought for the following research questions:This research determines the current status of online assessment systems, the need for which was felt more in the ERC period, by examining their features. To determine the prominent online assessment systems, this research systematically analyzed the articles published in 2020 on online assessment systems. The list of online assessment systems has been obtained by following the procedures proposed by Kitchenham . These pThe features of the prominent online assessment systems that were used or mentioned by the published articles are the data of this research. The data sources to identify these prominent systems are the articles published in 2020, and the data sources to determine the characteristics of the systems are the documents/texts published on the websites of these systems. To collect and analyze the data, the following five stages proposed by Kitchenham were perFirstly, to identify the prominent online assessment systems in 2020, a search strategy was determined, including the selection of a search tool and the identification of search keywords. The authors chose Google Scholar since (a) it is the most comprehensive search tool for finding academic studies supported platforms, RQ2) security features, and RQ3) common features. Frequency (f) values shown in the figures and tables show how many of the 22 examined systems have the relevant characteristics.Features of the platforms supported by online assessment systems are synthesized under five categories considering the study conducted by Foster and Layman , namely:Figure\u00a0Even though students tend to use mobile devices in online assessments do not have mobile operating system support yet , Blackboard , Canvas , and D2L . Other supported e-Learning platforms are as follows: Sakai , Open edX , Schoology , Opigno , other , and undefined .This result shows that Moodle, Blackboard, and Canvas are the most frequently used e-learning systems that work well with prominent online assessment systems. According to Yorulmaz and Can , the reaThe second theme determines the security features of the online assessment systems. The concept of security in online assessment is about the features included in the system to detect/prevent cheating and/or fraud. Security features have been synthesized and discussed under the following five categories: 2.a) Authentication, 2.b) Restrictions, 2.c) Monitoring, 2.d) Data Analysis, and 2.e) Intervention Methods.Authentication methods enable the online assessment system to verify the identity of the user/student to ensure the correct person has taken the exam. This category exhibits authentication methods used in the analyzed online assessment systems, and these authentication methods have been classified according to the time when they were applied, namely: before, during, and after the exam. Table Table When the authentication methods are evaluated from a broad perspective, it can be seen that the systems mostly use the biometric data of the users/students for authentication. Moini and Madni stated iOne of the least preferred authentication methods in the research is security (f\u2009=\u20093) questions. Similarly, Renaud and Just have\u00a0revOnline assessment systems use restriction methods and disable various features on a user's device in order to prevent cheating. Table According to Table Examining the disabled features in online assessment systems has revealed that some restrictions require users to install an additional application. However, the need to set up a program or a web browser plugin may prevent an assessment system from operating platform-independently. Therefore, future studies can consider the restriction methods synthesized in this research to deal with fraud and cheating.Security-enhanced online assessment systems enable instructors to monitor participants to ensure security in exams. This category has synthesized the monitoring/surveillance methods used in the analyzed online assessment systems under four sub-categories, namely: a) Semi-automatic Monitoring (Machine and Human Proctoring), b) Full-automatic Monitoring (Machine Proctoring), c) Live Monitoring (Human Proctoring), and d) Record Monitoring. These four classifications can be explained as follows: In a semi-automated monitoring system, the assessment system detects fraud and then a human decides whether the students are cheating or not. In a fully automated monitoring system, both detection and decision of cheating processes are performed by the machine (online assessment system). Live monitoring systems enable a human proctor to watch/follow all students\u2019 personal data during the exam. Record monitoring systems enable the human proctor to monitor recorded personal data of students at any time after the exam. Table Table On the other hand, this research has revealed a misunderstanding between semi-automatic monitoring and full-automatic monitoring. Some of the analyzed online assessment systems state on their web pages that the surveillance is done fully automatically, but when the working principles of these systems are examined, it is understood that these systems just collect students' personal data and then present those data to a human (not the system) to decide whether there is cheating or not. To be called full-automatic monitoring/proctoring, it is not enough to receive image data from the user; it should also be determined whether there is a fraudulent situation in that image data.To provide security in online exams, online assessment systems can analyze various data during the online exam. This category has classified the data collected for this purpose in the analyzed assessment systems, and Table Table In some countries, collecting/analyzing/storing users' biometric data is a sensitive subject that requires high attention by law. Therefore, it would be better for researchers/developers to pay attention to laws when using biometric data for authentication in an online assessment system. For instance, according to the Personal Data Protection Law of Turkey , if userRemote intervention features empower proctors or systems to intervene with users when security breaches are detected during the exam. This category has classified the remote intervention methods used in the analyzed assessment systems, and Table Table The third theme contributes to literature by determining the common features of the prominent online assessment systems. Common features have been synthesized under the following four categories: (3a) question types, (3b) support services, (3c) hardware or software requirements, and (3d) integration methods.This category has been classified into the types of questions supported by the leading online assessment systems. Table Table Richardson et al. drew attOnline assessment systems include various support services to help students during the online exams. Table When Table The possibility of live chat with the lecturer/supervisor has been seen in semi-supervised online assessment systems and mostly in technical matters, warnings, etc. with the supervisor, has been used at times. The low frequency of live chat with a lecturer/supervisor may be due to its low usability, especially in crowded classrooms. When the literature is examined, it has been found that students want to get feedback from the instructors, but they complain that online exams reduce their interaction with the instructor supported platforms, (2) security features, and (3) common features of the online assessment systems.The first research question has revealed that some of the existing online assessment systems are not mobile-friendly. However, the ability to attend assessments online via mobile devices can ensure flexibility in education. In addition, considering the socio-economic status of the students and mobile phones are more affordable than computers: it can be said that the rate of owning a mobile phone can be higher than the rate of having a computer. On the other hand, faculty can benefit from LMS integration while transferring data such as student identification and grade transfer. Therefore, developers should take into account that future online assessment systems would (i) provide mobile device support and (ii) enable e-Learning platform integration, and decision-makers would prefer the systems that have (iii) mobile device support and (iv) common web browser support that students frequently used.The second research question helps to better understand the security features of the online assessment systems. The security features of these systems towards detecting and preventing cheating have been synthesized in this research, and it is understood that the majority of these systems authenticate users/students through government-issued IDs; disable copy/paste functions; use semi-automatic monitoring methods; and analyze the video, image, voice, and screen records. The findings synthesized in the previous section can guide decision-makers, researchers and system developers working on security in online assessment systems, and can help them to better understand what features should/will online assessment systems include in order to ensure security.The third research question focuses on common features of the prominent online assessment systems, and synthesis of the common features has revealed the following characteristics: question types they support are multiple-choice, essay, and true/false questions; modules they include are frequently asked questions module, help documents module, and technical support module; requirements are webcam, microphone, and Internet connection; and data sharing method they support is API. Hereby, these features can be expressed as the basic features of an online assessment system and can be taken into account by future studies.This research examines online assessment systems as a tool, and findings of this research (characteristics of online assessment systems) can guide practitioners, decision-makers, researchers, and system developers in the process of selecting and/or developing an online assessment system in the future. However, since students are the users of online assessment systems, it is important to understand students' views, how online assessment systems reflect on students and support their learning. Therefore, future studies can address the effects of online assessment systems on various academic factors.The findings of this research are limited to the features of online assessment systems mentioned in the studies indexed by Google Scholar in 2020. The studies on online assessment systems were identified by using search queries in January 2021.Various contradictory pieces of information were encountered on the web pages of some online assessment systems. Therefore, researchers sent the data collection form/tool to the online assessment system companies via email. However, if a company did not respond, then researchers collected data from its web page (including videos and help documents) and social media accounts. When contradictory or confusing statements were found in the data sources, the researchers filled in the data collection form by adding their comments."} +{"text": "Motor impairment is one early feature of preclinical Alzheimer\u2019s disease and strongly predicts future dementia. Whether older persons with dementia who had prior motor impairment present specific characteristics is less clear. In the Baltimore Longitudinal Study of Aging, we compared characteristics of participants with cognitive impairment or dementia who had slow gait on average 6 years before symptom onset (<1.0m/sec) to those who did not . Early cognition and key biomarkers on average 7 years before onset were compared between groups using multivariable linear regression, adjusted for demographics. Those with prior slow gait had early unfavorable metabolic panel , lower manual dexterity and processing speed, and lower lysophosphatidylcholine 18:1 and 18:2, critical in the synthesis of cardiolipin-an essential component of mitochondrial membranes. Strategies to prevent metabolic dysfunction at an early stage may slow down dementia progression."