Citrobacter gillenii FP75 was identified by using biochemical tests and 16S ribosomal gene analysis. Nucleotide and amino acid sequences of the qnrB89 gene exhibited an identity to qnrB of 81.24% and 91.59%, respectively. The genetic environment of qnrB89 was characterized by the upstream location of a sequence encoding for a protein containing a heavy metal-binding domain and a gene encoding for a N-acetylmuramoyl-L-alanine amidase protein, whereas downstream to qnrB89 gene were detected the csp and cspG genes, encoding cold-shock proteins. The qnrB89 gene was located on a large chromosomal contig of the FP75 genome and was not associated with the 10-kb plasmid and class 1 integron harbored by the FP75 strain. This study reports for the first time the carriage of a qnrB gene by the C. gillenii species, and its detection in a bacterial strain isolated from farmed salmon in Chile.The main objective of this study was to characterize using whole-genome sequencing analysis, a new variant of the  Piscirickettsia salmonis [In Chilean salmon farming, the low availability of efficient vaccines and the high prevalence of bacterial infections, such as Piscirickettsiosis caused by the intracellular pathogen salmonis ,2,3, havsalmonis . During salmonis ,5.qnr genes, encoding proteins that protect DNA gyrase and topoisomerase IV against quinolones, thus preventing their activity [Fluoroquinolones belong to a class of synthetic antimicrobial agents with a broad spectrum of activity ,7,8 and activity ,16.qnr-carrying bacteria exhibit only a low-level resistance to fluoroquinolones, but these bacteria can facilitate the emergence of resistant strains through the acquisition of other mechanisms of quinolone resistance, such as topoisomerase mutations and efflux action [It is well known that x action ,17,18.qnr genes is widespread, thus their occurrence among environmental bacteria is of high concern, but only a few studies addressing the occurrence of qnr genes among bacteria associated with aquaculture settings [Currently, various studies report that prevalence of plasmid-mediated quinolone resistance, mainly encoded by the settings ,20,21, osettings  have beeqnr gene carriage by bacteria isolated from reared fish in Chilean salmon farming has never been previously reported. Thus, this study adds important information on the detection of a qnr gene carried by bacteria of animal origin, and reporting for the first time the detection of a qnr gene in Citrobacter gillenii species.It must be noted that It could be concluded that quinolone resistance genes could be detected among bacteria from reared salmon in Chile but not necessarily associated with plasmid elements with potential for horizontal transfer, thus not prompting an important risk to human public health.qnrB gene and its genomic environment carried by the multidrug resistant C. gillenii FP75 strain recovered from mucus of salmon reared in a Chilean salmon farm.The main aim of the study was to describe a new variant of the Citrobacter gillenii by 16S ribosomal gene amplification (EMBL database accession number KX279662.1). The 16S rRNA gene sequence of strain FP75 was compared with all sequences currently available for members of the genus Citrobacter and related taxa. The results are presented as a phylogenetic dendrogram developed with the neighbor-joining method showing that FP75 strain is a member of the genus Citrobacter, where the most related species was C. gillenii (16S rRNA gene similarity 99.79%), as is depicted in C. gillenii was confirmed by specific biochemical characteristics, exhibiting important differences to the C. freundii species .A qnr gene was detected in the genome of qnrB89 showed a high identity to the sequences reported for the founding members of qnrB and qnrE genes included in the GenBank database , a gene encoding a N-acetylmuramoyl-L-alanine amidase protein, which is a peptidoglycan hydrolase (NALAP) and a gene encoding for a NAD(P)-dependent oxidoreductase protein (NAD(P)-DOP), whereas downstream of the qnrB89 gene were detected two genes encoding cold shock proteins (csp and cspG), a gene that encodes a lipoprotein (LP), and the artP gene, encoding an arginine transport ATP-binding protein . As showC. gilleni genomes, corresponding to C. gillenii UMG736 (SUQN00000000.1) and C. gillenii MBT-C3 (QVEK00000000.1), composed of 24 and 37 contigs, respectively, no qnr gene was detected. However, it is interesting to note that in both genomes the qnrB89 neighborhood was present, but instead of the qnrB89 gene, gene appA, encoding for an AppA family phytase/histidine-type acid phosphatase protein and gene gnsB, encoding for an addiction module toxin, GnsA/GnsB family protein, were observed, located in contigs 1 and 9 of C. gillenii UMG736 and C. gillenii MBT-C3 genomes, respectively  [Previous studies evidence rB genes ,32. Citrnd water . Furthers mykiss ,36,37,38symptoms , and froidellus) ,40.qnr gene detected in the C. gillenii species, and prompts the necessity to investigate the carriage of qnr genes in farmed salmonid microbiota, and to provide a scientific basis of the prevalence of this opportunistic pathogen in farmed salmon to prevent potential secondary infections.To the best of our knowledge, this is the first report of a qnrB89 with the qnrB allele, when compared to the qnrE sequences, determined that this gene was classified as qnrB89. As was stated by Jacoby et al. [qnrB is the most common of the five qnr families and has the greatest number of allelic variants, being mostly detected in the Citrobacter genus with several of them located on the bacterial chromosome. Among the qnrB alleles, qnrB89 variant exhibited the highest degree of amino acid (93.46%) sequence identity to the qnrB12 sequence from Citrobacter werkmanii isolated from poultry [The higher similarity of nucleotide and amino acid sequence of y et al. , qnrB is poultry .qnrB89 gene is very different to those reported for most of the other qnrB alleles, which consistently include the pspF (encoding a phage shock protein) and sapA  genes upstream and downstream of the qnrB genes, respectively [qnrB89 background. Furthermore, the qnrB89 genetic background is also very different to those of the qnrE1 gene, which is flanked by the genes araJ (encoding a arabinose efflux permease) and ppk (encoding a polyphosphate kinase) or tnp (encoding a transposase) [qnrB genes, observing eight different conserved genetic platforms for closely related qnrB genes carried by different Citrobacter species, demonstrating an association between the qnrB platforms carrying closely related qnrB genes and specific Citrobacter species. However, these genetic environments are highly different to that observed for the qnrB89 gene of C. gillenii FP75. However, it should be noted that C. gillenii species was not included in the referenced study.However, the genetic environment of the ectively , which asposase) ,42. Ribesposase)  characteqnrB genes have been commonly associated with integrons in clinic isolates belonging to the Citrobacter genus [qnrB62 gene carried by a C. freundii clinical isolate, observing an association with a complex class 1 integron. Furthermore, Ferreira et al. [C. freundii strain carrying a qnrB gene associated with a class 1 integron inserted in a large plasmid. In another study, several ciprofloxacin-resistant C. freundii recovered from wastewater treatment plants carried a qnrB gene as part of a complex integron [C. gillenii FP75 carries a class 1 integron [qnrB89 gene, which is in agreement with other previous studies of aquaculture associated bacteria carrying qnrB genes [In previous studies, er genus . Lee et er genus  studied a et al.  isolatedintegron . Despiteintegron , this strB genes ,49.qnrB89 gene, many reported qnrB alleles are associated with plasmid elements and are able to be horizontally transferred [qnrB variants have previously been described as non-transferable, such as the qnrB12 variant carried by Citrobacter werkmanii and a qnrB variant carried by Rhodococcus sp., which were unable to be horizontally mobilized [C. gillenii FP75, which are associated with the 10-kb plasmid [qnrB89 gene in the salmon mucus microbiota.Otherwise, unlike nsferred ,51,52,53obilized ,54. Howe plasmid , suggestqnrB gene by C. gillenii FP75, this strain is susceptible to quinolones. It is well known that resistance to quinolones is mainly due to chromosomal point mutations rather than being carried on any mobile genetic elements, but although qnr genes only confer low-level resistance to quinolones, these genes could favor the selection of additional chromosome-encoded quinolone resistance mechanisms. Furthermore, the study of qnr genes has an epidemiological relevance to advance a comprehensive understanding of the resistome associated with environmental settings, and to know their evolution and spread in these environments.Despite the carriage of a variant of the C. gillenii FP75 strain leading to amino acid substitutions in the quinolone-resistance-determining regions of bacterial protein targets of quinolones were only a conserved change (Ser-83 to Thr) in the gyrase A protein, but not conferring resistance or low level susceptibility to quinolones, whereas the detected amino acid substitutions in GyrB (Leu-417 to His) and ParE  are located in protein regions not associated with changes in the susceptibility to quinolones.The chromosomal mutations exhibited by the qnrA, qnrB, and qnrS, were described in strains isolated from uncontaminated sediments and sectors adjacent to a site affected by salmon farming [qnr gene detected in a bacterium directly associated with Chilean salmon farming, and most importantly, from farmed salmon mucus. The finding of this gene in a bacterial strain exhibiting susceptibility to quinolones suggest that incidence of qnr genes in these systems could be strongly underestimated when they are not associated with fluoroquinolone-resistant bacteria. Furthermore, these qnr-carrying bacteria are most frequently resistant to other antimicrobials, such as florfenicol and oxytetracycline, intensively used in Chilean aquaculture, thus favoring the prevalence of qnr genes in Chilean salmon farming, constituting a potential threat for salmon handlers and consumers, mostly considering that the chromosome of Citrobacter is the likely source of plasmid-mediated qnrB, as was suggested by Jacoby et al. [In Chile in 2014 and 2015, the genes  farming ,54. Howey et al. .qnr genes must be elucidated, prompting the necessity of detecting qnr genes in a high number of representatives of reared salmon microbiota, including isolates exhibiting various levels of susceptibility to quinolones.Thus, the role of Chilean salmon farming as a potential reservoir of TM vials , was grown in Trypticase soy agar  at 30 °C for 24 h prior to use.The FP75 strain was recovered from mucus of reared salmon fingerling cultured in a freshwater farm located at the Puyehue Lake, in the South of Chile . The purCitrobacter freundii species [The phenotypic tests Gram staining, cell morphology, oxidase production and oxidation/fermentation (O/F) of glucose were determined according to the procedures described in Buller . In addi species , using t species .Furthermore, FP75 strain was identified by bacterial 16S rRNA gene sequence analysis. DNA extraction and the amplification of the 16S ribosomal gene of the FP75 strain was performed as was previously described . The seq® Genomic DNA Purification kit , following the indications of the supplier. The characterization of the qnr gene and its genetic environment was carried out through the complete sequencing of the bacterium’s genome. The whole genomic DNA was sequenced by Macrogen USA . The analysis of the qnr gene sequence and its genetic environment was based on the contigs derived from genomic sequencing. This analysis was performed with the BioEdit 7.2.5 software [qnr gene was complemented with the group of experts led by Dr. George Jacoby.Genomic DNA was extracted and purified using the commercial Wizardsoftware  and subsC. gillenii FP75, E. coli DH5α and transconjugant strain E. coli FP75T was determined using a disk diffusion test according to the Clinical and Laboratory Standards Institute (CLSI) guideline VET3-A [E. coli ATCC 25922 was used as a quality control strain. All strains were re-examined to check the reproducibility of the assay.The antimicrobial susceptibility to 16 antimicrobials of strains e VET3-A  and preve VET3-A . Brieflye VET3-A , and stre VET3-A ,63. As re VET3-A , the refC. gillenii FP75 and E. coli UC238 were determined by a microdilution method, as recommended by the CLSI guideline M07-A10 [4 colony-forming units per well, and incubated for 24 h at 28 °C. The reference strain E. coli ATCC 25922 was included as quality control, as was recommended [Minimum inhibitory concentrations (MICs) of flumequine and ciprofloxacin of  M07-A10  and prev M07-A10 . Conicalommended . All assEscherichia coli and Salmonella spp. (≤2.0 µg/mL for susceptible).A breakpoint of ≤1.0 µg/mL was used to consider susceptibility to ciprofloxacin as stated by the CLSI  for enteqnrB variant (qnrB89) by a bacterial strain isolated from reared fish in Chilean salmon farming, and the detection of a qnr gene in the Citrobacter gillenii species. The detection of a qnr gene carried by a quinolone susceptible strain strongly suggests that farmed fish is an important reservoir of these genes but are significantly underestimated considering that qnr genes are almost exclusively investigated among quinolone-resistant bacteria. Furthermore, the uncommon genetic environment of the qnrB89 gene, compared to other qnrB genes, and its non-association to integrons or plasmids suggest a most probable environmental origin, not related to a clinical source. This study shows important information on the characteristics of a qnr gene carried by quinolone susceptible bacteria from salmon farming, thus providing an important basis to advance the performing of genetic epidemiology studies on quinolone resistance genes in fish farm associated environments in Chile.In conclusion, the results of this study demonstrated for the first time the carriage of a new It can be concluded that quinolone resistance genes can be detected among bacteria from reared salmon in Chile but not necessarily associated with transferable elements, reducing their ability to be horizontally transferred, thus not significantly contributing to spread of these genes in these environments and not prompting an important risk to human public health.


