HER2 amplification and TP53 and APC mutation rates were lower, whereas SMAD4, PIK3CA, ACVR2A, KMT2D, LRP1, TGFBR2, GRIN2A, BRAF V600E, PTEN, and BRCA2 mutation rates were higher in MC than in AC. The mutation frequencies in MAPK, PI3K, and TGF- pathways were higher, whereas those of cell cycle proteins and Wnt were lower in MC and AMC than in AC. The proportion of hypermutated tumors was significantly higher in MC and AMC than in AC. As MC has a distinct molecular signature from AC, immunotherapy can be potentially applied in treating MC. Similar molecular profiles of AMC and MC suggest that treatment strategies for MC, but not AC, can be used for AMC treatment.Colorectal mucinous carcinoma (MC) is associated with inferior prognosis and response to treatment compared to adenocarcinoma (AC). The molecular landscapes of MC and adenocarcinoma with mucous composition (AMC) are not well-defined. We aimed to describe the genomic landscape of MC and AMC in a large colorectal cancer cohort. Tumor samples from patients with MC, AMC, or AC were analyzed using next-generation sequencing. MC had a molecular signature distinct from that of AC; genomic features were similar between AMC and MC but not between AMC and AC.  According to the 2018 global cancer statistics released by the International Cancer Research Institute of the World Health Organization (WHO), colorectal cancer (CRC) has the third highest incidence rate and second highest mortality rate, and an increasing annual prevalence rate , 4. MC cMC is clinically more prevalent among women, frequently located in the proximal colon, and associated with young age, high malignancy grade, tumor infiltration, lymph node metastasis, and peritoneal metastasis , 7. CompColorectal MC has unique molecular characteristics. Most early studies focused on protein expression levels and reported that MUC2 and MUC5AC are upregulated in MC tumors , 11. RecIn this study, we aimed to perform comprehensive targeted next-generation sequencing (NGS) to detect the two pathological subtypes of CRC, MC and AMC, and gain deep insights into their molecular characteristics, through the evaluation of the landscape of genetic alterations, pathway analysis, and analysis of biomarkers for immunotherapy to provide a molecular basis for the establishment of a precise treatment strategy for MC and AMC.Tumor specimens of patients with CRC involved in this study from January 2018 to September 2019 were sent for NGS analysis. Of 2,115 patients with CRC, 1,226 with a confirmed pathological diagnosis of MC, AMC, or AC were selected and recruited. Patients with an uncertain diagnosis of the pathological subtype or those with other special pathological subtypes, such as signet-ring cell carcinoma, undifferentiated carcinoma, and squamous cell carcinoma, were excluded. Of 2,115 patients with CRC, 1,226 with a confirmed pathological diagnosis of MC, AMC, or AC were selected and recruited. Patients with an uncertain diagnosis of the pathological subtype or those with other special pathological subtypes, such as signet-ring cell carcinoma, undifferentiated carcinoma, and squamous cell carcinoma, were excluded. MC was defined as extracellular mucus secretion accounting for >50% of the tumor volume. AMC was defined that accounted for 50%. And AC was defined as tumor with no extracellular mucus secretion. All tumor tissues were assessed independently by two experienced pathologists before sample disposal to pathologically confirm the diagnoses.This study was approved by the Institution Review Board of the Sixth Affiliated Hospital of Sun Yat-sen University in accordance with the Declaration of Helsinki. Written informed consent was obtained from all enrolled patients.NGS analysis was carried out at OrigiMed , a College of American Pathologists-accredited and Clinical Laboratory Improvement Amendments-certified laboratory, using a 450-gene comprehensive assay . At leasAll types of genetic alterations, including single-nucleotide variant (SNV), short and long indels, copy number alterations (CNAs), and gene rearrangement, were called using a suite of bioinformatics pipelines. Analysis of SNVs and indels began with the alignment of raw reads to the human genome reference sequence (hg19) with the Burrows-Wheeler Aligner , followed by polymerase chain reaction (PCR) duplicates removal using MarkDuplicates algorithm from Picard . Local realignment and base quality recalibration for SNVs were performed using GATK  and subsequently called by MUTECT . The CNAs included: (1) amplification, defined as an increase in the number of gene segment copies by 8, and (2) homozygous deletion, defined as decrease of complete loss of gene segment copies in samples with 20% purity. To identify these alterations, tumor cellularity was estimated by allele frequencies of sequenced single-nucleotide polymorphisms (SNPs). For detection of gene rearrangement, aligned reads with abnormal insert size of 2,000 or zero bp were collected and used as discordant reads, that is, paired-end reads that could not be closely mapped to a genome reference, with each read of paired reads aligned to the same chromosomes or different chromosomes. Originally, the discordant reads with the distance less than 500 bp formed clusters were further assembled by fermi-lite to identify potential rearrangement breakpoints. The breakpoints were double confirmed by BLAT, and the resulting chimeric gene candidates were annotated. For germline mutations, common single nucleotide polymorphisms, defined as those from