} +{"text": "ICC value was between 0.860 and 0.966 (p < 0.001). Since the Cohen\u2019s value and ICC were above 0.6 for all raters, the ratings performed by all six raters were used in the analysis. The ICC values between raters were between 0.899 and 0.902. Conclusions: We came up with a set of reference photos that can be used for submental fat rating scale applicable to Korean subjects. Level of Evidence: II.Background: This aim of this study was to develop an objective tool for rating submental fat applied to Koreans. Methods: The study was conducted between April 2019 and October 2019. A total of 92 subjects were enrolled in the study. Clinical photos of the subjects were categorized using validated CR-SMFRS by three plastic surgeons and one dermatologist. The categorized photos were then shown to six different plastic surgeons for evaluation. Results: The Cohen\u2019s kappa value for the six raters were 0.830, 0.742, 0.703, 0.907, 0.862, and 0.793 with statistical significance ( In an era of global population aging, people are becoming more interested in rejuvenating their facial cosmetic appearance . SubmentThe purpose of this study was to develop and validate an objective submental fat rating tool for Koreans. For this purpose, the Clinician-Reported Submental Fat Rating Scale (CR-SMFRS) used for the trial of ATX-101 was used as a reference to evaluate the inter-rater and intra-rater reliability of a submental fat rating scale conducted on Korean subjects.The study was conducted in accordance with the Declaration of Helsinki and Korean Good Clinical Practice (KGCP). All aspects of the study were approved by the Institutional Review Board (IRB No. B-1901/514-303). Prior to their participation, subjects were informed of the purpose and potential risks associated with the trial through a consent form approved by the IRB. The consent form was signed, and a copy was given to the subject accordingly.The study was conducted between 1 April 2019 and 18 October 2019. A total of 92 subjects were enrolled in this study. Subjects included any male or female patients between the ages of 15 and 65 years who agreed to sign the consent form. The exclusion criteria are listed in The tools used for the trial of ATX1-1, such as the Clinician-Reported Submental Fat Rating Scale (CR-SMFRS), Patient-Reported Submental Fat Rating Scale (PR-SMFRS), Subject-Self Rating Scale (SSRS), and Patient-Reported Submental Fat Impact Scale (PR-SMFIS), were translated with linguistic validation and renamed for the study. The renamed tools are presented in On the first day of the visit, the subjects signed the consent form. Height and body weight measurements of the subjects were also taken. Two photographers took two- and three-dimensional photographs of all the enrolled subjects. Five two-dimensional photos were taken in front, left/right lateral, and left/right oblique views, as shown in The subjects completed the SSSS, SR-SMFPIS, DAS 24, BIQLI, blind SR-SMFRS, and unblind SR-SMFRS. If the clinical photograph taken was considered inappropriate for evaluation, the subject was asked to revisit for a new set of clinical photographs. The clinical photos taken were used to evaluate the subjects\u2019 submental fat using the ATX-101 validated SMFRS used as a reference. Thus, clinical photos were taken using standardized methods from two photographers.The research team was composed of three plastic surgeons and one dermatologist from one institution. The evaluation team was composed of six plastic surgeons from three different institutions. The photos, without any patient information, were sent to the research team of three plastic surgeons and one dermatologist. After being familiarized with the validated CR-SMFRS used in the ATX-101 trial, the research team categorized the set of photos into the different grades used in CR-SMFRS. Categorization was performed so that at least 10 subjects were included in each grade. This set of photos was then provided to the rating team.The rating team, composed of six plastic surgeons, was informed about the validated CR-SMFRS used in the ATX-101 trial. The blind raters were given clinical photos of subjects, graded by the research team, in a random arrangement and were instructed to stratify them based on the validated CR-SMFRS guideline. Four weeks later, the rater team, which was still blinded, evaluated the same set of photos that were rearranged randomly. The rating scale utilized by the rater team was ER-SMFRS. The intra-rater reliability was analyzed using Cohen\u2019s kappa coefficient, and any rater with a kappa value < 0.6 was excluded. Inter-rater reliability was analyzed using intraclass correlation coefficient (ICC). Data derived from a rater with values < 0.6 were excluded from the analysis.All statistical analyses were performed using R software (version 4.0). For continuous variables, the mean, standard deviation, and minimum and maximum values were calculated. For categorical variables, the frequencies and percentages were calculated. Data with missing values were excluded from the analysis. All test statistics used in the analysis of this data were the results of a two-sided test, and the statistical confidence level was based on a multiplicity correction of 0.05.The correlation of unblind SR-SMFRS with blind SR-SMFR; ER-SMFRS with blind SR-SMFRS, unblind SR-SMFRS, SSSS, SR-SMFPIS, DAS24, BIQLI, and cervicomental angle; blind SR-SMFR with SSSS, SR-SMFPIS, DAS 24, BIQLI, and cervicomental angle; unblind SR-SMFR with SSS, SR-SMFPIS, DAS 24, BIQLI, and cervicomental angle; and SSSS with SR-SMFPIS, DAS 24, BIQLI, and cervicomental angle was evaluated.Initially, 92 patients agreed to participate and were enrolled in the study. Among them, one was excluded due to a violation of selection/exclusion criterion (the subject\u2019s age was above 65 years). Based on the unblind SR-SMFRS, 15 subjects were classified as grade 0, 20 as grade 1, 28 as grade 2, 14 as grade 3, and 14 as grade 4, respectively. The photography team excluded 16 subjects due to quality issues of photos including poor subject position, poor image quality, inadequate Frankfort horizontal plane, and insufficient light exposure. The research team excluded eight subjects: three due to low inter-rater match, two due to acne scar, one due to lax chin skin, one due to short chin, and one due to mandibular prognathism. Three additional subjects were excluded due to inappropriate cervicomental angle in the three-dimensional analysis. The hyoid bone and sternal notch were marked on the subjects before 3D scanning. However, the markers were inadvertently deleted during the scanning process for the three subjects excluded from the cervicomental angle analysis. The exclusion process is shown in p < 0.001). The ICC values were between 0.860 and 0.966 (p < 0.001). Since the Cohen\u2019s values and ICC were above 0.6, for all raters, the ratings performed by all six raters were used in the analysis. The ICC values between the raters were between 0.899 and 0.902.The Cohen\u2019s kappa values for the six raters were 0.830, 0.742, 0.703, 0.907, 0.862, and 0.793, respectively (p < 0.001). Age, BMI, and sex information are outlined in p = 0.001, p < 0.001, respectively). The ratio of female subjects was highest in grade 0. The ratio of male subjects was highest in grade 4, and the ratio increased with an increase in the grade (p < 0.003).The gradings performed by the rater team, corresponding to ER- SMFRS, were 0.11 \u00b1 0.12 for grade 0, 1.01 \u00b1 0.27 for grade 1, 2.06 \u00b1 0.26 for grade 2, 3.01 \u00b1 0.31 for grade 3, and 3.75 \u00b1 0.23 for grade 4. The difference between grades was statistically significant . Regarding sex, the ratio of women was the highest in grade 0, and the ratio of men was the largest in grade 4. Based on the results, it was found that when the submental fat grade increased, the ratio of men was high. Therefore, when evaluating the degree of submental fat, it is necessary to consider the factors of age, sex, and BMI.By comparing the various demographic information that may affect submental fat among the subjects, it was confirmed that there were meaningful differences in submental fat depending on the subject\u2019s age, BMI, and sex. Age was highest in subjects categorized as grade 2. BMI was highest in grade 4, and both age and BMI increased with increasing submental fat grade (p < 0.001). No evaluators were excluded because there were no raters with Cohen\u2019s kappa values below 0.6. The ICC of the results of submental fat rating among raters also ranged from 0.899 to 0.902 with a 95% confidence interval, showing almost perfect agreement. Both the 1st and 2nd evaluations conducted 4 weeks apart were found to have high reliability and reproducibility: 0.11 \u00b1 0.12 for grade 0, 1.01 \u00b1 0.27 for grade 1, 2.06 \u00b1 0.26 for grade 2, 3.01 \u00b1 0.31 for grade 3, and 3.75 \u00b1 0.23 for grade 4. The difference between grades was statistically significant (p < 0.001). Clinicians in the esthetic field who have been trained with this highly reliable evaluation method are expected to be able to objectively evaluate the degree of improvement of submental fat during clinical studies and procedures.The rater team, independent of the research team, was composed of six plastic surgeons at three different institutions. The team was trained and informed about the validated CR-SMFRS used in the ATX-101 trial, and the team evaluated the front, left/right lateral, and left/right oblique view clinical photos of 64 subjects using ER-SMFRS. The intra-rater reliability within the six raters ranged from 0.703 to 0.907, which were in \u201cexcellent agreement\u201d and \u201cperfect agreement.\u201d The Cohen\u2019s kappa values for each rater were 0.830, 0.742, 0.703, 0.907, 0.862, and 0.793, respectively, with statistical significance . This implied that the subject could objectively self-evaluate the submental fat with a certain precision.The correlation coefficient between blind SR-SMFRS, which was performed while maintaining the blindness of subjects by not providing information on validated CR-SMFRS used in the ATX-101 trial, and unblind SR = SMFRS, which was performed without blinding the subjects by providing a photobook of validated CR-SMFRS used in the ATX-101 clinical trial, was 0.859 with statistical significance (p < 0.001). The correlation coefficient between the ER-SMFRS and unblind SR-SMFRS was 0.757 (p < 0.001). Although ER-SMFRS correlated with both blind and unblind SR-SMFRS with statistical significance, the correlation was stronger with unblind SR-SMFRS. Thus, if validated CR-SMFRS were provided to subjects for reference, self-evaluation of submental fat would be more objective.The correlation coefficient between the ER-SMFRS and blind SR-SMFRS was 0.689 (p < 0.01), 0.426 (p < 0.001), and 00.497 (p < 0.001), respectively. The average cervicomental angles were 113 \u00b1 5.12\u00b0 for ER-SMFRS grade 0 subjects and 139.8 \u00b1 13.22\u00b0 for ER-SMFRS grade 4 subjects. The difference between grade 0 and 4 subjects varied from 8 to 44\u00b0. Due to the variability in the anatomical shape of each subject, there was a limitation in arriving at a reliable, objective cervicomental angle range for each submental fat grade.The correlation between submental fat grading and cervicomental angle, a representative index of esthetic submental fat assessment, was evaluated using three-dimensional photo analysis. The correlation coefficients between the cervicomental angle and ER-SMFRS, blind SR-SMFRS, and unblind SR-SMFRS were 0.694 (There are several limitations to the study. First, all evaluations were made using clinical photos only. Thus, skin laxity was evaluated only by photo. The study would have been much more precise if patients were evaluated in person. The study excluded any patient older than 65 years old. As submental laxity occurs more frequently in aged patients, a study including patients older than 65 years old would be a more comprehensive tool that can be used for patients in a wider range of age. Furthermore, the scale may not be generalizable to the entire East Asian population since the study included only Korean patients. At last, this study excluded patients who received any type of non-surgical or surgical treatment of submental fat. Thus, it may not be accurate in evaluating the effectiveness of any rejuvenation therapy to reduce submental fat.Based on the results derived from this study, it is possible to produce an evaluation guideline that includes reference photos and criteria for a reliable submental fat rating scale applicable to subjects, especially Koreans, to evaluate the improvement of submental fat. Furthermore, a survey that evaluates the quality of life associated with submental fat can also be developed. This validated submental fat rating scale can be applied as a tool for evaluating the effectiveness of clinical trials for the improvement of submental fat. It can be utilized as a diagnostic index that can be applied in the field of cosmetic plastic surgery to provide customized yet reliable information on submental fat to both patients and medical staff."} +{"text": "Journal of Healthcare Engineering has retracted the article titled \u201cNeuroprotective Effects of Probiotic-Supplemented Diet on Cognitive Behavior of 3xTg-AD Mice\u201d [AD Mice\u201d due to cFollowing an investigation conducted by the Hindawi Research Integrity team , signifiThe authors do not agree with the retraction."