Pancreatic cancer (PC) is one of the most malignant tumors. Despite considerable progress in the treatment of PC, the prognosis of patients with PC is poor. The aim of this study was to identify potential biomarkers for the diagnosis and prognosis of PC. First, the original data of three independent mRNA expression datasets were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas databases and screened for differentially expressed genes (DEGs) using the R software. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the DEGs were performed, and a protein-protein interaction (PPI) network was constructed to screen for hub genes. The hub genes were analyzed for genetic variations, as well as for survival, prognostic, and diagnostic value, using the cBioPortal and Gene Expression Profiling Interactive Analysis (GEPIA) databases and the pROC package. After screening for potential biomarkers, the mRNA and protein levels of the biomarkers were verified at the tissue and cellular levels using the Cancer Cell Line Encyclopedia, GEPIA, and the Human Protein Atlas. As a result, a total of 248 DEGs were identified. The GO terms enriched in DEGs were related to the separation of mitotic sister chromatids and the binding of the spindle to the extracellular matrix. The enriched pathways were associated with focal adhesion, ECM-receptor interaction, and phosphatidylinositol 3-kinase (PI3K)/AKT signaling. The top 20 genes were selected from the PPI network as hub genes, and based on the analysis of multiple databases, MCM2 and NUSAP1 were identified as potential biomarkers for the diagnosis and prognosis of PC. In conclusion, our results show that MCM2 and NUSAP1 can be used as potential biomarkers for the diagnosis and prognosis of PC. The study also provides new insights into the underlying molecular mechanisms of PC. Pancreatic cancer (PC) is one of the most common malignant tumors, with a 5-year survival rate of only 9% . CurrentThe main potential biomarkers of PC identified in the past two decades are CA19-9, DUPAN-2, CAM17.1, TPS, SPan-1, TAT1, POA, YKL-40, TUM2-PK, and matrix metalloproteinases . AlthougOPN and CISD2 has been shown to play a key role in the progression of PC [Increasing evidence indicates that abnormal expression of and mutations in certain genes are closely related to the occurrence and development of PC. Abnormal expression of on of PC , 8, whilon of PC –11. Theron of PC , 13. At http://www.ncbi.nlm.nih.gov/geo) is a public repository of various high-throughput experimental data [http://www.cancergenome.nih.gov) is a large-scale cancer genetic information database [GEO , based database  that prohttp://r-project.org/) was used to read the raw data (CEL file) of the three datasets and then convert the original data format, fill the missing values, and apply background correction. The samples were then subjected to differential expression analysis, and the DEGs were exported using the limma package [P value < 0.05 and ∣log2FC∣ > 1 criteria were considered. Finally, the intersection of the DEGs from the three datasets was obtained using the FunRich software [http://funrich.org/index.html).The affy package  in the R package . DEGs thsoftware  (versionhttp://string-db.org) [https://www.networkanalyst.ca/faces/home.xhtml) was used to display the coexpressed gene network of DEGs; cBioPortal  [http://syslab4.nchu.edu.tw) [The Search Tool for the Retrieval of Interacting Genes database   was used-db.org) . To bett-db.org)  was usedtal.org)  was used.edu.tw)  was usedGO covers three aspects of biology: cellular component (CC), molecular function (MF), and biological process (BP) . KEGG ishttps://david.ncifcrf.gov; version 6.8) provides a comprehensive set of gene and protein functional annotation information [The Database for Annotation, Visualization, and Integrated Discovery  [To further screen for potential biomarkers for the diagnosis and prognosis of PC, a comprehensive analysis of the 20 hub genes was performed. We used cBioPortal to analyze genetic variations in the hub genes. The GEPIA database (-pku.cn)  was used-pku.cn) . Potentihttps://portals.broadinstitute.org/ccle) [https://www.Proteinatlas.org/) [EGFR, ERBB2, and KRAS, which are important therapeutic targets in PC [To minimize the bias and improve the accuracy of the results of analysis, multiple online databases, including CCLE (rg/ccle) , the Humas.org/) , and GEPts in PC –36, usints in PC .The results of normalization of the sample data from the GSE15471 and GSE16515 datasets are presented in box plots Fig. . SamplesMELK, MAD2L1, ATAD2, PBK, CDK1, NUSAP1, HMMR, FANCI, TPX2, BUB1, KIF23, DTL, CDKN3, RAD51AP1, KIF20A, MCM2, CCNB1, SMC4, CENPE, and ANLN , including missense, start lost, initiator codon, frameshift, stop lost, and stop gained mutations, which suggested that these mutations might be involved in the occurrence and progression of PC. To investigate the prognostic value of the hub genes in PC, we performed a survival analysis using the GEPIA database. As shown in BUB1, CDK1, FANCI, KIF20A, HMMR, KIF23, MCM2, NUSAP1, and TPX2 were associated with a poor OS and were also closely related to a poor DFS of patients  = 0.954) and NUSAP1 (AUC = 0.93) had the highest diagnostic value for PC  values are shown in MCM2 and NUSAP1 in cell lines derived from different tissues. The results showed that MCM2 and NUSAP1 were expressed in various cell lines, but their mRNA levels were low in pancreatic cell lines (Figures MCM2 and NUSAP1 in 21 experimental PC cell lines and found that both were upregulated in most of the PC cell lines (Figures To ensure the accuracy of the analytical results, we used multiple databases to validate the mRNA and protein expression of MCM2 and NUSAP1 in PC at the tissue and cell levels. We used the GEPIA , ICGC F, and TCG Figures . We also Figures . At the  Figures . Further Figures . The MCM Figures .The lack of methods for early screening for and detection of PC leads to late detection of the disease and a high mortality rate in patients . Hence, In this study, we downloaded three PC-related mRNA expression datasets from the GEO and TCGA databases and screened the data for DEGs. In total, 248 DEGs were identified, and it was found that the GO terms enriched in DEGs were mainly related to “mitotic mitosis”, “centromere region”, “mitotic spindle”, and “extracellular matrix binding”; the enriched pathways were mainly associated with “focus adhesion”, “ECM-receptor interactions”, and “PI3K/Akt signaling pathway”. ECM and focal adhesion have been shown to be important components of tumorigenesis and cancer progression –43. DysrFANCI and RAD51AP1 in tumors are limited, and hence, these genes deserve further investigation.In addition, we constructed a PPI network for DEGs and selected the top 20 genes as the hub genes. The hub genes were analyzed using GO and KEGG pathway enrichment and were found to be mainly enriched in the GO terms “apoptotic process” and “cell cycle” and in the KEGG pathways related to the “cell cycle” and “p53 signaling pathway”. Previous studies have reported that the apoptotic process and cell cycle are closely related to the development and progression of PC , 48. As MCM2 and NUSAP1 as potential biomarkers for the diagnosis and prognosis of PC. To minimize the bias, we used multiple databases to verify the mRNA and protein levels of MCM2 and NUSAP1 at the tissue and cell levels. The results showed that the mRNA and protein levels of MCM2 and NUSAP1 were significantly upregulated in PC cells and tissues. In addition, gene correlation analysis showed that the expression levels of MCM2, NUSAP1, EGFR, ERBB2, and KRAS significantly correlated, and thus, MCM2 and NUSAP1 are likely to become potential therapeutic targets for PC.To further screen for potential biomarkers for the diagnosis and prognosis of PC, we analyzed genetic variations, verified the differential expression, and evaluated the survival, prognostic, and diagnostic value of the hub genes. Finally, we identified MCM2 is an important DNA replication initiation factor in humans. It is present in the nucleus and is highly expressed in proliferating cells. MCM2 is not expressed or is poorly expressed in quiescent or well-differentiated cells, suggesting that it can be used as a specific marker for proliferating cells. Studies have reported that MCM2 is a key molecule involved in the pathogenesis of non-small-cell lung cancer and may be a novel therapeutic target for lovastatin in the treatment of this cancer . ProteomNUSAP1 is a tubulin that is involved in the assembly of the spindle to ensure a normal cell cycle, thus playing an important role in mitosis . An abnoMost bioinformatics studies have reported analysis of a single mRNA expression dataset. In this study, we selected three mRNA expression datasets from two databases, thereby increasing the sample size and confidence level. We used different bioinformatics methods to mine the data deeper and used multiple databases to perform multidimensional verification of MCM2 and NUSAP1 expression in PC, providing diverse perspectives. However, this study has certain limitations. First, a certain degree of heterogeneity was present in the datasets selected for this study, and only four normal samples were included in TCGA. Although we removed the batch data and performed quality control and standardization of the raw data, a larger sample size and a higher-quality dataset are still required to verify the reliability of the results. Second, our study involved a second round of mining and analysis of previously published datasets. Although some previous data were consistent with those of our analysis, further molecular biology experiments are required to verify the accuracy of our results. As a future research direction, validation of MCM2 and NUSAP1 as diagnostic and prognostic markers of PC is needed in a large number of clinical PC samples and PC cell lines.MCM2 and NUSAP1, which were confirmed, using multidimensional verification, as potential biomarkers for the diagnosis and prognosis of PC. However, the results need to be validated in a larger number of clinical samples using additional experimental methods before using MCM2 and NUSAP1 as effective diagnostic and prognostic markers for PC.In conclusion, using bioinformatics analysis of three mRNA expression datasets, a total of 20 hub genes were identified, which may play key roles in the occurrence and progression of PC. Analysis of the clinical significance of the 20 hub genes in PC revealed two abnormally regulated genes,


Escherichia coli, the toxin–antitoxin system (TA-system) has been described as a programmed cell death pathway in which mRNA and ribosome organizations are modified, favoring the production of specific death-related proteins, but also of a minor portion of survival proteins, determining the destiny of the cell population. In the eukaryote Saccharomyces cerevisiae, the ribosome was shown to change its stoichiometry in terms of ribosomal protein content during stress response, affecting the relative proportion between ohnologs, i.e., the couple of paralogs derived by a whole genome duplication event. Here, we confirm the differential expression of ribosomal proteins in yeast also during programmed cell death induced by acetic acid, and we highlight that also in this case pairs of ohnologs are involved. We also show that there are different trends in cytosolic and mitochondrial ribosomal proteins gene expression during the process. Moreover, we show that the exposure to acetic acid induces the differential expression of further genes coding for products related to translation processes and to rRNA post-transcriptional maturation, involving mRNA decapping, affecting translation accuracy, and snoRNA synthesis. Our results suggest that the reprogramming of the overall translation apparatus, including the cytosolic ribosome reorganization, are relevant events in yeast programmed cell death induced by acetic acid.Programmed cell death involves complex molecular pathways in both eukaryotes and prokaryotes. In  The main phenomenon are associated to the activation of complex molecular pathways that lead to membrane depolarization, DNA fragmentation and breakdown of the plasma membrane1. Specific events involving translation processes during PCD have also been reported. In Homo sapiens, it has been shown that, during apoptosis, there is a reduction in cap-dependent translation, because of the cap cleavage by caspase 3, or by changes in the phosphorylation state of canonical initiation factors 6. In addition, certain mRNAs, coding for proteins that are essential to the apoptotic process (including Apaf-15 and c-Myc7), are translated by the recognition of the internal ribosome entry site (IRES). In Caenorhabditis elegans changes in protein synthesis act as a physiological signal to initiate cell death in germ cells. In particular, a modification of the balance between the two eIF4G isoforms (the long and the short ones) disrupts cap-dependent mRNA translation in favor of a cap-independent mechanism. Indeed, the depletion of the long eIF4G isoform promotes the cap-independent translation, inducing the translation of pro-apoptotic proteins8. Lastly, in Drosophila melanogaster, the pro-apoptotic protein Reaper binds specifically the 40S subunit, affecting the AUG recognition, thereby inhibiting cap-dependent translation9, and facilitating the selective translation of specific pro-apoptotic mRNAs thanks to the recognition of IRES segments10. Interestingly, the involvement of the translational machinery in Escherichia coli PCD has been clearly described by the MazEF module11, in the toxin–antitoxin system. Stress conditions  reduce MazE levels in the cell, favoring the release of the MazF toxin13. This endoribonuclease can produce leaderless mRNAs14 and modify 16S rRNA structure in the ribosomes, producing the “stress ribosomes”17. These events dramatically affect the overall translational process18, determining the parallel production of “death”  and of “survival”  proteins, promoting a higher propensity to cell death19.Programmed cell deaths (PCDs) are a group of diversified processes that are triggered by stress or developmental events, occurring in both prokaryotes and eukaryotesSaccharomyces cerevisiae (yeast) can undergo PCD triggered by aging, drugs, heat stress, UV radiation, heterologous expression of pro-apoptotic human genes, and acetic acid21. The process shows the canonical apoptosis hallmarks, such as phosphatidylserine externalization, chromatin condensation and DNA fragmentation, cytochrome c release from mitochondria, and dissipation of the mitochondrial transmembrane potential22.23. The yeast genome is reported to encode 137 ribosomal protein genes (RPGs), ∼150 rDNA repeats, and 76 small nucleolar RNAs (snoRNAs), that are mainly involved in the post-transcriptional modifications of the rRNAs25. Changes in the expression of each single components participating in the overall process may affect the ribosome biogenesis27. RPGs in yeast are characterized by couples of paralogs28. In particular, among the 137 RPGs annotated in the S. cerevisiae genome, 116 were identified to be ohnologs, i.e., duplicated genes, or paralogs, originated by a whole genome duplication29. In 2019, Ghulam et al.30. demonstrated that the stoichiometry of ohnolog RPGs changes in response to variations of environmental conditions. In particular, under physiological conditions, the ribosome population is dominated by the presence of one of the two paralogs that was defined as the major paralog. When cells are exposed to stress conditions, decreased expression of the major paralog and/or overexpression of the minor one, in ohnolog pairs of ribosomal proteins, reduce the relative proportion of the major versus the minor paralog content in the cell30.The ribosome structure organization in yeast requires a fine-tuning in the gene expression of many of its components, involving ∼200 assembly factors. The 60S ribosomal subunit contains three rRNAs  and ∼46 ribosomal proteins, while the 40S subunit is composed of a single rRNA (18S) and ∼33 proteins31. Our results show changes in the gene expression of RPGs, with differential patterns between pairs of ohnologs when compared to control conditions. Moreover, snoRNA genes are also differentially expressed, revealing additional changes in contributors to ribosomal biogenesis. We show also a differential expression of genes coding for mitochondrial ribosomal proteins (MRPGs). A different trend, moreover, characterizes the expression of the proteins of the mitoribosome versus the ones of the cytosolic ribosome.We exploited RNA-seq data of yeast undergoing PCD when treated with acetic acid. The experiment considers three different time points after exposure This is the first time that changes in ribosome organization are described in yeast PCD, and these events, accompanied by the differential expression of genes involved in the post-transcriptional maturation of the rRNAs and in mRNA translation accuracy, suggest an overall reprogramming of the translation machinery as a hallmark of the early stage of the process.S. cerevisiae exposed to acetic acid31 resulted in the list of significant differential expressed genes (DEGs)  enrichment at each time point is in the Supplemental Table SThe list of pathways enriched during the three treatments is in the Supplemental Table S33, is upregulated at 200 min , that is involved in mRNA decapping35 focused on the role of RPL1A and B paralogs. They found that ribosomes containing RPL1B are more efficient in translation of respiration-related proteins, and that this specific form is required for proper mitochondrial function, morphology, and for cell wall integrity. Moreover, they also showed that the upregulation of RPL1A did not replace the functionalities of RPL1B.Among the differentially expressed genes, 127 RPGs over 137 total RPGs resulted to be DEGs in at least one stage of the treatments by acetic acid. In particular, at 45 min there are 6 RPGs downregulated and 12 RPGs upregulated. At 120 min and at 200 min there are only downregulated genes, 60 and 122 per stage, respectively . Moreover, considering the remaining couples of paralogs, that were not classified in ref. 30, the ratio changes also in 8 additional couples. In particular, at 45 min the ratio is changed in 3 couples, at 120 minutes in 4, and at 200 min in 6 couples, respectively , and snR128 (box C/D), that are all downregulated at 120 min, are also involved in the cleavage of the primary rRNA transcript39. Furthermore, loss of snR10 results in slow growth, impaired production of ribosomal small subunits, loss of pseudouridylation at positions 2919 (which occurs in the A loop), and a reduced rate of protein synthesis26. This gene is also associated to cells with higher susceptibility to osmotic and cold stresses25. In addition, also snR191 is downregulated at 120 min. This H/ACA snoRNA guides pseudouridylation at positions 2258 and 2260 in the large subunit rRNAs. These two modified bases are the most conserved pseudouridines from bacteria to human rRNAs. In yeast, the presence of this snoRNAs and of the conserved pseudouridines is not essential for viability, but it is discussed to provide a small growth advantage in cells placed in competition with cells that lack these features40. Finally, also snR5, that is upregulated at 200 min, is reported to be a guide for rRNAs pseudouridylation in eukaryotes41. It is assembled with the core domain of Cbf5p, Gar1p, Nop10p, and Nhp2p proteins42, and is known to be involved in modifications of uridines, with a relevant role in the process43.The snoRNAs are involved in different biological processes including rRNA modifications, maturation, and splicing. Interestingly, although all of them are known to be involved in the ribosome biogenesis, specific roles for some of them have been reported. snR17B, that is revealed upregulated at 45 min, is one of the two box C/D snoRNAs U3  that are required for cleavage of the primary rRNA transcript31 and PCD in yeast20. Here we show that transcriptomic changes extensively involve genes associated with ribosome biogenesis and rRNA maturation, also inducing mRNA decapping and affecting translation accuracy, indicating a reprogramming of the cell translational apparatus during the exposure. Indeed, the upregulation of EDC1 (YGL222C), a gene involved in mRNA decapping, highlights the possible role of this mechanism in response to exposure to acetic acid, a phenomena associated also to the yeast response to heat stress33. In addition, the downregulation of ASC1 (YMR116C), that causes higher translational activity44, but favors frameshifting34, suggests that the response affects the rate and the quality of the protein synthesis during the response.Acetic acid induces transcriptomic changes30, that demonstrated that RPGs ohnologs relative content changes in cells that are exposed to stress. They also show that the repression of the major paralogs and/or the upregulation of the minor paralogs reduce the ratio of the major versus the corresponding minor ohnolog in the cells, increasing the frequency of the minor paralogs in the overall ribosome population. Our results confirm the overexpression of the minor paralogs in 3 of the couples of ohnologs described also in ref. 30. This highlights that the changes of the ratios in favor of the minor paralogs occur also in the response to acetic acid  induces the activation of the toxin MazF under stress response. MazF cleaves single-stranded mRNAs, near the AUG start codon, generating leaderless mRNAs and 16S rRNA at the decoding center. These events lead to the formation of a subpopulation of ribosomes able to translate leaderless mRNAs18. In mycobacteria, ribosomes containing the alternative small ribosomal subunit protein RpsR2 generate distinct translational landscapes compared to canonical ribosomes. Moreover, this alternative small ribosomal subunit protein is necessary for growth in iron-depleted medium, highlighting that alternative bacterial ribosomes may play a crucial role during nutrient depletion stress48.Changes in the translational machinery are known to occur during stress response or PCD in several species from prokaryotes to eukaryotes. In 30.In yeast, the formation of a “stress ribosomes” was demonstrated in stress responses, with the involvement of the differential usage of paralogsArabidopsis thaliana, RPS5A is highly expressed in dividing cells, whereas RPS5B is expressed in cells undergoing differentiation10. Furthermore, the ribosomal protein RPL11B is highly expressed in all actively dividing cells, whereas the expression of its paralog, RPL11A, is tissue-specific10. Moreover, deletion of RPL23AA, but not of RPL23AB, impedes growth and leads to morphological abnormalities49.Interestingly, preferential expression of paralogs among ribosomal proteins have also been described in plants. In D. melanogaster two genes encode the ribosomal protein S5, RpS5a and RpS5b. In 2019, Kong et al.50 demonstrated that females lacking RpS5b produce ovaries which undergo apoptosis during oogenesis, whereas females lacking RpS5a are fully fertile. This suggested that RpS5b-containing ribosomes are specifically essential during oogenesis50.In H. sapiens, ribosomal proteins changes have been demonstrated to be involved in ribosome dysfunctions, leading to ribosomopathies, including Diamond-Blackfan anemia51, 5q-syndrome52, dyskeratosis congenita, cartilage hair hypoplasia, Treacher Collins syndrome54, and Shwachman-Diamond syndrome55. Moreover, alterations of RPL5, RPL10, RPS15, RPL11, and RPL22 ribosomal proteins have been described in 10 to 40% of tumor types. This highlights that the somatic ribosomal proteins mutations influence the ribosomal functioning, resulting in what is called an oncogenic rewiring of the protein expression mechanisms56. In addition, changes in translation mechanisms favoring IRES-dependent protein synthesis where shown in human apoptosis, promoting apoptotic proteins formation60.In 61. Moreover, differential usage of ribosomal protein paralogs resulted to be a remarkable event in stress or dysfunctions, as shown in plants49, in D. melanogaster50, in bacteria48 and in yeast30.In conclusions, studies from animals, plants and prokaryotes suggest that ribosomal proteins asset plays a crucial role in stress and dysfunctionsHere we show that the exposure to acetic acid in yeast determines a differential usage of ribosomal protein paralogs, with an overexpression of mitoribosomal proteins. Although more in-depth analyses are required to confirm the compositional modifications at ribosome level, also to shed further light on the molecular mechanisms inducing the differential expression patterns among ohnologs, our