} +{"text": "Incarcerated people are at a disproportionate risk of contracting HIV. We estimated the prevalence and correlates of HIV testing among incarcerated people with a history of HIV-related high-risk behaviours in Iran.Data for this analysis were obtained from three consecutive nationwide bio-behavioural surveillance surveys of a random sample of incarcerated people in 2009 (n\u2009=\u20095953), 2013 (n\u2009=\u20095490), and 2017 (n\u2009=\u20095785). History of testing for HIV in the last 12\u2009months was the primary outcome variable. HIV testing was examined among those with a history of HIV-related high-risk behaviours . The outcome variable was divided into three categories: Never tested for HIV, ever tested for HIV inside the prison\u00a0in the last 12 months, and ever tested for HIV outside the prison\u00a0in the last 12 months. We used multivariable multinomial logistic regression models to examine factors associated with HIV testing.Overall, 8,553 participants with a history of HIV-related high-risk behaviors with valid responses to the HIV testing question were included in the analysis. Although HIV testing inside prison has increased , the prevalence of HIV testing outside prison has decreased over time. Our multivariable multinomial regression model showed older age , history of the previous incarceration , currently receiving methadone maintenance therapy inside prison , having access to condoms inside prison and sufficient HIV knowledge were significantly associated with an increased probability of having an HIV test in the last 12\u2009months inside prison.HIV testing among high-risk Iranian prisoners has increased from 2009 to 2017. However, HIV testing remains considerably low, and half of the incarcerated people with a history of HIV-related high-risk behaviours had never tested for HIV inside prison. Evidence-based programs are needed to optimize HIV testing inside and outside prisons and identify those at greater risk of HIV.The online version contains supplementary material available at 10.1186/s12879-022-07897-z. Incarcerated people are at a higher risk of contracting or transmitting HIV than the general population, primarily due to their engagement in HIV-related high-risk behaviours and PWID \u201325. HarmThree national BBSS were conducted among Iranian incarcerated people in 2009, 2013, and 2017. The design, sampling, and data collection approaches were harmonized across the three surveys and have been described elsewhere . A multiThe eligibility criteria across all surveys were being incarcerated for at least one week, having not participated in a similar study in the previous two months, and providing verbal informed consent . Gender-\u201chaving completed an HIV test in the last 12\u2009months\u201d. The primary outcome had three options (never tested vs. tested inside a prison in the last 12\u2009months vs. tested outside a prison in the last 12\u2009months). Incarcerated people were questioned: \u201cHave you ever tested for HIV?\u201d If no, they were coded as \u201cnever tested.\u201d If yes, they were asked, \u201cWhen did you last test for HIV?\u201d and \u201cWhere did you last test for HIV?\u201d. Participants who had a test during the last 12\u2009months and inside the prison were considered to be \u201ctested inside a prison in the last 12\u2009months\u201d. Participants who had a test during the last 12\u2009months and outside the prison were considered to \u201ctest outside a prison in the last 12\u2009months\u201d.As routine HIV testing is recommended for people at a high risk of HIV, we restricted the analysis to those with a history of HIV-related high-risk behaviours, including a history of injection drug use, multiple-sex partnerships (two or more partners at the same time), and having had a tattoo (ever). The primary outcome variable in this study was Socio-demographic factors of interest were sex , age at interview \u2264\u200929 vs. >29) , 29, cur vs. >29 Data were also recorded on drug use and sexual risk behaviours, history of previous incarcerations (yes vs. no), lifetime history of alcohol consumption (yes vs. no), lifetime history of illicit drug use (yes vs. no), current receipt of MMT (yes vs. no), having access to sterile syringe inside prison (yes vs. no), age at first sex (<\u200918 vs. \u226518\u2009years),\u00a0and having access to condom inside prison (yes vs. no). We also measured HIV knowledge and self-perceived risk of HIV (yes vs. no).Descriptive statistics, including frequencies, percentages, and 95% confidence intervals (95% CI), were reported for HIV testing inside and outside of prison in the last 12\u2009months. People living with HIV who knew their HIV status were excluded from the analysis. Bivariable and multivariable multinomial logistic regression models were built to compare the probability of having an HIV test in the last 12\u2009months among different subgroups of incarcerated people after merging the data from three rounds of surveys and adjusting for the year of data collection. Variables with a p-value less than <\u20090.2 in the bivariable multinomial logistic regression model were entered into the multivariable multinomial logistic regression model. The final model was chosen through the backward elimination method using Wald statistics. Survey analysis using the Svy package in Stata /SE 14.0 was used to analyze the data. Analyses were adjusted for prison\u2019s sample sizes by applying appropriate sampling weights.\u00a0A sensitivity analysis was conducted to assess correlates\u00a0of HIV testing across different surveys in 2009, 2013, and 2017 through separate regression analyses participants had a history of HIV-related high-risk behaviours . Additionally, of the 5490 participants in 2013, 3,449 , and out of 5785 participants in 2017, 3,353 had a history of HIV-related high-risk behaviours and were included in this analysis. Among the 2009 survey participants, 97.6% (n\u2009=\u20092811) were male, 52.1% (n\u2009=\u20091500) were >\u200929, 45.7% (n\u2009=\u20091309) were single, and 55.5% (n\u2009=\u20091598) had secondary or high school education. In the 2013 survey, 98.8% (n\u2009=\u20092770) were male, 69.7% (n\u2009=\u20091945) were in >\u200929\u2009years, 46.2% (n\u2009=\u20091294) were married, 51.9% (n\u2009=\u20091451) were secondary or high school education. In the 2017 survey, 96.1% (n\u2009=\u20092756) were male, 72.9% (n\u2009=\u20092086) were in >\u200929\u2009years, 41.3% (n\u2009=\u20091185) were single, 61.8% (n\u2009=\u20091770) had secondary or high school education participants had tested for HIV inside\u00a0the prison in the last 12\u2009months. Also, 223 participants had tested for HIV outside\u00a0the prison in the last 12\u2009months. Of the 2,804 included participants in 2013, 603 had a history of HIV test inside\u00a0the prison in the last 12\u2009months, and 211 had a history of HIV test outside\u00a0the prison in the last 12\u2009months. In 2017, of the 2,869 included participants, 1,442 participants had a history of HIV test inside\u00a0the prison in the last 12\u2009months and 119 participants with an HIV test outside the prison in last 12\u2009months : 1.24, 95% CI: 1.05, 1.47), history of previous incarceration , current receipt of MMT inside prison , having access to condoms inside prison , and sufficient HIV knowledge were significantly associated with an increased probability of having an HIV test in the last 12\u2009months inside prison. No covariate was significantly associated with the probability of having an HIV test in the last 12\u2009months outside prison , 32, andWe also found that incarcerated people with a history of previous incarceration, currently receiving MMT inside prison, access to condoms inside prison, and sufficient knowledge about HIV transmission had a higher probability of HIV testing inside the prison. This finding highlights that harm reduction services inside prisons might have been impactful in encouraging incarcerated people with a higher risk of drug use and sexual behaviour profiles to engage in HIV testing; as shown by our findings in 2009, the prevalence of HIV testing among the incarcerated people has remained low . HoweverWe acknowledge the limitations of our study. First, the cross-sectional nature of our surveys limited the drawing of any causal inference in the observed associations. Second, the self-reported nature of our data makes it prone to social desirability and recall biases. As some of the behaviours asked in the interview were sensitive , some people may not have disclosed their behaviors. Third, we did not measure the availability of antiretroviral treatment after HIV testing for people living with HIV, which could be explored in future studies. Lastly, we did not measure the barriers to HIV testing inside and outside the prisons. We recommend future quantitative and qualitative studies to examine the obstacles to HIV testing among incarcerated people.While the increasing prevalence of HIV testing inside prisons in Iran is encouraging, our findings indicate that incarcerated people\u2019s HIV testing practices are still considerably low both inside and outside prisons in Iran. Our findings highlight the need to revisit and re-evaluate existing HIV testing policies and services inside and outside prison settings to help improve HIV testing uptake among this subpopulation. Scaling up rapid tests and routine opt-out HIV testing services could help encourage incarcerated people to further use the available harm reduction facilities inside and outside prisons.Additional file 1.\u00a0Multivariable Multinomial logistic regression on HIV testing in thelast 12\u2009months among incarcerated in 2009, 2013, and 2017."} +{"text": "While first gene-drug pairs have emerged to be clinically actionable in the treatment of major depressive disorders (MDD) , genomic studies have not yet been successful in identifying replicable and valid biomarkers of pharmacological treatment outcome. While some trials suggest that candidates such as CYP2D6, CYP2C19, CYP1A2, SLC6A4 and HTR2A polymorphisms may improve the prediction of response/remission, these results should be interpreted cautiously and required confirmation in larger samples. This presentation will cover state of the art of pharmacogenomics for MDD as well as the emerging field of pharmacotranscriptomics and functional genomics analyses in MDD. Specifically, pharmacotranscriptomics in combination with genomics may be a promising avenue in overcoming some of the current limitations in treatment response prediction research. More recently, the combined genetic effect of polygenic risk scores has shown promising results in predicting treatment response. Importantly, adequately large and well phenotyped clinical trials are required to be conducted with pharmacogenomics/-transcriptomics prospectively in mind.No significant relationships."} +{"text": "Several biomarkers have been proposed to predict the occurrence of acute kidney injury (AKI); however, their efficacy varies between different trials. The aim of this study was to compare the predictive performance of different candidate biomarkers for AKI.In this systematic review, we searched PubMed, Medline, Embase, and the Cochrane Library for papers published up to August 15, 2022. We selected all studies of adults (>\u200918\u00a0years) that reported the predictive performance of damage biomarkers , kidney injury molecule-1 (KIM-1), liver-type fatty acid-binding protein (L-FABP)), inflammatory biomarker (interleukin-18 (IL-18)), and stress biomarker ) for the occurrence of AKI. We performed pairwise meta-analyses to calculate odds ratios (ORs) and 95% confidence intervals (CIs) individually. Hierarchical summary receiver operating characteristic curves (HSROCs) were used to summarize the pooled test performance, and the Grading of Recommendations, Assessment, Development and Evaluations criteria were used to appraise the quality of evidence.We identified 242 published relevant studies from 1,803 screened abstracts, of which 110 studies with 38,725 patients were included in this meta-analysis. Urinary NGAL/creatinine , urinary NGAL , and serum NGAL had the best diagnostic accuracy for the risk of AKI. In subgroup analyses, urinary NGAL, urinary NGAL/creatinine, and serum NGAL had better diagnostic accuracy for AKI than urinary IL-18 in non-critically ill patients. However, all of the biomarkers had similar diagnostic accuracy in critically ill patients. In the setting of medical and non-sepsis patients, urinary NGAL had better predictive performance than urinary IL-18, urinary L-FABP, and urinary TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3. In the surgical patients, urinary NGAL/creatinine and urinary KIM-1 had the best diagnostic accuracy. The HSROC values of urinary NGAL/creatinine, urinary NGAL, and serum NGAL were 91.4%, 85.2%, and 84.7%, respectively.Biomarkers containing NGAL had the best predictive accuracy for the occurrence of AKI, regardless of whether or not the values were adjusted by urinary creatinine, and especially in medically treated patients. However, the predictive performance of urinary NGAL was limited in surgical patients, and urinary NGAL/creatinine seemed to be the most accurate biomarkers in these patients. All of the biomarkers had similar predictive performance in critically ill patients.Trial registrationCRD42020207883, October 06, 2020.The online version contains supplementary material available at 10.1186/s13054-022-04223-6. Acute kidney injury (AKI) is associated with a higher risk of chronic kidney disease (CKD), end-stage renal disease (ESRD), and long-term adverse cardiovascular effects , 2. Due We conducted this pairwise meta-analysis according to the Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) statement and usedThe primary outcome was incident AKI. Electronic searches were performed on PubMed (Ovid), Medline, Embase, and Cochrane library from inception to August 15, 2022 clinical studies that included participants over 18\u00a0years of age and of any ethnic origin or sex; (2) studies that reported candidate AKI biomarkers including NGAL, KIM-1, L-FABP, IL-18, and