analysis highlights that this phenomenon is also accompanied by rRNA modifications, mRNA decapping, affecting translation accuracy and putatively, triggering the synthesis of proteins involved in PCD Fig. . This scS. cerevisiae exposed to acetic acid at three different time points  plus control data at each stage31 were downloaded from sequence read archive (SRA)62 (SRP075510). Raw reads were trimmed with Trimmomatic (release 0.38)63 and aligned to the S. cerevisiae strain S288c genome (version R64-1-1) using STAR (release 2.6.0)64 . Reads counting was performed using FeatureCounts (release 1.6.3)65 . Read counts were normalized by counts per million (CPM), filtering genes with CPM ≥ 1 in each replicate per treatment and control. The differential expression analysis was done using edgeR66. Only genes with |log2Fold change | ≥1 and FDR < 0.05 were considered as significant DEGs.RNA-seq data (fastq files) from https://yeastmine.yeastgenome.org/yeastmine/begin.do)67, filtering enriched GOs at p-value < 1 × 10−4.GO enrichment analysis was performed using YeastMine (http://cpdb.molgen.mpg.de/YCPDB)68, filtering enriched pathways at p-value < 1 × 10−4.Enriched biochemical pathways were obtained by ConsensusPathDB-yeast (S. cerevisiae were downloaded from the Saccharomyces Genome Database (https://www.yeastgenome.org/)69 and cross-confirmed by Byrne et al.29.Ohnolog genes in 30.The classification of major and minor paralogs in ohnolog ribosomal protein genes is from Ghulam et al.http://genesilico.pl/modomics/)70.List of snoRNAs and their action (site and type of modification) were downloaded from Modomics (Supplementary informationSupplemental Figure S1: Ribosomal protein genes expression.Supplemental Figure S2: Mitochondrial ribosomal protein genes expression.Supplemental Table S1: List of differential expressed genes.Supplemental Table S2: Gene ontology enrichment.Supplemental Table S3: Pathways enrichment.Supplemental Table S4: Ribosomal protein genes details and paralog couples expression patterns.Supplemental Table S5: Mitoribosomal protein genes expression patterns.


Enterovirus B was the most detected species worldwide, while the other species showed continent-specific differences, with Enterovirus C more detected in Africa and Enterovirus A more detected in Asia. Echovirus 30 was by far the most detected type, especially in studies conducted in Europe. EV types in species Enterovirus B—including echovirus 30—were often detected in patient groups with neurological infections and in cerebrospinal fluid, while Enterovirus C types were often found in stool samples.Enteroviruses (EVs) are highly prevalent viruses world-wide, causing a wide range of diseases in both children and adults. Insight in the global prevalence of EVs is important to define their clinical significance and total disease burden, and assists in making therapeutic decisions. While many studies have been conducted to describe epidemiology of EVs in specific (sub)populations and patient cohorts, little effort has been made to aggregate the available evidence. In the current study, we conducted a search in the PubMed and Embase (Ovid) databases to identify articles reporting EV prevalence and type distribution. We summarized the findings of 153 included studies. We found that EVs are highly prevalent viruses in all continents.  Enterovirus A, B, C or D. These species vary in size; species Enterovirus B contains more than half of all known EVs (n = 59), while only 4 Enterovirus D types have been defined (Enteroviruses (EVs) are among the most prevalent viruses infecting humans worldwide. There are 106 types currently known to infect humans, of which dozens are frequently detected globally ,3,4. Mos defined .EVs are known to cause a wide spectrum of diseases, including encephalitis, meningitis, myocarditis, hand-foot-mouth disease (HFMD), conjunctivitis, respiratory disease and gastro-intestinal disease, but the majority of infections remain asymptomatic ,10,11,12Over the years, prevalence and surveillance studies have made use of different methods to detect and type EVs. Whereas cell culture has been applied for many years to detect presence of EVs in clinical samples, this changed with the introduction of sequencing. The currently recommended method of detection is by a PCR targeting the 5’ untranslated region (UTR) of the virus, which has proven to be more sensitive than culture ,14,15. TEnterovirus B was the most prevalent species, with E30 and several other Enterovirus B types as the most detected types. Enterovirus A appeared to be the predominant species in Asia, mainly resulting from large outbreaks of HFMD caused by CVA16 and EVA71 [Though many studies on EV prevalence have been conducted in either specific (sub)populations or patient cohorts, few efforts have been made to aggregate the epidemiological evidence to clearly display the worldwide prevalence, related type-distribution of EVs and their clinical relevance. A review by Janes et al. including several large surveillance studies—mainly applying virus isolation and serotyping methods—showed that globally, On 12 June 2020, we performed a literature search in the PubMed and Embase (Ovid) databases , to idenArticles were included for analysis on EV prevalence or analysis on EV type distribution, or both. We extracted data on the number of samples tested and typed, continent and country where the study was conducted, symptoms of the included participants, age of the participants, sample type, EV prevalence and EV types detected from the included articles. When no data on number of samples was available, number of included patients was taken as a proxy. Sample type was recorded as one of five categories; gastrointestinal , cerebrospinal fluid (CSF), respiratory , blood (including serum and plasma) and multiple sample types. Clinical complaints were recorded as one of five categories; gastrointestinal, neurological, respiratory, other or no symptoms. When the age limit of a pediatric population was not specified, it was assumed individuals up to 18 years of age were included. The quality of all included articles was assessed using quality control checklists for cross sectional and case–control studies .Median EV prevalence and interquartile ranges (IQRs) weighted for the number of tested samples were calculated overall and for different continents, symptom groups, age groups and sample types. The proportion of each EV species was calculated for continents for which at least ten studies describing EV type distribution were available. Proportions corrected for the number of types per species  were calA total of 10,132 unique studies were identified in the search in the PubMed and Embase (Ovid) databases. Eleven papers were added during the in- and exclusion through chain-referral sampling. Screening on title and abstract and full text resulted in exclusion of 9636 and 354 articles, respectively. A total of 153 studies were included for the current study, of which 135 were included for analysis on EV prevalence and 47 were included for analysis on EV type distribution . The quaOf the total number of studies, most were conducted in Europe (35.9%) and Asia (37.3%), followed by Africa (13.1%) and North and South America (both 5.9%) A. The duOf the 153 included studies, 135 reported on EV prevalence. Overall, the range in prevalence reported in the studies was wide, ranging from 0% in a Portuguese study on respiratory samples obtained from elderly with acute respiratory infections  to 89.9%Most included studies were conducted on the Northern hemisphere, and reported peaks in incidence mainly in summer and fall (June–November) ,33,34,35Enterovirus A, 47 types in species Enterovirus B, 17 types in species Enterovirus C and one type in species Enterovirus D. Species distribution was calculated overall, and for Africa, Asia and Europe   , Enterovd Europe C,D and En Africa B. The prn Europe C,D.Enterovirus B—were especially prevalent, with echovirus 30 (E30) ranking highest. CVA6, CVA16 and EV-A71 were the most prevalent types in species Enterovirus A. In species Enterovirus C, CVA13, CVA24 and EV-C99 were the three types with highest prevalence. Species Enterovirus D was represented only by type EV-D68. E30 was prevalent in Europe, while it was rarely found in Africa. In contrast, Enterovirus C types CVA13, CVA20 and EV-C99, rarely found in Asia and Europe, were prevalent in Africa  compared to studies including children up to three (10.8%) or eighteen years of age (7.9%). The median prevalence in studies including adults was lowest at 6.2% C. EV preEnterovirus A and Enterovirus B were detected in a variety of sample types, often including both respiratory and gastrointestinal samples. Types in Enterovirus C were detected almost exclusively in gastrointestinal samples, while Enterovirus D type EV-D68 was detected mainly in respiratory samples  cohorts’ characteristics of the included studies.In the current review, we presented an overview of worldwide EV prevalence and type distribution. We found that EVs are highly prevalent globally, with comparable EV prevalence rates across continents. Enterovirus D in Europe may be partially explained by the high number of studies conducted in Europe that included respiratory samples specifically, as EV-D68 is known to cause predominantly respiratory disease. Several studies conducted on the African continent included asymptomatic participants (often children) of whom stool samples were obtained. This could have skewed the detections towards species Enterovirus C, as types within this species cause predominantly gastrointestinal symptoms, and the prevalence in children specifically is high. However, these findings are in line with findings in African environmental samples in which Enterovirus C strains are highly prevalent [Enterovirus B strains are often predominant, in line with the results in this review [Enterovirus C strains are also often found in environmental samples in Europe, suggesting a widespread circulation of this species in the European continent, which is in contrast with the low detection rate of Enterovirus C shown in this review.The high prevalence of revalent ,50. Simis review ,52,53,54Enterovirus A in Asia, it was not the predominant species. This is in contrast with a previous review where Enterovirus A was the predominant species in this continent [Enterovirus A types.Although showing a high prevalence of ontinent , and repontinent ,56. As wEnterovirus B appears the predominant species, followed by Enterovirus D and Enterovirus A. CVA21 was the only reported Enterovirus C type in these reports, while CVA9, CVB1-5, E6, E9, E11, E18, E25 and E30 were the most frequently reported types in Enterovirus B. EV-A71 and CVA16 were most frequently reported in Enterovirus A and EV-D68 was the only reported type in Enterovirus D.The information available on North and South America was limited. Though the USA has an EV surveillance system in place and frequently reports the results ,14,57,58An abundance of types was found to circulate globally. Type E30 showed an extraordinary peak in the proportion of genotypes, mainly in Europe, and was the most frequent EV detected in patients with neurological symptoms. This is in line with frequent reports of outbreaks of E30 related meningitis, both in Europe and in other continents ,60,61,62Enterovirus C were rarely found in the studies conducted in Europe, while types belonging to this species appear to be abundant in Africa. While many EV types in Enterovirus A and B are detected in different sample types, most Enterovirus C types were found almost exclusively in stool samples, and Enterovirus D type EV-D68 was found almost exclusively in respiratory samples. Possibly, this indicates that the Enterovirus C and D types are limited to infecting a single organ system or cell type, while viruses belonging species Enterovirus A and Enterovirus B have a less exclusive cell tropism.EVs belonging to species Surprisingly, the mean prevalence in asymptomatic subjects was higher than the mean prevalence in studies including specific patient groups. The EV infections detected in cohorts of healthy subjects may represent not only asymptomatic infections, but also reflect prolonged asymptomatic viral fecal or respiratory shedding after symptomatic infections. The high prevalence can further be explained by the fact that most studies on cohorts of asymptomatic subjects tested EVs in stool samples, which have a higher prevalence rate than other sample types. A total of twelve case-control studies were included in the current review. Ten of those case-control studies reported a lower EV prevalence in the control group than in the case group ,73,74,75Although EVs are prevalent in children and adults, their prevalence in young children is higher as compared to adults.Though much effort has been put into describing the epidemiology of EVs, the studies performed differ in many aspects, such as patient cohorts and collected sample types as highlighted in this review. The limited comparability between these studies, hampers the possibility to draw conclusions from aggregated information. To improve this, studies are needed that combine different clinical specimens or compare different patient groups. Specifically, asymptomatic and control matched cohorts are of interest. Most studies performed to date included patient groups with specified symptoms, though the majority of EV infections remains asymptomatic. Studies on asymptomatic participants or studies that include a control group will be important in estimating the real circulation of these viruses. By obtaining multiple sample types from these cohorts, the differences between the rates of and the EV types involved in respiratory and gastro-intestinal circulation can be further elucidated.In this review, we chose to only include studies reporting on EV prevalence by PCR directed at the 5’UTR and studies reporting EV type distribution by typing based on VP1 sequences. Usage of these specific methods for studying EV epidemiology is advised by the European Non-Polio Enterovirus Network (ENPEN) , and exc


Although there are numerous publications about surgical treatment of proximal humeral fractures (PHFs), few assess conservative treatment, which is the most common approach. The aim of this systematic literature review was to assess criteria for indications, treatment protocols, and outcomes obtained with the conservative treatment of 3-part and 4-part PHF.We searched the PubMed and Cochrane databases for clinical studies published between 2000 and 2019 on conservative treatment for 3-part and 4-part PHF that included patients older than 18 years, a minimum follow-up of 1 year, fracture classification, and description of outcomes with assessment scales.The search yielded 26,660 records. We reviewed 44 of them in full, and finally 6 studies were included. We obtained a population of 133 patients (79% women), with a mean age of 74.3 years (range 25 to 98) and mean follow-up of 32 months (range 12 to 68.8). According to the Neer classification system, there were 41% (55) three-part fractures and 59% (78) four-part fractures; 5.81% of the patients were lost to follow-up. The mean Constant score was 64.5 for three-part fractures and 54.9 patients with four-part fractures. Consolidation was achieved in 95% of the three-part fractures and 91% of the four-part fractures. Loss of mobility varied according to the type of fracture. Regarding complications, the most frequent was malunion (21%), followed by avascular necrosis (9%).Our data show that most three-part PHFs treated conservatively achieve fracture consolidation even noting a negligible rate of malunion got fair–good functional results with few complications, while the orthopedic four-part PHF treatment presents high rate of consolidation with less rate of malunion than the three-part PHF but achieve poor functional results with few complications.Level IV, Systematic Review There is a high incidence of proximal humeral fractures (PHFs) in the current population due to the increasing life expectancy and the consequent rise of osteoporotic bone fractures . These fThere are numerous publications on the different surgical treatment options for these fractures, but few studies assess conservative treatment, which is the most frequently used , since mIt should also be mentioned, when treating this type of fracture, the difference between referral hospitals, with well-established shoulder units, and smaller hospitals where the orthopedic surgeons are not subspecialized. Probably, due to the technical complexity of these surgeries, conservative treatment is chosen more frequently in these latter hospitals, while in shoulder units, surgical treatment is more often preferred. For this reason, it is even more necessary to find a consensus when treating PHFs.This systematic literature review has the following aims: to assess criteria for indications, treatment protocols, and outcomes obtained with conservative treatment of 3-part and 4-part PHFs.We performed a systematic review of publications on conservative treatment for three-part and four-part PHFs, in compliance with the PRISMA protocol . Two revThe inclusion and exclusion criteria were agreed by the authors to obtain a minimal homogeneous information in order to carry out a review.Inclusion criteria for the articles were 1) studies of three-part and four-part PHFs of traumatic origin  treated conservatively; (2) patients older than 18 years; (3) studies with levels I–IV evidence; (4) studies with minimum follow-up of 1 year; (5) studies with more than 15 participants; (6) studies using a fracture classification system (Neer or AO) (7); studies reporting outcomes on an assessment scale; and (8) studies describing range of motion, pain, and complications (Table  studies We excluded studies that (1) focused exclusively on surgical treatment for PHF, (2) were systematic reviews and meta-analyses, and (3) reported level V evidence.We classified all records retrieved in the search as relevant, potentially relevant, and irrelevant. We extracted from the relevant studies the following data: study design, year of publication, journal of publication, type of study, level of evidence, number of patients, mean age, sex, laterality of fracture, indication for conservative treatment, type of fracture, follow-up, losses, type of conservative treatment, Constant scale, other scales, subjective results, consolidation, and complications. This piloted form was evaluated in duplicate by independent authors.Studies reporting on patients treated surgically were included only if data on participants treated conservatively were clearly differentiated, and those with inconsistent or ambiguous data were excluded. We resolved any disagreements with two independent reviewers by consensus or adjudication by the senior author.All institutional and author information were concealed to minimize reviewer bias. The risk of bias was evaluated as suggested by Furlan et al. . The risOur search strategy yielded 26,660 records. After screening abstracts, the number of potentially relevant records dropped to 44. Full-text assessment further reduced the number of articles that met the inclusion criteria to 6 Fig. . These sk = 0.66) and moderate interobserver reliability (k = 0.50) [To classify the fractures, all studies used the well-known system proposed by Neer in 1970 , which h = 0.50) . Accordi = 0.50) , 13.With respect to the indications for conservative treatment, all studies applied conservative treatment to three- and four-part fractures, defined as having displacement > 1 cm or angulation > 45°, according to the Neer criteria . Van denAll of the conservative treatment protocols included a period of immobilization with a sling or Velpeau for 2 to 4 weeks –16. SubsWith regard to outcomes, all studies used the Constant score system (CS) , 20, whiIn respect to subjective outcomes, there was greater variability in the type of assessment scales used. The most frequent was the visual analog scale (VAS)  and the Concerning complications, 7.5% of patients experienced pseudoarthrosis, 21% malunion, 9% avascular necrosis, 4.5% post-traumatic arthrosis, 0% glenohumeral instability, and 0% torn rotator cuff  . One of Good results of orthopedic treatment in minimal displaced or two-part FHPs are widely known to the community , 33–35. The reason for the discrepancy between the Constant score versus the Eq-5D, DASH, and pain score is unclear. A possible explanation may be that the Constant score is not self-reported and is therefore less sensitive to subjectively experienced, yet important, differences in outcomes .With regard to the range of motion and Constant score, we found differences between three- versus four-part fractures. The former , 14, 15 Although the outcomes achieved with conservative treatment for three- and four-part fractures were worse than those obtained for minimally displaced and two-part fractures , the surIn terms of the imaging studies of the PHFs, 50% of the studies used only simple radiographic projections, while the rest employed computerized tomography (CT) imaging to complete the diagnosis. Currently, classifying complex PHFs without CT, which is generally available in all hospital emergency departments, implies some risk of selection bias, as evaluation of radiographic images alone is imprecise , 26–29.About conservative treatment protocols, and even rehabilitation, the scarce interest among orthopedic surgeons for this treatment modality is well-known once it is indicated. All studies employed a shoulder immobilizer  during 2 to 6 weeks followed by a rehabilitation program that began with passive exercises at 1 or 2 weeks after immobilization. Better findings at the evaluation of the Constant score happened in treatment protocols that began before 2 weeks of passive exercises as we have observed at Van den Broek et al.  who obtaThis systematic review is the first after the PROFHER study , which gTo our knowledge, the present systematic review is the second on conservative treatment of PHFs, updating the review published by Iyengar et al. in 2011  and inclThe pooled rate of consolidation following conservative treatment in the included studies was 92.5%. This high percentage shows that PHFs tend to consolidate, even in patients with poor bone quality . It woulOne frequent complication of conservative treatment of PHFs is loss of mobility. While this limitation could be used to argue in favor of surgical treatment, authors such as Olerud et al.  comparedWith regard to avascular necrosis, our review found a mean rate of 9%. The correlation between risk of necrosis and severity of the fracture is well-known , 41, so This review is limited by the weaknesses of its included studies. Our work includes only six studies because the others did not include data by subgroups of fractures with regard to outcomes on range of motion, assessment scales, and complications, as the overall results may be deceptive. Moreover, the inclusion of retrospective studies always confers the risk of selection bias, as injuries treated nonoperatively tend to be less severe and therefore have better outcomes. Furthermore, it is difficult to compare results between studies using different assessment scales.Focusing on the risk of bias, 1 of 6 studies had high risk of bias. It did not report about randomization, allocated interventions, and groups similar at baseline. The remaining 5 studies had a low risk of bias (Table Future research is needed to improve knowledge around the possibilities of conservative treatment of PHFs, performing studies of subgroups of fractures and comparing diverse treatment protocols.Our study shows that most three-part PHFs can be treated conservatively, with a high rate of consolidation, fair–good functional outcomes in accordance with the severity of the fracture and few complications compared to surgical treatment. Conservative treatment for this type of fracture could be preferable in elderly patients with PHFs, as they should obtain acceptable functional outcomes and good control of pain while avoiding the potential risks of surgery. If patient functional expectations are high and have few comorbidities, more aggressive treatment could be indicated.Otherwise, orthopedic treatment of four-part PHFs provided a high rate of consolidation with few complications, but worse functional results despite a less rate of malunion than three-part PHFs. This modality of treatment is advisable to four-part PHFs with low functional expectation or with severe comorbidities because poorer results are expected.About conservative treatment protocols, we observed that an early start of passive exercises achieved better results and should be recommended.