TIMP-2\u2009\u00d7\u2009IGFBP-7; and (3) studies that assessed the occurrence of incident AKI. The exclusion criteria were as follows: (1) studies including patients who had previously received dialysis; (2) studies including pregnant or lactating patients; (3) letters, conference or case reports; and (4) studies that lacked data on sensitivity or specificity of biomarkers to predict the occurrence of AKI. Only regular full papers were selected for quality assessment and data synthesis. We contacted the authors of abstracts for further detailed information, if available.Six investigators independently reviewed the search results and identified eligible studies. Any resulting discrepancies were resolved by discussion with a seventh investigator (Vin-Cent Wu). All relevant data were independently extracted from the included studies by eight investigators according to a standardized form. Extracted data included study characteristics and participants\u2019 baseline data (mean age (years), gender (%), comorbidities, severity of illness). When available, odds ratios and 95% confidence intervals (CIs) from cohort or case-controlled studies were extracted. Other a priori determined parameters included the type of intensive care unit (ICU) setting , criteria used to diagnose AKI and severe AKI, cohort size, and the presence of sepsis. Any disagreements were resolved by discussion with the investigators (Heng-Chih Pan and Vin-Cent Wu).The Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool was used to assess the quality of each included study , 21. TheWe hypothesized that the following factors could have high impacts on patient outcomes observed among different studies: clinical setting (ICU/non-ICU), patient population , whether the studies only included patients with sepsis or not and different AKI criteria ; Acute Kidney Injury Network (AKIN); Kidney Disease: Improving Global Outcomes (KDIGO)).P value\u2009<\u20090.05 was considered statistically significant. The bivariate model was conducted using SAS version 9.4 with the \u201cMETADAS\u201d macro (version 1.3) which is recommended by the Cochrane Diagnostic Test Accuracy Working Group. The HSROC analysis and funnel plots were performed using R software version 3.6.3 with the \u201cmeta4diag\u201d package (version 2.0.8) based on Bayesian inference.A 2 by 2 table reporting the patient number of true positive, false positive, true negative, and false negative findings for the cutoff point given by the included studies was used to generate sensitivity, specificity, and diagnostic odds ratio (DOR) for each study. The sensitivity, specificity, and DOR for all of the included studies were combined using a bivariate model. DOR was defined as the endpoint of primary interest in this study because it combines the strengths of sensitivity and specificity with the advantage of accuracy as a single indicator . The senThe study selection process is summarized in Additional file All 110 studies provided quantifiable results for AKI. Seventy-nine studies exclusively enrolled ICU patients, and 31 studies enrolled non-ICU patients. Fifty-seven studies exclusively enrolled surgery patients, and 55 studies enrolled patients from mixed surgical/medical settings. Only 8 studies enrolled patients with sepsis, and therefore, analysis of sepsis was not conducted. Of the enrolled studies, 44 used the KDIGO classification as the only definition for AKI, 23 used AKIN, 21 used RIFLE, 6 used two or more definitions, 6 used a 50% increase in SCr, 1 used an increase in SCr from normal to >\u20093\u00a0mg/dL, 3 used a 0.5\u00a0mg/dL increase in SCr within 48\u201372\u00a0h, and 6 were at the discretion of the attending physicians.The studies were published over 18\u00a0years and varied in sample size from 22 to 1635 patients was numerically highest for NGAL/creatinine (NGAL/Cr) , which was reported in 9 studies. The results demonstrated that urinary NGAL had high diagnostic accuracy , which was significantly better than IL-18 , and TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 for the occurrence of AKI , followed by L-FABP/Cr and urinary NGAL. The diagnostic accuracy of urinary NGAL was significantly better than TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 , which was significantly better than IL-18 , IL-18/Cr , KIM-1 , L-FABP , and TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 for the occurrence of AKI in the setting of medical/mixed patients had significantly better diagnostic accuracy for AKI than IL-18 , L-FABP , and TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 , and therefore, the analysis was not conducted. In the 100 studies which adopted standard AKI criteria, NGAL/Cr had the highest diagnostic accuracy , followed by KIM-1 , and urinary NGAL . Urinary NGAL had significantly better diagnostic accuracy for AKI than IL-18 and TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 , followed by IL-18 , and TIMP-2\u2009\u00d7\u2009IGFBP-7: 2 . Among the other 80 studies that diagnosed AKI without using urine output criteria, NGAL had the highest diagnostic accuracy , followed by urinary NGAL/Cr . Urinary NGAL had significantly better diagnostic accuracy for AKI than IL-18 , IL-18/Cr , KIM-1 , and L-FABP , followed by urinary NGAL/Cr , and serum NGAL . Urinary NGAL had significantly better diagnostic accuracy for AKI than IL-18 , L-FABP , and TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 . Among the low- or middle-quality studies, KIM-1/Cr had the highest diagnostic accuracy , followed by KIM-1 , and IL-18 . Both KIM-1 and IL-18 had significantly better diagnostic accuracy for AKI than NGAL, while IL-18/Cr had significantly worse diagnostic accuracy for AKI than NGAL , and urinary NGAL . Urinary NGAL had significantly better diagnostic accuracy for AKI than IL-18 , L-FABP , and TIMP-2\u2009\u00d7\u2009IGFBP-7: 0.3 . Among the other 32 studies conducted in middle- or low-income countries, L-FABP had the highest diagnostic accuracy , which was significantly better than urinary NGAL , and the diagnostic accuracy was numerically highest for L-FABP , serum NGAL , L-FABP/Cr , and urinary NGAL , and the diagnostic accuracy was numerically highest for TIMP-2\u2009\u00d7\u2009IGFBP-7: custom , and serum NGAL . Serum NGAL and urinary NGAL were the most commonly used biomarkers for AKI (Table 0/38725. There is an unmet need for the early detection of AKI due to an increase in the incidence of AKI in hospitalized patients , 135. InThe complexity of the pathogenesis of AKI due to factors such as hemodynamics, inflammatory status, genetic background, the use of nephrotoxic compounds, and interventions means that the clinical course of AKI differs in different clinical situations . In critIn non-critically ill or medical patients, patient stratification for the risk of AKI should be applied to the entire hospital population before any scheduled elective intervention. In order to minimize unnecessary impacts due to these scheduled treatments, the specificity should outweigh the sensitivity . In our However, the sensitivity and specificity in the enrolled studies were heterogeneous because they depended on the circumstances and the threshold effects of the biomarkers. Considering the potential threshold effects and the correlation between sensitivity and specificity, HSROC analysis proved the good predictive performance of L-FABP/Cr and the NGAL series Fig.\u00a0A. There Although the damage and stress biomarkers in this study had good predictive performance, unlike troponin in acute coronary syndrome, none of the reported biomarkers are completely specific for AKI. Previous studies have reported that NGAL, IL-18, and KIM-1 may be elevated in the setting of sepsis and CKD \u2013146. Of The strength of our analysis is the extensive literature search of related studies. We used standard Cochrane protocols and included the largest cumulative study sample size to date in comparison with previous reports. The strength of our meta-analysis also lies in the comprehensive data search with subgroup analyses across several clinical scenarios. We used the GRADE approach to rate the certainty of evidence .Besides limitations in the meta-analysis, there were several limitations in the individual studies. First, most studies had a small sample size, and this contributed to the high heterogeneity of the meta-analysis. Second, our funnel meta-regression and Cochrane Collaboration tool analysis showed significant publication bias (Additional file Based on our pairwise meta-analysis of biomarkers to predict AKI, NGAL series had the best diagnostic accuracy for the prediction of AKI, regardless of whether or not they were adjusted by urinary creatinine, especially in medical patients. However, the predictive performance of urinary NGAL was limited in surgical patients, and NGAL/Cr seemed to be the best biomarkers in these patients. All of the biomarkers had similar predictive performance in critically ill patients. Future pragmatic clinical trials are warranted to evaluate the real-world predictive accuracy of AKI biomarkers.Additional file 1: Supplementary appendix."} +{"text": "Incisional hernias are more frequent in adults than in children. It is hypothesized that a more efficient healing process in pediatric patients could explain this difference in incidence. Certain elements of healing such as neovascularization, degree of inflammation, percentage of mature and immature collagen, the proliferation of fibroblasts, and expression of certain genes could explain why healing in children is more efficient when compared to the adult and elderly populations.Seventy-one rats of\u00a03\u00a0different age groups underwent surgery with\u00a03\u00a0different incisions . During the procedure, the skin and abdominal wall of the animal were sectioned and only the skin was sutured to mimic incisional hernia in the animals. Four weeks after surgery, the rats were euthanized, their skin was removed, and the extent of scar tissue formed in the muscle opening was measured. In addition, samples of the scar tissue were collected for histological, immunohistochemical, and molecular analyzes. Nine rats served as controls.p\u00a0=\u00a00.03). There was a greater proliferation of fibroblasts in rats in the younger age groups, regardless of the type of incision. The Lox gene was more expressed in weaning rats with vertical and oblique incisions.Shorter-length hernias were formed in weaning rats when compared to old ones when the surgical incision was horizontal (These differences could explain the better healing and lower incidence of hernias in the pediatric population, although this aspect requires further studies. Incisional hernia is a condition that affects patients who undergo surgical procedures, especially in the abdominal region, and subsequently presents an opening in the muscle layer allowing the passage of organs and other internal tissues, without exceeding the skin limit.These hernias usually affect adults but are not so common in the pediatric population. A study conducted at a Japanese hospital with\u00a02049\u00a0patients aged less than\u00a015\u00a0years who underwent surgery reported an overall rate of\u00a00.68% of incisional hernia, an incidence rate significantly different from that of adults, estimated at\u00a05%\u00a0to\u00a050%.,,Collagen has a major role in the wound healing process, being the main element of the new cellular matrix and responsible for the ability of the new tissue to resist tension.The histological and genetic mechanisms behind the lower incidence of incisional hernia in the pediatric vs. adult population are still unknown. The objective of the present study was to investigate and analyze these crucial elements in the scar tissue of rats of\u00a03\u00a0different age groups using histological, immunohistochemical, and molecular analyzes. This study also sought to understand how incisions in different orientations can interfere with the formation of incisional hernias in rats.The animals were cared for following the criteria set forth in the \u201cGuide for Care and Use of Laboratory Animals\u201d from the National Academy of Sciences. The study protocol was reviewed and approved by the Animal Ethics Committee of the present study's institution.Seventy-one rats of\u00a03\u00a0different ages were subjected to abdominal incisions in\u00a03\u00a0different orientations and nine additional rats that did not undergo any procedure were used as controls. The control animals had the same age as the experimental animals. Once the incision was made, only the skin was closed with continuous Mononylon\u00a04.0\u00a0sutures, while the muscle remained open to mimic the condition of an incisional hernia. Four weeks after the procedure, the animals were euthanized and two samples of scar tissue from the muscle layer were collected from each animal. In addition, the extension of hernia regression was measured in all groups, by removing the skin of the euthanized animal and measuring the largest dimension of the scar formed at the muscle opening. During the entire time between the procedure and euthanasia, the rats were kept in controlled-environment cages, under analgesia, with water and food. The results of the measurements of groups were compared.For histological