The impact of a broiler chicken diet that is supplemented with canola meal (CM) on the cecal microbiota, growth, and metabolome of broiler chickens was examined. CM did not affect feed consumption, weight gain, nor the richness, evenness, or diversity of the cecal bacterial community. However, CM affected the structure of the bacterial microbiota, the concentrations of short-chain fatty acids (SCFAs) were elevated, and the abundance of bacterial taxa known to ferment dietary fiber were more abundant in the ceca of birds fed the CM diet. Nuclear magnetic resonance spectroscopy metabolomics was applied, and a number of metabolites that were associated with SCFA metabolism were differentially regulated in cecal digesta. The supplementation of diet with CM also affected the metabolic profiles of the pancreas, liver, and breast muscle . In summary, broiler chickens that were fed a diet supplemented with CM showed equivalent feed consumption and growth, but CM affected the composition and function of the cecal microbiota, and the metabolome of the pancreas, liver, and breast muscle of birds indicating the possibility of an increased disease risk. The study also demonstrated the utility of using metabolomics to ascertain impacts of diet on the microbiota and host tissues, and identified biomarkers to facilitate subsequent evaluation in production settings.Methanobacteriaceae, Atopobiaceae, Prevotellaceae, Clostridiales Family XIII, Peptostreptococcaceae, and Succinivibrionaceae), and concentrations of SCFAs were higher in the ceca of birds fed the CM diet. Moreover, concentrations of isoleucine, isobutyrate, glutamate, and 2-oxoglutarate were higher, whereas concentrations of phenyllactic acid, indole, glucose, 3-phenylpropionate, and 2-oxobutyrate were lower in the digesta of chickens that were fed CM. The metabolic profiles of pancreas, liver, and breast muscle tissues of birds fed the CM diet differed from control birds. Metabolites that were associated with energy production, protection against oxidative stress, and pathways of amino acid and glycerophospholipid metabolism had altered concentrations in these tissues. Some of the observed changes in metabolite levels may indicate an increased disease risk in birds fed the CM diet , and others suggested that birds mounted metabolic response to offset the adverse impacts of CM . The inoculation of one-day-old broiler chicks with the cecal contents from a mature broiler breeder resulted in a highly diverse and uniform cecal bacterial community. CM did not affect feed consumption, weight gain, nor the richness, evenness, or diversity of the cecal bacterial community. However, the structure of the bacterial community was altered in birds fed the CM diet. Although the CM diet was formulated to contain equivalent metabolizable energy to the control diet, it contained more dietary fiber. The abundance of bacterial families, including those that are known to contain species able to metabolize fiber was altered (e.g., bacteria within the families,  Brassica napus) is a major annual field crop grown in North America, with >5.0 million ha in production annually in Canada [Canola ; establish a uniform microbiota that is representative of broilers in a production setting; measure feed efficiency in broilers over a 35-day period; apply Illumina technology and microbiota bioinformatics pipelines to ascertain the structure of the cecal microbiota; measure SCFAs in cecal digesta; and, apply proton NMR-based metabolomics in order to comparatively examine the metabolome within cecal digesta, pancreas, liver, and breast muscle.The study was carried out in strict accordance with the recommendations that were established in the Canadian Council on Animal Care Guidelines. The project was reviewed and approved by the Agriculture and Agri-Food Canada (AAFC) Lethbridge Research and Development Centre (LeRDC) Animal Care Committee .https://www.pleasantvalleyoilmills.com/ (accessed on 17 February 2021)) at a concentration of 20% (w/w) using the same starter, grower, and finisher schedule as the control diet. The canola cultivar used to generate the CM was not genetically modified . Care was taken to ensure that the two diets were energetically equivalent (The experiment was arranged as a completely randomized design with two levels of diet (±CM supplementation) and six levels of replicate. The starter (10 days), grower 14 days), and finisher (11 days) control diets used were corn-based formulations that are commercially used in Canada . For theuivalent . All of  days, anOne-day-old broiler chicks (Ross 308) were obtained from the Sunrise Hatchery  in two groups of 30 that were hatched six days apart; the two groups of birds that were obtained on separate occasions comprised different replicates and they were treated identically with respect to the experimental design . All of the chicks were hatched from eggs that were sourced from the same broiler breeder farm. Chicks were transported from the hatchery to the Livestock Containment Unit (LCU) at LeRDC. Upon arrival at the facility, each chick was identified with a numbered ankle bracelet, and they were weighed. The ambient temperature within the LCU animal rooms was set to 30 °C. From each hatch, 15 randomly selected chicks were placed in one custom stainless steel elevated enclosure , and the remaining 15 chicks were placed in another identical enclosure. The enclosures were arranged so that there was no possibility of contact between birds in adjacent enclosures. The enclosures were sanitized before placement of the chicks, and brown kraft postal paper was placed on the slotted floor of the enclosures. Each cage was equipped with a 30 × 30 cm brooder , a 24″ chick feeder , and a Bell waterer (United Farmers of Alberta Co-operative Ltd.). Chicks in one of the enclosures were provided a control starter diet, and chicks in the remaining enclosure were provided a starter diet that was supplemented with CM.2 captive bolt device , and immediately transferred to LeRDC. Individual birds were placed into a sanitized plastic container, and the feathers were removed. The skin was sanitized with Peroxigard™ , a laparotomy was conducted, and the ceca were ligated using sterile cable ties  to prevent infiltration of air. The ceca were incised and transferred into a Thermo Forma 1025 anaerobic chamber  containing a 85% N2: 10% CO2: 5% H2 atmosphere. Within the chamber, ceca were placed on a sterile large petri-dish (20-cm-diameter), the cable ties were removed, and, using a sterile wooden splint, the cecal contents were pushed into a sterile pre-weighed 500 mL Pyrex™ medium bottle . The digesta was suspended in reduced phosphate buffered saline (PBS) at a 3:1 ratio (PBS: digesta v/w), transferred in 1.5 mL aliquots into 2.0 mL screw cap tubes, snap frozen in liquid nitrogen, and then stored at −80 °C until used.All of the chicks were administered cecal contents that were harvested from healthy mature broiler breeder male donors (≈30 weeks-of-age). The donors were humanely euthanized on farm using a compressed COCecal digesta was thawed in an anoxic atmosphere, the slurry was transferred into sterile 3 mL syringes (1 mL per syringe), and the syringes were capped in order to prevent oxygen infiltration and placed in sealed plastic bags (eight syringes per bag). Each plastic bag was placed in a thermos containing a pre-heated (37 °C) ThermoSafe PolarPack (Fisher Scientific), and the syringes were at transported to the LCU. Chicks (two-day-old) were orally inoculated with the cecal digesta slurry within ≈20 min. of removal of the syringes from the anoxic atmosphere. Each syringe was fitted with a flexible disposable PTFE feeding needle  . Individual chicks were gently restrained, the beak was carefully opened by inserting the feeding needle, and the feeding needle was carefully advanced into the mouth and then guided into the esophagus. Once in place, 1 mL of the cecal slurry was delivered into the esophagus, and the needle was carefully removed. Chicks were observed for 30 min., and none of the birds showed any evidence of distress.n = six replicates per diet treatment). The floor of the enclosure was papered; however, the flooring under the area around the feeder was left unpapered in order to allow unconsumed feed to fall through the enclosure slots onto plastic trays situated on the animal room floor below the enclosures. Feed that was provided in the morning was weighed using a Pelouze scale , and residual feed within the feeder, on the paper within the enclosure, and on the plastic tray below the enclosure at the end of the 24-h period was weighed daily using a Mettler scale . Care was taken to ensure that feces was removed from the residual feed. Waterers were filled, changed, and washed as needed. Identification ankle bracelets were regularly checked to ensure that they were not becoming too tight as the birds grew. Bracelets were typically replaced once per week if needed. The birds were weighed at seven-day intervals for 35 days.The chicks were retained in the two groups for the first five days, at which point five randomly-selected chicks per diet treatment were placed into three new sanitized enclosures containing a brooder, a feeder, and a water. These individual cages represented a replicate with five observations  per replicate , and stored at −80 °C until processed. The birds were then humanely euthanized by decapitation . Feathers on the breast area were removed, the skin was sanitized with Peroxigard™, an incision in the skin was made, and a sample of breast muscle (middle portion of the right breast) was removed for metabolomic analysis. A ventral laparotomy was performed, viscera was exposed, ceca were removed, incised, and cecal contents were carefully removed for the analysis of SCFAs, characterization of bacterial communities by Illumina sequencing, and metabolomics analyses. In addition, the pancreas  and liver (lower left lobe) were removed for metabolomics analysis. Cecal digesta for bacterial community characterization and metabolomics, and inferior pancreas, part of the inferior left lobe of the liver, and breast muscle samples for metabolomics were snap frozen. All of the samples were maintained at −80 °C until they were processed. The sex