analysis, two samples of the scar tissue were taken from each rat and kept for\u00a024\u00a0hours in\u00a010%\u00a0formaldehyde. After fixation, the specimens were dehydrated and then embedded in paraffin; 4-\u03bcm\u00a0thick sections were stained with hematoxylin-eosin and picrosirius red.The hematoxylin-eosin staining allowed us to analyze the general morphology of the tissue. The picrosirius red staining observed under polarized light was helpful to show the structural arrangement of collagen fibers. Histologically, type\u00a0I collagen present in mature fibrotic lesions is strongly birefringent, showing a yellow or red color when exposed to polarized light after staining with picrosirius red. Type\u00a0III fibers present in the weaning animals\u2019 granulation tissue, also called reticular fibers, are histologically individualized, forming thin networks with low refringence and a greenish color.The histological characteristics analyzed in the slides stained with hematoxylin-eosin were the total number of vessels and inflammatory infiltrates. To analyze these characteristics, 5\u00a0random fields were selected per slide (under\u00a0400\u00a0\u00d7\u00a0magnification). The total number of vessels was a quantitative measurement, while the analysis of inflammatory infiltrates was performed using a semi-quantitative method based on scores .The collagen present in the analyzed fields was measured using the NIS Elements BR\u00a03.2\u00a0software . A selection of red or orange birefringent shades (corresponding to type\u00a0I) or greenish collagen fibers (corresponding to type\u00a0III collagen fibers) was performed.The immunohistochemical analysis to evaluate fibroblast cell proliferation was performed using the Ki-67 antibody . The number of immunopositive cells (stained in brown) was counted using the NIS Elements BR\u00a03.2\u00a0software . Ten random fields were analyzed per slide (under\u00a0400\u00a0\u00d7\u00a0magnification) and the number of immunopositive cells was quantified per field. Therefore, the final value for comparison between the groups was expressed as the number of stained cells per field.The molecular analysis assessed the expression of the Lox gene by quantitative Real-Time PCR (qRT-PCR). This is a fluorescence technique for monitoring DNA amplification within each cycle. The number of copies of the gene was determined using Sybr Green\u00a0I, a fluorescent dye that intersperses with double-stranded DNA. Fluorescence is captured by the thermocycler at each new PCR cycle reaction and allows the equipment to draw an amplification curve for each sample.The analysis of gene amplification by qRT-PCR was performed using relative quantification, in which the gene expression of one sample is described in relation to another. In this case, samples from control animals not submitted to the procedure were used, and only muscle specimens from these animals were collected.The statistical comparison between groups was done by age and by the orientation of the incision . In the comparison between these groups, the variables analyzed were hernia size, neovascularization, inflammatory infiltrates, percentage of immature and mature collagen, the proliferation of fibroblasts, and expression of the Lox gene.p\u00a0\u2264\u00a00.05.Continuous quantitative data were analyzed using the one-way ANOVA method to compare three or more groups. Nonparametric data were analyzed using the Kruskal-Wallis test to compare three or more groups. The tests were performed using the SPSS version\u00a018.0 for Windows and the equality hypothesis was rejected for\u00a0p\u00a0=\u00a00.03), despite the fact that horizontal incisions were always proportionally smaller than the vertical incisions (25%\u00a0of the size).Hernia size analysis: in all groups of rats subjected to a specific incision , a reduction of the opening in the muscle created surgically was noted four weeks after the procedure. Rats of different ages subjected to the same type of incision did not present statistically significant differences regarding the reduction of the initial incision . The onlp\u00a0=\u00a00.003) and the AV group exhibited less neovascularization vs. the OV group (p\u00a0=\u00a00.001). After horizontal incision, there was a statistically significant difference between the WH and AH groups, with a higher number of new vessels found in the AH group (p\u00a0=\u00a00.001). Regarding the level of inflammation, there was no statistical difference between groups. . The WO group exhibited a lower percentage of immature collagen vs. the AO and OO groups . Concerning mature collagen, the quantification revealed a higher percentage of this protein in the AV group vs. the WV and OV groups (p < 0.001 in both comparisons). The WO group displayed a higher degree of mature collagen vs. the AO group (p\u00a0=\u00a00.006) .Fig. 3Plp < 0.001). With oblique incisions, the only statistically significant difference was found between the WO and the OO groups, with weaning animals presenting more ki67-stained fibroblasts. Similarly, in the horizontal incision groups, younger animals displayed more stained cells, with differences found between the WH and AH groups (more stained fibroblasts in the WH group) and between the AH and OH groups (with AH displaying more stained fibroblasts) . With oblique incisions, a similar phenomenon occurred, with the younger WO group showing higher expression of the Lox gene vs. the AO and OO groups .Fig. 5ExThe results of the current investigation show that certain elements present in the scar tissue may help to explain the difference in the incidence of incisional hernia observed between adult and pediatric patients. The advantage of the chosen model is the fact that the same elements are found in the scar tissue of both rats and humans, namely mature and immature collagen, fibroblasts, blood vessels, and inflammatory infiltrates. Thus, the authors can analyze these elements and infer that their behavior in rats is analogous to that of the human species. However, the differences in anatomy between rats and humans, namely the quadrupedal position, make it difficult to actually measure the hernia formed and casts doubt on whether the measurements can be extrapolated to humans.,All groups exhibited a reduction in the size of the hernia\u00a04\u00a0weeks after the procedure, but only the horizontal incision groups presented statistically relevant differences, with shorter hernias in the WH group vs. the OH group. These results are consistent with those found in the current medical literature, i.e., a higher degree of scar tissue contraction in younger vs. older animals (rats and humans).Based on vessel quantification, younger animals subjected to vertical incisions had more intense neovascularization than adults, while among those subjected to horizontal incisions, adult animals exhibited greater neovascularization than weaning animals. This finding could be explained by the fact that the histopathological arrangement may change with age depending on the incision or by imperfections of the methods used. Some have shown increased neovascularization in the scar tissue of young rats, while other studies found a reduction in such a process.The analysis of mature and immature collagen also had unexpected results. Initially, the percentage of immature collagen (green stain), was lower in the AV group vs. the OV group, yet such percentage was higher in the AH group vs. the OH group. The mature collagen analysis also displayed divergences, with a higher degree of red areas in group AV in comparison to the other groups, and a statistical difference between the WV and OV groups. However, in the oblique incision groups, the WO group had a higher degree of red areas than the AO group. Again, that could be explained by the fact that the histopathological arrangement may change with age, depending on the incision or by imperfections of the utilized methods. Nevertheless, the averages observed in the groups tended to not show statistical differences. These findings agree with the conclusions of Swift et al.The immunohistochemical analysis indicated a clear increase in fibroblast proliferation in younger animals with any type of incision. Ki-67 is a marker of cell proliferation used in this study to support the authors\u2019 hypothesis that the staining of fibroblasts in younger animals would be more intense than in older animals.In the current investigation, the authors studied a gene that encodes a member of the lysyl oxidase family of proteins (Lox gene). A higher expression of this gene was observed in younger rats subjected to vertical and oblique incisions. Until now, no association was found between this gene and the cicatricial tissue of incisional hernias, even though in some studies an intense expression of Lox was observed in the extracellular matrix remodeling after injury to skin tissues.In conclusion, the current study found that smaller hernias after horizontal incisions, higher fibroblast proliferation, and higher Lox gene expression in weaning rats were important differences between the younger and older animals. Such findings could be associated with a lower incidence of incisional hernias in children when compared to older individuals. The literature still lacks comparative studies of scar tissue in different age groups which might corroborate the conclusions of this study.Adult Horizontal (AH); Adult Oblique (AO); Adult Vertical (AV); Old Horizontal (OH); Old Oblique (OO); Old Vertical (OV); Quantitative Real-Time PCR (qRT-PCR); Weaning Horizontal (WH); Weaning Oblique (WO); Weaning Vertical (WV)Josiane de Oliveira Gon\u00e7alves, Suellen Serafini performed all laboratory studies and the statistical analyses; Uenis Tannuri was responsible for the final approval of the investigation and for the manuscript revision. All authors have read and approved the manuscript.Rafael Nogueira do Amaral, Ana Cristina Aoun Tannuri, Junia Marielle Teixeira Rodrigues Neri, and Hugo de Souza Reis performed the animal experiments; 10.13039/501100001807Funda\u00e7\u00e3o de Amparo \u00e0 Pesquisa do Estado de S\u00e3o Paulo, process number\u00a02019/10,347\u20134.The project had financial support from The authors declare no conflicts of interest."} +{"text": "Background\u2003Hypothenar free flaps (HTFFs) have been widely used for reconstructing palmar defects. Although previous anatomical and clinical studies of HTFF have been conducted, this technique still has some limitations. In this study, we describe some tips for large flap design that allows for easy harvesting of HTFFs with minimal donor site morbidity.Methods\u2003A total of 14 HTFF for hand defect reconstruction were recorded. The oblique flap was designed in the proximal HT area following relaxed skin tension line along the axis between fourth web space and 10\u2009mm ulnar side of pisiform. A flap pedicle includes one or two perforators with ulnar digital artery and HT branch of basilic vein. In addition, innervated HTFF can be harvested with a branch of ulnar digital nerve. Electronic medical records were reviewed to obtain data on patients' information, operative details, and follow-up period. In addition, surgical outcome score was obtained from the patient, up to 10 points, at the last follow-up.Results\u2003Mean harvest time was 46\u2009minutes, and two perforators were included in 10 cases. The mean flap area was 10.84\u2009cm2. There were no problems such as donor site depression, scar contracture, keloids, wound dehiscence, numbness or neuroma pain at donor sites, and hypersensitivity or cold intolerance at flap site, either functionally or aesthetically.Conclusion\u2003Palmar defect reconstruction is challenging for hand surgeons. However, large HTFF can be harvested without complications using the oblique axis HTFF technique. We believe our surgical tips increase utility of HTFF for palmar defect reconstruction. The principle of soft tissue reconstruction in plastic surgery is to replace the defects with the most similar tissues and minimize donor site morbidity. Reconstruction of soft tissue hand defects is technically challenging for hand surgeons. The use of similar and adjacent tissues for reconstruction is important as the characteristics of the dorsum and palmar tissues differ.Historically, the tissue in the hypothenar (HT) area, which exhibits more tissue redundancy than tissues in other areas, has been used for the reconstruction of hand defects.However, some of the disadvantages of the HTFF include a high learning curve due to the small perforator diameter, limited flap size, short pedicles, and scar contractures at donor sites.The experimental protocol was approved by the institutional review board of Korea National Institute for Bioethics Policy (P01-202112-21-022). Patients provided informed consent for the publication of the clinical photographs included in this article.In our study, the medical records of a total of 14 HTFF surgeries for hand defect reconstructions were extracted. After the participants were selected, sex, age, operative techniques, flap size, harvest time, injury mechanism, and the number of perforators included in the flap were obtained from the electrical medical records.The procedure was performed on patients under brachial plexus block