of all birds was determined at necropsy .When birds reached 35 days-of-age, they were anesthetized within an induction chamber with isoflurane  using an E-Z anesthesia machine . Once anesthetized, the birds were transferred to an anesthesia mask system that was located within a fume hood. Under anesthesia, blood was intracardially collected, serum was separated using BD Vacutainerw/v). Meta-phosphoric acid  was added to the homogenate at a 1:4 ratio (v/v), and then incubated at room temperature for 30 min. The samples were centrifuged at room temperature for 75 min. at 16,100× g, and the supernatants were collected and stored at −20 °C. Concentrations of acetate, propionate, isobutyrate, butyrate, valerate, isovalerate, and caproate concentrations were quantified with a gas chromatograph  according to established protocols [Concentrations of SCFAs were determined, as described previously ,16. Brierotocols ,18. In a® PowerFecal® Pro DNA kit  according to manufacturer’s protocols. This kit includes a bead beating step, and is designed to effectively extract genomic DNA from both Gram positive and Gram negative bacteria. The extracted DNA was quantified using a Qubit 4 fluorometer (Thermo Fisher Scientific) and PCR amplified for 16S rRNA gene metagenomics analyses using the protocol developed by Kozich et al. [The total DNA from cecal contents was extracted from 200 mg ± 20 mg of cecal digesta using the QIAamph et al. , which tQuantitative Insights Into Microbial Ecology 2  , and rep2PO4, 0.5 M K2HPO4, 0.00375 M NaN3, and 0.375 M KF; pH 7.4). Liver and pancreas tissue as well as cecal digesta were homogenized using a Bullet Blender tissue homogenizer  with 150 mg of 2-mm-diameter zirconium oxide beads (Next Advance) at setting 8 for 5 min. The breast muscle samples were homogenized with 150 mg of 6-mm-diameter steel beads for 10 min. using a Qiagen TissueLyser LT at 50 Hz (Qiagen Inc.). All of the samples were then centrifuged at 14,000× g for 5 min. Each supernatant was passed through a 3000 MWCO Amicon Ultra-0.5 filter  that had been rinsed with Millipore water 10 times immediately prior to use [g for 30 min. at 4 °C. From each sample, 360 μL of the filtrate was mixed with 140 μL of deuterium oxide containing 0.05% v/v trimethylsilylpropanoic acid (TSP) and 200 μL of metabolomics buffer ; TSP was used as a chemical shift reference for 1H-NMR spectroscopy. The solution was vortexed and then centrifuged at 12,000× g for 5 min. at 4 °C to pellet any particulate matter. Following centrifugation, a 550 μL aliquot of the supernatant was loaded in a 5 mm NMR tube, and then run on a 700 MHz Bruker Avance III HD spectrometer  for spectral collection.The samples (150 mg) were suspended in metabolomics buffer  were treated as a random effect. The mixed procedure of SAS  was used to analyze feed consumption and weight gain. Before analysis, the normality of variance was confirmed. Variables that were not independent  were treated as a repeated measure; the appropriate covariance structure was utilized according to the lowest Akaike’s Information Criterion. In the event of a significant main effect, the least squares means (lsmeans) test was used to compare the treatments within factors. p ≤ 0.05).Alpha diversity of the cecal microbiota was analyzed by pairwise comparisons of Kruskal–Wallis test. The beta diversity of the microbiota  was analyzed by pairwise permutational multivariate analysis of variance (PERMANOVA) . A BenjaMetabolomics analysis was performed using MATLAB (Math Works) and the Metaboanalyst R package . Spectrap < 0.001) over time, but there was no difference (p = 0.514) between the diet treatments (n = 28) and male (n = 26) birds differed over time (p < 0.001), and male birds were heavier (p ≤ 0.008) than female birds at day 21 and thereafter (p = 0.118) of the diet treatment on body weights.The rate of feed consumption increased (eatments A. Increaereafter B. There p ≥ 0.161) between the sexes and diet treatments , weighted UniFrac (p = 0.021), Jaccard (p = 0.001), and Bray Curtis (p = 0.001) analysis indicated that the structure of the cecal microbiota of broilers that were fed the CM diet differed from that of the control birds (phylum-family) abundance showed that Archaea-Methanobacteriaceae, Actinobacteria-Atopobiaceae, Bacteroidetes-Prevotellaceae, Firmicutes-Clostridiales D4 Family XIII, Firmicutes-Peptostreptococcaceae, and Proteobacteria-Succinivibrionaceae were more abundant (p ≤ 0.047), and Archaea- Methanocorpusculaceae, Bacteroidetes-D4 uncultured, Firmicutes-Christensenellaceae, Firmicutes-Clostridiales vadin BB60 group, and Proteobacteria-Desulfovibrionaceae were less abundant (p ≤ 0.041) in the cecal contents of broilers that were fed the CM-supplemented diet (Firmicutes-Peptostreptococcaceae (p = 0.005), there were no differences (p ≥ 0.118) between males and females.The composition of the microbiota was dominated by bacteria within the reatment A. Withinrominent B. Withinol birds . A quantted diet . With thp ≥ 0.549) in the cecal concentrations of SCFAs between males and females (p = 0.025), propionate (p = 0.013), butyrate (p = 0.018), valerate (p < 0.001), and total SCFAs (p = 0.017) were higher in the ceca of birds fed the CM diet. In contrast, there was no difference (p ≥ 0.069) in concentrations of isobutyrate, isovalerate, and caproate between the two diet treatments. A higher (p = 0.043) ratio of two carbon to three carbon SCFAs was only observed in female birds fed the CM diet.Dietary fiber was higher in the CM diet . There w females . Howeverp ≤ 0.007) in both male and female birds fed the CM diet included isoleucine, isobutyrate, glutamate, and 2-oxoglutarate  and male (195 significant bins) broilers A,B. Indilutarate A. Whereap < 0.001; Q2 = 0.63; R2 = 0.78), and in male chickens, nine bins were affected . In the liver of female chickens, 56 metabolite bins differed  between birds that were fed the CM and control diets  between diets  and 50  metabolite bins were altered between diets for female and male broilers, respectively. A number of specific metabolites were differentially abundant in the pancreas, liver, and breast muscle tissues of male and female chickens fed the two diets  are able to convert isobutryate to butyrate [Metabolomics was applied to obtain a direct functional readout of the physiological state of microbial activity  within tbutyrate . The low-product . In thisbutyrate ; thus, tThe pancreas has both exocrine and endocrine functions that are crucial in the digestion  and reguIn male birds, five metabolites that were involved in energy production were affected by the diet. Lactate is an important energy metabolite that is a product of glycolysis, an energy producing pathway, and it can also be re-used as an energy source by being re-converted to glucose via gluconeogenesis. The fact that both lactate and glycerol, which are also involved in gluconeogenesis, were less abundant in the pancreases of chickens fed the CM diet suggests that rates of gluconeogenesis were increased as the birds produced more glucose for energy. Creatine was observed to be more abundant in male birds that were fed the CM diet. Creatine is produced from arginine, glycine, and methionine in the pancreas , and it The liver is crucial for nutrition in chickens, including bile formation, the metabolism of carbohydrates, lipids, and protein, and storage of glycogen, fat, and fat-soluble vitamins. Moreover, the liver plays an essential role in the detoxification of ingested substances, and evidence of pathologic changes to liver that were linked to glucosinolates in RM have been observed previously . We obseProtein metabolism in the liver is linked to amino acid synthesis, including glutathione metabolism. Glutathione is upregulated, and it is an antioxidant that is used to scavenge free radical and reactive oxygen species (ROS). Moreover, glutathione is synthesized from glutamate, cysteine, and glycine. Thus, the reduced abundance of glutamate and corresponding decrease in abundance of glutathione suggests that the livers of birds fed the CM diet is either working more efficiently to protect the body from ROS, or it is having to work harder as a result of more ROS being present. The liver is involved in lipid metabolism, including glycerophospholipid metabolism, as indicated previously. The increased abundance of sn-glycero-3-phosphocholine and reduced abundance of acetylcholine in birds fed the CM diet are consistent with increased glycerolphospholipid metabolism; sn-glycero-3-phosphocholine is an upstream metabolite that is converted into choline and then to acetylcholine. Acetylcholine receptors are expressed throughout the liver and are connected to parasympathetic nerve fibers , and nicAlthough male broiler breast muscle tends to be larger and have higher nutrient requirements to reach larger mass , we did The inclusion of expeller-pressed CM in a broiler diet did not affect the feed consumption or weight gain of broiler chickens that were housed in a controlled setting. However, the CM diet influenced the structure of the cecal microbiota selecting for bacterial taxa involved in fiber catabolism, which was associated with the increased production of SCFAs. Even though the energy intake was equivalent for the two diet treatments, the metabolic profiles of cecal digesta, pancreas, liver, and breast muscle of birds fed the CM diet significantly differed from birds that were maintained on the control diet. Some of the altered metabolites were linked to the action of enteric bacteria. Others were linked to energy production, protection against oxidative stress, and changes to pathways of amino acid and glycerophospholipid metabolism. Importantly, the study demonstrated the power of using metabolomics to study diet impacts in poultry science, and to identify biomarkers of bird health that may be used as the baselines for evaluating chicken responses to diets, including those that are associated with potential adverse impacts of CM and other feed supplements. The combined metabolomic changes that were observed in the current study indicate that further investigation into the potential health effects of CM supplementation of diets on female and male broiler chickens in production settings is warranted.