in the supine position by a single plastic surgeon (S.H.O.). First, a line between the midpoint of the metacarpophalangeal (MCP) crease of the little finger and the center of the pisiform was drawn. The dominant perforators are located slightly on the ulnar aspect of this line. One or two dominant perforators were usually found within a distance of 10\u2009mm proximally from the distal palmar crease using a handheld Doppler. The flap was designed along the axis from the fourth web space to a point 10\u2009mm from the ulnar side of the pisiform. The proximal margin of the flap was not passed along the proximal margin of the pisiform bone. The perforator was positioned at one-third of the HTFF .After the recipient vessel was prepared, an incision was made on the ulnar side of the flap, and the HT branch of the basilic vein was located in the subdermal layer. To the best of my knowledge, this vein is typically located along the line extending from the ulnar side of the little finger to a point 15\u2009mm from the ulnar aspect of the pisiform. The maximum possible length of this superficial vein was harvested. After an incision was made on the radial side of the flap extending to the deep fat layer, a meticulous dissection was performed to find the perforators. When the perforators were found, the dissection was continued in the retrograde direction to check the site where the perforator branched from the ulnar digital artery (UDA) of the little finger. Since the ulnar digital nerve (UDN) of the little finger usually travels together with and along the UDA, flaps can be harvested by including one or two UDN branches together. Considering the recipient vessel and flap insetting position, the UDA was harvested with 1 to 2 perforators in the distal or proximal direction .At the donor site, a Penrose drain was inserted and primary closure was performed. Usually, suprafascial dissection is automatically performed to harvest the HT branch of the basilic vein to facilitate tension-free closure.After HTFF, patients are discharged from our clinic at 2 weeks postoperatively if there are no complications. All patients are reviewed in our outpatient clinic at 1, 3, and 6 months postoperatively. Patients lost to follow-up before 6 months were excluded from the present study. At the last follow-up visit, the durability and elasticity of HTFF, flap contour, cold intolerance, and complications at the donor site were evaluated. In addition, surgical outcomes at the recipient site were scored by patients on a 10-point scale as a composite visual analogue scale at the last follow-up visit. The results of the 10-point scale demonstrated very satisfactory surgical outcomes indicating HTFF may be recommended for patients with volar defects in the hand.2. Overall, 13 HTFF were elevated with the digital nerve branch, and one HTFF was elevated without the digital nerve branch due to the absence of an innervated digital nerve branch.A 58-year-old man presented with a volar defect of the right thumb after infective tenosynovitis due to a paint gun gunshot injury . A 4\u2009\u00d7\u2009A 65-year-old man presented with pulp defects in the ring and little fingers after a compression injury at his workplace. A 5\u2009\u00d7\u20093\u2009cm ipsilateral innervated HTFF with an oblique axis was planned for simultaneous reconstruction of the entire pulp tissue. Two little finger UDA perforators were anastomosed with the RDA of the ring finger, and the HT branch of the basilic vein was anastomosed with the superficial vein of the thumb. One branch of the UDN was coapted with the RDN in an end-to-side fashion. The donor site was closed without tension. All of the flaps were viable with no complications such as depression scars at the donor site. At 6 months postoperatively, no complications were observed at the donor or flap sites. The patient was satisfied with the aesthetic appearance of the donor site .A 55-year-old man presented with a pulp necrosis of the left index finger following a replantation. A 3.5\u2009\u00d7\u20092.5\u2009cm ipsilateral innervated HTFF with an oblique axis was planned. One branch of the UDN was coapted with the UDN in an end-to-side fashion. The donor site was closed without tension. All of the flaps were viable without any complications. At 6 months postoperatively, there were no complications at the donor site such as depression, keloids, pain, or hypersensitivity. In addition, the flap contour, durability, and elasticity were acceptable with a full range of motion achieved .The essentials for a new flap design contain that is easy to reproduce, has pedicles at a consistent location, and does not cause donor site morbidity. The advantages of the HTFF design that we introduced in this study, include its simplicity and a consistently located pedicle. The perforator in the distal ulnar HT area was used for the pedicle of the flap and was located in the distal third of the flap. Studies by Omokawa et al, Uchida et al, and Han et al, have shown that the most appropriate perforator for the flap is located in the distal ulnar HT, and almost all perforators that originated from the UDA were located in the distal ulnar HT area.In our study, the HT branch of the basilic vein was always included in our flap design. This vein had a large diameter and constant location, and therefore, we propose the pathway of the superficial vein. More studies, such as cadaver studies, are needed in the future that would identify suitable locations on the superficial veins. The HT branch of the basilic veins in the patients in this study were all located along the line joining the ulnar side of the MCP joint of the little finger with the ulnar side 15\u2009mm from the center of the pisiform . FurtheRecently, many methods of treating fingertip and pulp amputations with a simple method have been introduced. However, for better functional and aesthetic results, reconstruction using flaps is required. In order to use flap coverage as the first choice in fingertip or pulp reconstruction, flaps should be easily harvested and anastomosis should be performed. Many studies are trying to suggest flaps with these features. When reconstructing palmar and pulp defects of the hand and finger, various flap options, such as second toe pulp transfer, radial artery superficial palmar branch (RASP) flap, free tissue transfer flaps, homodigital reverse island flaps, thenar flaps, and cross-finger flaps are available as local flaps.The first drawback is the limitation in flap size. In most cases of hand trauma, such as workplace crush injuries, reconstruction of the entire pulp or multiple-digit pulp defects are frequently needed. Therefore, a larger flap may be required for expanding the indication of HTFF to allow sufficient coverage of palmar defects.Second, the HTFF has a thin perforator diameter and a short pedicle. Microsurgical techniques have recently been developed to deal with vessels that are \u2264 1\u2009mm, which have helped widen the indications for microsurgery. However, a thin vessel diameter may be challenging for a beginner flap surgeon. Furthermore, the learning curve is high, and vessel spasm can easily occur.Finally, donor site morbidity can occur. Although the skin of the HT area has redundancy, it can cause scar contracture by generating a donor site scar perpendicular to the relaxed skin tension line (RSTL) and HT crease and donor site depression after harvesting the large flap vertically in the HT area.To overcome these limitations, HTFF should be used to harvest larger flaps with a large pedicle diameter under single anesthesia and operative field. A number of anatomical and clinical studies have been conducted on HTFF. In 2005, Hwang et alFirst, the advantages of using the oblique axis for HTFF are the flap position and the ability to close the donor site without tension. By positioning the perforator on the distal third of the HTFF, most of the flap area is located above the proximal HT muscle belly. The proximal HT area is more mobile than the distal HT area and has glabrous tissue that is similar to the distal HT area. This allows reduced tension at closure, a depressed scar with palmar sliding of dorsal skin along the RSTL, and postoperative scar contracture due to being parallel to the RSTL of the HT area and the elasticity of dorsal hand tissue. In our study, donor site closure was possible up to a size of 3\u2009cm without tension in our study. A more mobile donor site allows the harvesting of larger flaps. This method also results in satisfactory aesthetic outcomes, with all patients found to be satisfied with the operative results with minimal donor site scars . In addSecond, the use of a large diameter vessel for the pedicle has a number of advantages. The vena comitans of the digital artery can be used for venous drainage; however, this vein is not always present and can be very thin.Recently, to expand indications and overcome the limitations of HTFF, Kodaira and FukumotoKodaira and Fukumoto's methods have the advantages of ensuring longer and thicker pedicles, but are limited in the distal HT area. They can also result in scars that are perpendicular to the distal HT crease leading to scar contractures. In addition, superficial veins in the distal HT area are thinner than those in the proximal area. Yamamoto et al's methods are advantageous for harvesting larger flaps, but require additional transposition flaps to achieve donor site closure. The donor site scar of the transposition flap will be formed longitudinally on the margin of the HT area, which can potentially become a painful scar.We agree with Kodaira and Fukumoto's study, wherein they included a perforator with UDA and superficial vein, and Yamamoto et al's study, wherein the necessity of a larger flap was suggested. In our study, we incorporated both these while eliminating the disadvantages. At the last follow-up of the patients in our study, the surgical outcome score by patients was fine. It showed that the long-term operative result was fine either functionally and aesthetically. With our design, we can easily harvest a larger flap, with a thick and long pedicle with minimal donor site morbidity. HTFF can be considered as the ideal flap choice to cover almost all palmar digit defects.Further study is needed to gain further insights into HTFF. Innervated HTFF, which included the branch of the UDN, was used in almost all the patients in this study. The branch of UDN included in the HTFF is thin; thus, long-term follow-up is necessary to see how much nerve reinnervation is required to recover sensory deficits. In addition, since little fingers have both digital arteries, a sacrifice of the UDA will not affect circulation in the little fingers. Nevertheless, long-term follow-up is needed to determine any other complications, such as cold intolerance. Further studies should be conducted on the innervated HTFF and the use of the main vessel of the UDA of the little finger. It could allow for wider use of HTFF and result in more favorable results when used for palmar defect reconstructions.In conclusion, our suggested tips with a new concept for HTFF elevation may have utility in the reconstruction of palmar defects, thereby increasing the application of HTFF and improving operative results. Future studies on innervated HTFF with long-term follow-up will provide further insight and determine the efficacy and long-term outcomes of HTFF in reconstructing palmar defects."} +{"text": "Anopheles arabiensis and Anopheles funestus sensu stricto mosquitoes are major East African malaria vectors. Understanding their dispersal and population structure is critical for developing effective malaria control tools. Three mark-release-recapture (MRR) experiments were conducted for 51 nights to assess daily survival and flight range of An. arabiensis and An. funestus mosquitoes in south-eastern, Tanzania. Mosquitoes were marked with a fluorescent dye as they emerged from breeding sites via a self-marking device. Mosquitoes were collected indoors and outdoors using human landing catches (HLC) and Centers for Disease Control and Prevention light traps (CDC-LT). In total, 4210 An. arabiensis and An. funestus were collected with 316 (7.5%) marked and recaptured (MR). Daily mean MR was 6.8, standard deviation (SD\u2009\u00b1\u20097.6) for An. arabiensis and 8.9 (SD\u2009\u00b1\u20098.3) for An. funestus. Probability of daily survival was 0.76 for An. arabiensis and 0.86 for An. funestus translating into average life expectancy of 3.6\u00a0days for An. arabiensis and 6.5\u00a0days for An. funestus. Dispersal distance was 654\u00a0m for An. arabiensis and 510\u00a0m for An. funestus. An. funestus life expectancy was substantially longer than that of An. arabiensis. The MRR method described here could be routinely utilized when evaluating the impact of new vector control tools on mosquito survival. Both malaria cases and malaria deaths have increased in recent years due to population growth2, insufficient coverage of vector control tools3 and to some extent the increase of malaria vector resistance to insecticides used in vector control4. Malaria transmission is highly heterogeneous5 due to geographical differences including altitude, urbanization and vegetation6. For these reasons, the country has stratified malaria control deploying tools based on the intensity of malaria transmission7 as a response to the WHO high burden to high impact (HBHI) strategy8. Much of the finer scale (district) differences in malaria transmission intensity is attributable to the vector species composition in that area. To maximize resources, the deployment of malaria vector control needs to be targeted against those vectors that transmit most of the disease.Mainland Tanzania has been classified by World Health Organization (WHO) as a country with high malaria burden that requires targeted application of malaria control toolsAnopheles funestus sensu stricto and Anopheles arabiensis that differ markedly in their vectoral capacity9. An. funestus feeds on humans primarily indoors and late at night10 while An. arabiensis feeds on humans and cattle indoors or outdoors11. These differences in ecology result in different man-vector contact that to some extent explains their differing vectoral capacity12.In south-eastern Tanzania, malaria transmission is mediated by Plasmodium before they can transmit pathogens14. Vector control tools reduce mosquito infectivity rate15, density and daily survival, with the later having the greatest impact on malaria transmission17. In addition, the use of insecticides may also disproportionally increase mortality among older mosquitoes and can partially reduce the impact of insecticide resistance18. Other than old age, several other factors such as disease, predation or environmental factors contribute to mosquito mortality19. Therefore, it can be argued that vector population age structure is a more valid metric20 than mosquito density as an outcome in entomological trials of vector control tools, as has been elegantly demonstrated in early trials of Insecticide Treated Nets (ITNs)21.Adult survival is a critical component of vectoral capacity because adult females must survive the extrinsic incubation period of Anopheles mosquito range between few meters to several kilometres depending on resource availability i.e. larval habitats, sugar and blood hosts22 and species-specific environmental adaptability23. The range has been demonstrated to be bigger in rural areas, with relatively higher mobility compared to urban areas24. Laboratory-reared Anopheles have been found to disperse more than wild mosquitoes, possibly due to the presence of a memorised home range25. Anopheles mosquitoes actively disperse only or primarily during part of their gonotrophic cycle26. This is epidemiologically important as it influences the extent of malaria parasite acquisition and distribution that leads to transmission by female mosquitoes.Dispersal of 27. MRR is a technique where mosquitoes collected in the wild or reared in the laboratory, are marked, released then recaptured at a given distance and time interval from the releasing point30. MRR is an effective and low cost means of investigating adult mosquito dispersal and survivorship that can be utilized in most settings and has been evaluated against multiple species31. MRR experiments include a single mark and release of mosquitoes, followed by one or repeated recaptures32.Mosquito population parameters can be estimated through a number of morphological, biochemical, genetic and spectroscopic methods, each of which has limitations in reliability, specificity, validation, and requires high cost or the need for an expert\u2019s technical abilityAn. arabiensis and An. funestus in Ikungua village by marking mosquitoes as they emerged from their breeding sites33. Using mosquitoes as they emerge means that the technique is more reflective of natural dispersal and ethically less challenging as additional mosquitoes are not introduced and wild mosquitoes from the environment are recaptured. This data is needed to inform mathematical models used to optimize the selection of malaria control tools7. Furthermore, knowledge of survival and dispersal of malaria vectors is critical for planning, evaluating and implementing new tools which are intended to interrupt the pathogen\u2019s transmission34.This study investigated the survival and dispersal capabilities of An. arabiensis and An. funestus) were marked and released into the wild mosquito population (with correction factor of 86% marking efficiency33), over three separate releases and 17\u00a0days follow up for each release.A total of 4210 mosquitoes (both An. arabiensis and An. funestus) marked and unmarked mosquitoes were collected and morphologically identified as An. gambiae s.l. (7260) and An. funestus s.l. (6099). Polymerase chain reaction (PCR) showed all the An. gambiae s.l. tested to be An. arabiensis (50/50 succesful amplifications). Furthermore, for An. funestus group, 92% (46/50 successful amplifications) showed to be An. funestus s.s whilst 8% (4/50) of the samples did not amplify. Recapture rate was 3% (n\u2009=\u2009138) and 4% (n\u2009=\u2009178) with a daily average of 6.8 (SD\u2009\u00b1\u20097.6) for An. arabiensis and 9 (SD\u2009\u00b1\u20098) for An. Funestus, respectively. Daily recapture declined over time from 16 (5.3%) on the first day to 1 (0.3%) on the 15th day after each release. There were few recaptured male mosquitoes, 12 An. gambiae s.l. and 4 An. funestus s.l. because we used recapture methods that targeted host seeking female mosquitoes. Males were excluded from the analysis.A total of 13,359 metres for An. funestus. The probability of capturing a marked mosquito declined over distance (Table An. arabiensis and a lower proportion (75.8%) of An. funestus were recaptured within 500\u00a0m of the releasing point. Fewer mosquitoes were recaptured in the third annulus compared to the fourth annulus; 0.7% versus 3.6% for An. arabiensis and 2.2% versus 21.9% for An. funestus. There was a similarity between maximum recapture for An. arabiensis (20.5%) and An. funestus (20.0%) at 6\u00a0days after mosquito release, while the rate dropped dramatically from day 7 to 15 with 1.6% An. arabiensis and 6.4% An. funestus recaptured for An. arabiensis was estimated as 101,886 mosquitoes while that of An. funestus was estimated as 78,991 mosquitoes. There were approximately 443 An. arabiensis and 343 An. funestus per hectare of the study area.Population size for Plasmodium falciparum that requires\u2009>\u200912\u00a0days to be infective17. The longer lifespan of An. funestus may have therefore contributed to its relative efficiency as a vector compared to An. arabiensis despite its comparatively low abundance. More focused attention is needed on the control of An. funestus due to its efficiency in transmitting malaria35.This study was designed to investigate the mobility and life expectancy of two major malaria vector populations in south-eastern, Tanzania. Mosquito longevity is a critical aspect in transmission of An. funestus survives longer, and has a 10% higher daily survival probability than An. arabiensis. These findings corroborate with earlier studies done in the Kilombero valley37 as well as other studies from Tanzania38 and West Africa39 that reported An. funestus having higher survival rates and lower mortality than An. arabiensis. This higher survival may be as a result of its adaptation to readily available human hosts and reported resistance40 against pyrethroid insecticides used in ITNs implemented for malaria control in this area41. These adaptations (endophily and anthrophily), also make it extremely vulnerable to control with core tools using insecticides to which An. funestus is susceptible. It should be noted that An. arabiensis in the region are also highly pyrethroid-resistant42. However, An. funestus has been shown to be more efficient in malaria transmission than An. arabiensis which may be related to differences in resistance or behaviour35 whereas laboratory studies have reported similar survivorship for An. arabiensis44 and An. funestus43 under a standard culturing environment.Data from this study demonstrates that An. funestus in this study (0.84) is 2% lower than the study conducted in the same area in the 1990\u2019s (0.86)36 given the higher coverage (>\u200950%) of ITNs45 and other insecticide-based interventions than in the previous years, we hypothesize that pyrethroid resistance may be the reason for the maintained life expectancy of the species.Although, the estimated survival probability of 46 to measure the number of gonotrophic cycles that mosquitoes have undergone47 or the proportion of the proportion that have ever laid eggs48. The Detinova dissection technique is straightforward but requires dedicated technical staff, but very few people are skilled enough to routinely carry out the Polovodova technique49. Other age grading techniques include mid infra-red spectroscopy50, near-infra red spectroscopy51 as well as molecular methods such as transcription methods52. However, these more recent methods are still in development and are not used routinely53.Changes in mosquito population survival may also be evaluated through dissecting the ovariesAn. arabiensis and An. funestus was determined by measuring the distance travelled between the releasing point and the recapture house. An. arabiensis was found to have a similar dispersal distance to An. funestus. Similarly, Saddler et al.33 using the same MRR method, found the mean dispersal distance of An. arabiensis in Bagamoyo to be 579\u00a0m (95% CI 521\u2013636), which is similar to that obtained for An. arabiensis in the current study. However, other researchers have reported higher dispersal distances. Wada et al. recorded individual mosquitoes travelled 5100\u00a0m within a day of being released54. Thompson et al.24 recorded An. gambiae up to 1400\u00a0m from the releasing site. But, Midega et al. found no difference in the mean dispersal distance between An. funestus and An. arabiensis along the Kenyan coast55.The dispersal distance of 56, density and location of human hosts, availability of sugar sources, oviposition, breeding and resting sites22 as well as environmental factors like prevailing wind direction, humidity and temperature55. During the course of the study, one of the houses located in the 4th annulus registered an unlikely large number of recaptured An. funestus (17.4%). Further, investigations showed that the house was close to a seasonal breeding site and had twice (6 members) the average number of people compared to the other households (2.8 members). There is existing evidence supporting the occurrence of higher densities of malaria vectors in households located near breeding sites57 and in those with higher number of individuals due to increased levels of carbon dioxide, a long range attractant of host-seeking mosquitoes from the presence of more residents59.Differences in dispersal distances may be due to varying geographical terrainsAn. arabiensis and An. funestus) dispersed and recaptured at the furthest house were found 860\u00a0m from releasing site in the fourth annulus, but only a small proportion of mosquitoes (12.3%) reached this distance. Additional studies are needed with sampling more evenly distributed mosquitoes and carried out throughout the year to better understand the drivers of dispersal including population biomass and location of breeding sites in the study sites.In the current study, females of both species as they are more likely to survive longer which increases the chance of them being recaptured61. Use of young mosquitoes of a known age also allows determination of mosquito life span.In earlier investigations in Kikulukutu village, south-eastern, TanzaniaAn. funestus recaptured compared to An. arabiensis. This could be due to anthropophilic and endophilic nature of An. funestus62. Although widely used, MRR experiments are often limited by recapture rates below 5% of those released63. The overall recapture rate of 7.5% observed in the current study is double the average, 3% (1\u20139.5%) reported in the literature for Anopheles31. Fewer houses were located in the third annulus may explain why fewer mosquitoes were recaptured in the third annulus relative to the fourth annulus. Typically, more recaptures were made near the release point (first annuli) for both species and decreased as one moves further away from the releasing point. Therefore, greater sampling effort is required at greater distance from the releasing point.Most marked mosquitoes were recaptured in neighbouring houses several hours later after being released. The house closest to the releasing site which was 130\u00a0m away, had more An. Arabiensis has been shown to be resistant to pyrethroid42.The limitations to this study were refusal of some households to allow mosquito collections, resulting in unequal distribution of houses in each annulus. This was minimized by standardizing the study regions to accommodate all four annuli. Also, to simulate the natural environment, the release point was close to the natural breeding site located on the village periphery rather than centre in the study area. We did not collect data on the biomass in all houses or the location of all breeding sites in the village. A more comprehensive mapping effort at the beginning of the study would have allowed us to better understand mosquito dispersal. Another limitation was failure to get resistance