Participants. 30 hemiplegic patients with middle cerebral artery acute infarction of the dominant hemisphere. Interventions. 30 patients were divided into 2 groups randomly. 15 patients in the treatment group (TG) were treated with ISSA, needling at the parietal midline (MS5) and left anterior/posterior parietal-temporal oblique lines (MS6 and MS7), combined with western routine treatment. While another 15 patients in the control group (CG) received routine treatment only. Main Outcome Measures. (1) Functional connectivity (FC): patients received brain scan using 3.0 T MRI after the treatment for 1 week. Based on the Matlab2012a platform, SPM12 software and DPABI software were used to process the scanning data and finally the functional connectivity of the brain was obtained. (2) National Institute of Health Stroke Scale (NIHSS) score.  A parallel-group randomized controlled trial.  The difference in the NIHSS score between the two groups of patients before and after treatment was statistically significant , indicating that TG had a better effect. Centered to the seed region of the left supplementary motor area (SMA) , FC increased at the left middle cerebellar peduncle, left cerebellum posterior lobe (uvula and declive), vermis, fusiform gyrus, lingual gyrus, inferior occipital gyrus, calcarine, cuneus, precuneus, BA7, BA18 and BA19, etc. Centered to the seed region of the left parahippocampal gyrus (PG) , FC increased at the left precuneus, inside-paracingulate, inferior parietal gyrus, paracentral lobule, BA5, BA6, BA7, and BA40, right median cingulate, precuneus, BA19, BA23, and BA31, etc. ChiCTR-IOR-15007672. It is indicated that ISSA can regulate the brain functional connection in patients with middle cerebral artery acute infarction in the dominant hemisphere and specifically strengthen the connections between visual, cognitive, motor control, and planning-related brain regions, which may be related to the recovery of movement in the mechanism. This trial is registered with  Characterized by high morbidity, disability, and mortality rate, stroke has been ranked as the leading cause of motor disability across the world. Ischemic stroke is the most common type of stroke accounting for 85% of the total. The Global Burden of Disease  showed tThe popularization of mature and appropriate treatment methods such as thrombolysis and thrombectomy can recanalize the patient's blood vessels in time which may turn the crisis into safety and improve the AIS's survival rate . HoweverAcupuncture is one of the traditional rehabilitation techniques. A relevant meta-analysis found that acupuncture can improve the motor function of the defect in acute stroke patients without obvious serious adverse events . Among tIn recent years, the development of medical imaging has provided a new means for the study of acupoint specificity , 8 and aFC analyzes the functional connectivity intensity between brain regions by detecting the temporal correlations of blood oxygen level-dependent (BOLD) signals between spatially distant brain regions. FC obtains the changes in the overall brain functions of stroke patients, which is regarded as one of the hot spots in the study of mechanism of the therapeutic effect of acupuncture. Zhang et al.  believedTherefore, in order to further clarify the specific effect of ISSA on brain functional connectivity in AIS patients, this study observes the specific changes of brain functional connectivity in AIS patients after ISSA intervention by Rs-fMRI besides the observation of its curative effect.The study was a parallel-group randomized controlled trial, approved by the Ethics Committee of the China-Japan Union Hospital at Jilin University, on July 18, 2016 , and has been registered at the Chinese Clinical Trial Registry (registration no. ChiCTR-IOR-15007672). Based on the complexity and particularity of fMRI image acquisition and data statistics of stroke patients, this paper estimates the sample size of the fMRI study on the acupuncture effect mechanism. Referring to Desmond et al.  and the Thirty patients with acute infarction in the middle cerebral artery supply area in the dominant hemisphere were recruited from January 2017 to December 2017. The patients were randomly assigned into the TG of ISSA (15) and the CG of routine treatment (15).The diagnostic criteria: the researchers adopt the relevant standards of the Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2014 [The patients received antiplatelet, lipid-lowering and plaque-fixed, temperature, blood pressure, and blood glucose controls, nutritional support, and other treatments according to the needs of the disease.a) were inserted into the depth of 30 mm at the angles of 15–30° with respect to the bottom of the epicranial aponeurosis. After Deqi, the needles were twisted at a frequency of 200 turns per minute, causing a distending sensation. Twisting was repeated at 10-minute intervals during the 30-minute period. Two times a day for six days as one course, the trial was finished after a course of treatment.According to the International Standardization Scheme for Acupuncture Points of the Scalp –17, we sPatients of two groups were treated with routine medicine. TG patients were also treated by the same doctor who has obtained the license of the medicine practitioner and has been engaged in acupuncture clinical work for more than 3 years.b) before and after the treatment course. We conducted T1-weighted magnetization-prepared rapid acquisition of gradient-echo and fMRI-BOLD echo-planar images. Scanning precautions and the fMRI-BOLD scanning parameters are given in Brain scans were acquired using the MRI system  and comparable  . The genmparable .Compared to the NIHSS scores and motor function scores in NIHSS before and after treatment of the two intragroups, there were statistical differences, which indicated that 2 kinds of treatment had a certain effect in treating AIS and improving the motor function. Comparison of the NIHSS scores and motor function scores before and after treatment of the TG with the CG showed there was not any statistical difference. Comparison of the differences of NIHSS scores and motor function scores before and after treatment of the 2 groups showed that the former had a statistical difference, while the latter had no statistical difference. The results indicated that the complementary ISSA might have a better effect than routine therapy Tables .Centered to the seed region of the left SMA , FC increased at the left precuneus extending to left cuneus and BA19, left lingual gyrus extending to calcarine, and also increased at the left middle cerebellar peduncle, left cerebellum posterior lobe (uvula and declive), vermis, fusiform gyrus, inferior occipital gyrus, cuneus, precuneus, BA7, BA18, and BA19, etc. Centered to the seed region of the left PG , FC increased at the right median cingulate and inside-paracingulate (BA23) extending to precuneus, also increased at the left precuneus, inside-paracingulate, inferior parietal gyrus, paracentral lobule, BA5, BA6, BA7, and BA40, right BA19, BA23 and BA31, etc.  form the visual association cortex, while the calcarine is the main focus of the visual cortex, jointly involved in visual processing. Moreover, the cuneus is an important part of the visual cortex that regulates the vestibular-ocular reflex. The fusiform gyrus is the most important part of the “what” pathway for object category recognition , 26 and The precuneus (BA7) is adjacent to the somatosensory cortex and visual cortex in the sagittal position and has three main task domains . The antThe SMA is an important key of the motor system, which plays a considerable role in the planning, execution, and control of complex motor functions . SMA-proSensorimotor network (SMN) related brain regions: SMN is primarily concerned with motor planning, motor execution, and sensory processing and is responsible for sensorimotor integration , 31. TheVisual and tactile brain regions: BA19 is involved in visual processing. BA5 and BA7 form the somatosensory junction cortex that integrates various senses such as tactile and pressure or utilizes previously stored sensory experience.Brain regions correlated with cognitive function: the precuneus is involved in many advanced cognitive functions as well as the integration of sensorimotor. The cingulate cortex is closely related to cognition and default network. The inside-paracingulate , which is part of the executive control network, has strong bilateral connections with the lateral prefrontal, parietal, anterior motor, and SMA, responsible for cognitive function.Visual motor control area: the median cingulate (BA24), also known as cingulate motor area (CMA), is an advanced neural network regulatory executive function. BA24 has projections into the anterior part of the SMA, the primary motor cortex, the motor thalamus, and the spinal cord , 36. StuThe PG is an advanced center of cognition and emotion. It includes many complex functions, such as memory, sensory representation, spatial orientation, and object recognition , 30. TheIt showed that ISSA may stimulate the motor cortex of the brain by promoting the integration of perception and sensory information and establish the connection with cognition.The neural process by which the brain controls spontaneous movement can be divided into three stages : firstlyThere were some shortcomings in the lack of observed sample size in this study: the subject of study was limited to the patients with ischemic stroke in the left middle cerebral artery, with non-life-threatening acute phase; being required to complete 6 days of ISSA treatment; received 2 times of fMRI scans before and after treatment. Therefore, only a small number of samples was included, which needed further study.It was preliminarily found that, in the treatment for the patients with middle cerebral artery acute infarction in the dominant hemisphere, the application of ISSA could specifically strengthen the connections between visual, cognitive, motor control, and planning-related brain regions, which might relate to the recovery of voluntary movement.MS5 is located at the mid-sagittal line between Baihui (DU20) and Qianding (DU21), which are, respectively, located at 5 cun and 3.5 cun posterior to the midpoint of the anterior hairline. MS6 is located at the line joining EX-HN1(A), which is 1 cun anterior to DU20, and Xuanli (GB6), which is three quarters down the side of the hairline between Touwei (ST8) (0.5 cun above the corner of the hairline) and Qubin (GB7) . MS7 is located at the line joining DU20 and GB7.Each participant was laid supine on the examination bed with their head fixed in a coil. The researchers reduced the influence of visual and auditory stimulation by fitting the participants with eye masks and earplugs. The subjects were asked to avoid any mental activity. A single provocation echo-planar imaging sequence is in 8 mins, the repetition time is 2000 ms, echo time = 30 ms, flip angle = 90°, slice thickness = 3.5 mm, gap = 0.7 mm, voxel = 3.5 mm × 3.5 mm × 3.5 mm, field of view = 224 mm × 224 mm, phases per location = 240, matrix = 64 × 64, and number of slices = 37.https://www.fil.ion.ucl.ac.uk/spm/) and DPABI software (http://rfmri.org/dpabi) in Matlab 2012a. Preprocessing included slice time correction, motion correction, normalising, nuisance covariates regression , detrending, and filtering. Then, the researcher selected 116 brain regions in the AAL as seed regions. All seed regions were analyzed for consistency with the whole brain data to calculate the FC values of different seed regions in the two groups of patients. Using the DPABI software, with gender, age, last scanning time, systolic blood pressure, diastolic blood pressure, and head movements as covariates, two sets of abovementioned FC data were analyzed by two independent sample t tests to obtain statistical parameter maps. The statistical parameter map was identified and corrected to obtain the anatomical position and activation intensity of the brain region with significant difference in two groups' FC values of the different seed regions . Significant postintervention changes in functional connectivity based on anatomical knowledge and clinical experience were verified by experienced neurologists.Data were preprocessed using statistical parametric mapping 12 software (