profile of the two-mosquito species from the study area during the study however 64. The marking method was investigated during the development of the MRR method used here and found not to affect survival33 but it is not known if it can affect flight or predator response to marked insects.In further studies of MRR the use of indoor resting collections and mosquito abdominal status is recommended to measure if the dyes affect the ability of mosquitoes to feed. The validity of the MRR method rests on the assumption that marked individuals behave in every respect as the unmarked ones (wild), and that both marked and unmarked mix together in a homogenous, random way. MRR includes marking mosquitoes with a fluorescent dye, a procedure that has been reported by many investigators to not affect the survival and dispersal behaviour of mosquitoes provided it is applied correctlyAn. funestus has a substantially longer life expectancy than An. arabiensis in this setting, which may partially explain the greater efficiency of An. funestus in malaria transmission.MRR used as mosquitoes emerge from breeding sites is a simple and cost-effective method for measuring the dispersal and survival of mosquitoes. It can be deployed as part of routine entomological collections when evaluating vector control tools with active ingredients that that are designed to overcome resistance to existing classes of insecticides and consequently reduce mosquito population life expectancy when deployed at scale. This study has demonstrated that 65. The village had 347 houses and 984 inhabitants over 36 hectares. The village is surrounded by forests, water bodies, and agricultural areas. The temperature ranged from 23 to 32 \u00b0C during the study and the annual rainfall ranges from 1200 to 1800\u00a0ml. Residents are subsistence farmers, growing bananas and millet in the hillsides whilst growing rice in the valleys through an irrigation system, which provides breeding sites for malaria vectors. An. gambiae s.s populations have significantly declined in the study area35 leaving An. arabiensis and An. funestus as the main vectors with An. funestus mediating majority of the infections even though it is present in lower densities than An. arabiensis9. House structures in the village allow indoor entry through opened eaves, mud walls, thatch roofs, and doors not covering the whole entrance as well as windows66. National malaria control is implemented through Insecticide Treated Nets (ITNs) in the study area. Although, a field study of an indoor residual spray product was implemented in the villages shortly (about 1\u00a0year) before the study.Three experimental phases of MRR survey were performed during the study, which was conducted at the end of the rainy season between September and October 2020 in Ikungua village in South-eastern, TanzaniaAn. arabiensis and An. funestus pupae and larvae stage 2\u20134 were collected from multiple natural breeding ponds and puddles located within a one thousand meters radius from the releasing point using a larval dipper and one-millimetre bulb pipette. The colony was maintained in a field laboratory with 300 larvae per bowl reared at 25\u2009\u00b1\u20097 \u00b0C temperature and 40\u201399% relative humidity. These pupae and stage 4 larvae were maintained in a plastic bowl with some water and placed underneath the marking trap near a shelter in releasing area. After emergence, adult mosquitoes on their first flight out to seek for food and mate pass through pigment impregnated onto cloth strips where they would be marked with dye pigments.Wild Each release was conducted for 5\u00a0days consecutively with daily average of 281 pupae/larvae placed under the trap each day at 18:00\u00a0h. Recapture was conducted each night of the release and for 12\u00a0days after the last release, total 17 nights of recapture. There was then a wash out period of a further 3\u00a0days before the next round was conducted. In total, three rounds of MRR were conducted. The total mosquitoes released were: 794 in 1st, 2025 in 2nd, and 1435 in the 3rd release. A single releasing point, close to the mosquitoes\u2019 natural breeding site was used for all of the releases and 10\u2009\u00d7\u2009dissection microscope . A total number of 100 mosquitoes, 50 An. gambiae s.l. and 50 An. funestus s.l. were packed in Eppendorf tubed on silica and taken to the IHI Ifakara laboratory for PCR speciation70.Mosquitoes were recaptured from 20 houses located in four distance groups (annuli) from the releasing site; 215, 430, 645, and 860\u00a0m. Mosquito collection started on the day of the release and was conducted for 17 consecutive days between 18:00\u00a0h and 06:00\u00a0h using CDC-LT beside human-occupied bed net72, with the assumption of having different densities of traps in different annuli73. As the area of the annuli increases with greater distance from the release site a correction factor (CF) for each annulus was estimated by dividing the area of the annulus by the sum of the area of all four annuli and multiplying the result by the total number of traps in the specific annuli74. Area of each annulus was calculated using half of the distance from the releasing site as the radius. Then, the number of mosquitoes recaptured in each annulus was divided by the total number of traps in the annuli and multiplied by the correction factor of the annulus to get estimated recapture (ER). Using the ER, cumulative estimated recapture (CER) was calculated. The MDT was then calculated as the sum of the product of ER and radius of the annuli over the total CER.Mean distance travelled (MDT), was used to describe the distance travelled by the estimate the dispersal of the released mosquitoes. This method estimates movement of adult mosquitoes against the radius of the experimental area75 with recaptured mosquitoes adjusted in the model and for the average life expectancy was calculated according to Niebylski and Craig76.Survival was estimated with linear regression approach defined by Buonaccorsi et al., are simple methods of estimating population size29 in mark-release experiments. The method assumes that: (1) marked and wild mosquitoes mix homogeneously immediately after release in a random way, (2) random dispersal of the marked and wild population without loss or gain in the population, and (3) there is a constant mortality rate among released mosquitoes. Using this method, estimates of total population size (P) are determined by the numbers of mark-released mosquitoes (a), the number captured on the subsequent occasion (n), and the number of those recaptured which had been marked (r), when r is greater than 20. In this study, it was assumed half of the marked-released mosquitoes were An. arabiensis and half were An. funestus. The analysis was done using R statistical software v4.1.177Population size was estimated using the Lincoln Index Eq.\u00a0. The FisEquation\u00a0 showing 78 and were medically supervised79. I confirm that the recommended guidelines from the ministry of health were properly followed.Ethical approval was granted by the Institutional Review Board of the Ifakara Health Institute (IHI) and Tanzanian National Institute of Medical Research (NIMR) (NIMR/HQ/R.8a/Vol. IX/2894). Written informed consent was obtained from household heads of the twenty houses selected for mosquito collection and from volunteers who performed HLC. The volunteers were provided doxycycline prophylaxis as per Tanzania Ministry of Health guidelines"} +{"text": "Drosophila as a model, we uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). We also detected a robust immune overactivation in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, we showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, our findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases.TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using The online version contains supplementary material available at 10.1186/s12979-023-00354-8. ALS is an age-related neurodegenerative disease characterized by progressive loss of motor neurons . ProteinThe mitogen-activated protein kinase (MAPK) cascades play a vital role in transduction of extracellular signals and regulation of different cellular functions such as stress and inflammatory responses , 12, celImmune and inflammatory responses are known to impact on the pathogenesis and progression of several neurological disorders, including ALS, FTD and AD \u201329. And,Drosophila ERK gene rl was significantly upregulated in TDP-43 flies, and upregulation of the MEK/ERK pathway in fly neurons induced robust activation of the innate immunity. Moreover, neuron-specific downregulation of the MEK/ERK pathway or KD of the abnormally upregulated AMPs was sufficient to suppress the neurodegenerative phenotypes and extend the shortened lifespan of TDP-43 flies. Finally, feeding flies with trametinib, a small compound inhibitor of MEK, significantly ameliorated TDP-43-induced behavioral deficits in fly models, offering an opportunity for developing new therapeutic strategies aimed at the intervention of ALS and other TDP-43-related diseases.In this study, we discovered that the Drosophila eye, a well-established and convenient in vivo cytotoxicity model for identifying and investigating new factors that modulate neurodegeneration [hTDP-43) in fly eyes with a GMR-Gal4 driver , showed dramatic suppression of the age-dependent eye degeneration of the TDP-43 flies analysis driver, which led to age-dependent climbing decline , Raf, Dsor1 (dMEK), rl (dERK) and S6kII (dRSK), significantly suppressed the eye degeneration of the TDP-43 flies. Although previously reported to modify TDP-43 toxicity [lic (dMKK3/6) and MKK4, their downregulation enhanced the toxicity of TDP-43. Thus, these data suggested that, of the three main MAPK families, the MEK/ERK pathway was closely involved in TDP-43-mediated neurodegeneration.In addition to toxicity , downregrl, the Dsor1 downstream gene encoding the Drosophila ERK (dERK), showed the second strongest suppression of TDP-43-induced eye degeneration is a disease-hallmarked change of TDP-43 protein in ALS and FTD , 39. NexDsor1 or rl in fly neurons did not show a significant impact on TDP-43 phosphorylation, it raised an alternative hypothesis that the MEK/ERK pathway was misregulated in TDP-43 flies, which contributed to TDP-43-induced neurodegeneration and therefore could be rescued by RNAi-Dsor1 or RNAi-rl. Indeed, although the mRNA levels of Dsor1 were unchanged by TDP-43 was reported to be a potent negative regulator of the IMD immune pathway in flies [Dnr1 in adult flies led to substantial upregulation of immune AMPs as well as age-dependent decline of the motor function as a genetic suppressor of TDP-43 toxicity in a fly screen. Further examination uncovered that transgenic expression of hTDP-43 caused a remarkable upregulation of the fly ERK gene rl, leading to the aberrant elevation of the MEK/ERK pathway in TDP-43 flies. Upregulation of the MEK/ERK pathway in fly neurons activated the innate immunity, evidenced by a dramatic increase in the expression of the immune AMP genes, which was also observed in the elavGS\u2009>\u2009hTDP-43 fly brains. More importantly, we showed that KD of the upregulated AMPs such as AttC and DptB suppressed TDP-43-induced motor deficits, whereas immune overactivation by KD of Dnr1, a negative regulator of the IMD pathway, abolished the mitigating effect of RNAi-Dsor1 on TDP-43 toxicity (Fig.\u00a0In this study, we identified RNAi-ity Fig.\u00a0.Fig. 5A dERK were markedly increased in TDP-43 flies. And, we further showed that the abnormal elevation of the MEK/ERK pathway led to overactivation of the innate immunity, which contributed to TDP-43 pathogenesis.Abnormal TDP-43 protein inclusions in the patients with ALS or FTD are often associated with TDP-43 hyperphosphorylation and phosphorylation levels are positively correlated with TDP-43 toxicity , 59, 60.TDP-43 could activate inflammatory and immune responses via the cGAS/STING pathway by triggering mitochondrial DNA release in neurons . NeverthhTDP-43, downregulation of the MEK/ERK pathway, and genetic inhibition or overactivation of the innate immunity, were restricted to mature neurons in flies. In other words, if the immune responses were entirely secondary to TDP-43-induced neurodegeneration or only involved glia, neuronal KD of the immune AMPs would not have been able to rescue TDP-43 flies. Nevertheless, our data do not exclude the possibility that the initial cell-autonomous immune overactivation in neurons triggers a subsequent avalanche of immune responses from outside neurons, which together lead to the overall degenerative consequences. Collectively, our data indicate that the abnormal elevation of the MEK/ERK signaling promotes the innate immunity in fly neurons, which contributes to TDP-43 toxicity.It should be noted that, all the genetic manipulations in this study, including transgenic expression of The Ras/Raf/MEK/ERK cascade is an important multi-function signaling pathway and plays a critical role in tumorigenesis. The MEKi hence have been developed for treating cancers such as melanoma and non-small cell lung cancer \u201371. TramAdditional file 1.Additional file 2."} +{"text": \ No newline at end of file