the dbSNP database (Version 147), at a frequency of more than 1.5% from the Exome Sequencing Project 6500 (ESP6500), or at a frequency of more than 1.5% from the 1000 Genomes Project, were excluded. Furthermore, the variant allele frequency was adjusted with tumor purity estimated using FACETS.The TMB was estimated using the method of Chalmers etal. . In briet-test and non-normally distributed data were analyzed using the Wilcoxon rank test. All tests were two-tailed and significance was defined as a P value less than 0.05. All statistical analyses were performed using R software (Version 3.4.2).Qualitative variables were assessed using Fishers exact test. Normally distributed quantitative data were analyzed using the vs. 59 years, P = 0.037), and the incidence of MC in the right colon was higher than that of AC . Patients with MC accounted for a larger proportion of patients with stage III CRC (44.2%) than AC  and AMC ; but for AMC, the difference is only marginally significant. Furthermore, AMC was significantly more common in the right colon than AC was . No significant differences were observed between AMC and AC with respect to other clinical features.We defined MC as adenocarcinoma with mucous composition greater than 50% and AC as adenocarcinoma with no mucous composition. Adenocarcinoma with mucous composition but less than 50% is called AMC. KRAS (55.8%), TP53 (53.5%), APC (46.5%), SMAD4 (34.1%), ACVR2A (28.7%), PIK3CA (28.7%), KMT2D (22.5%), LRP1 (21.7%), TGFBR2 (20.2%), and ARID1A (19.4%)  and APC  displayed a significantly lower mutation rate, whereas SMAD4 , PIK3CA (28.7 vs. 19.2% P = 0.014), ACVR2A , KMT2D , LRP1 , TGFBR2 , and GRIN2A  displayed a significantly higher mutation rate in MC than in AC.Comprehensive targeted NGS revealed that the top 10 prevalent mutations in MC were Furthermore, pathway analysis revealed that the mutation frequencies in MAPK, PI3K, and TGF pathways were higher, whereas those of cell cycle proteins and the Wnt pathway were lower, in MC and AMC than in AC Figure 2BRAF V600E , PIK3CA , PTEN , and BRCA2  were significantly higher in MC than in AC. Although HER2 mutation rates were comparable between MC and AC , HER2 amplification occurred at a rate of 2.1% in AC but was not detected in MC or AMC. The mutation rate of KRAS was significantly higher in AMC than in AC ; however, it did not significantly differ between MC and AC or MC and AMC. The mutation frequencies of clinically actionable genes in MC, AMC, and AC are summarized in The mutation pattern of clinically actionable alterations in MC was different from that in AC but similar to that in AMC. The mutation rates of ETV6-NTRK3 fusion and the tumor was identified as MSI-H. NCOA4-RET and FGFR2-PIBF1 fusions were observed in patients with MC and AMC, respectively. The frequency of druggable fusions did not significantly differ among the three CRC pathological subtypes.Gene fusions in receptor tyrosine kinases have been recently identified as druggable targets in CRC . One patPOLE mutations that result in a dramatic TMB elevation. In general, the proportion of hypermutated tumors was significantly higher in MC and AMC than in AC .We defined hypermutated tumors as MSI-H tumors or those harboring vs. AC 6.8%, P < 0.001; AMC 17% vs. AC 6.8%, P = 0.001) and comparable between MC and AMC. Although the percentage of all POLE mutations among the three subtypes did not differ significantly, the proportion of POLE mutations resulting in a high TMB in MSS tumors was significantly higher in MC than in AC . The median TMB and median number of somatic mutations were also significantly higher in MC and AMC than in AC  than that in cases of POLE mutations in the exonuclease domain . Furthermore, POLE mutations detected in three patients harboring MSI-H tumors were not present in the exonuclease domain. Details of clinical and molecular characteristics of MC with POLE mutations are summarized in We further evaluated the relevant immunotherapy indicators in MC, which revealed that 29 of 129 patients harbored MSI-H tumors, among which three harbored utations Figure 4This study identified the comprehensive genomic features of MC and AMC by targeted NGS using a large cohort of patients with CRC. We comprehensively compared genetic differences between MC, AMC, and AC and identified the following major features. In general, MC had a molecular signature that was distinct from that of AC. The genomic features were similar between AMC and MC but different between AMC and AC. MC had a distinguished mutation pattern for prevalent gene mutations and biomarkers used clinically for CRC. Most importantly, the proportion of hypermutated tumors in MC and AMC was significantly higher than that in AC, indicating the higher applicability of immunotherapy for patients with these histological subtypes. Our results support developing more tailored treatment strategies for patients with CRC according to an individuals histological subtype.SMAD4, GNAS, BRAF, and KRAS occur at high frequencies in MC, whereas TP53, APC, and NRAS mutations are less common  did not significantly differ among the three groups. The SMAD4 mutation frequency was significantly higher in MC and AMC than in AC. Patients with a SMAD4 deletion have worse relapse-free survival and are resistant to chemotherapy with 5-fluorouracil  is more frequent in high-stage MC. The observation suggests that the variation in the mutation rates among the three cancer types is attributed to the different clonal evolution processes, from which MC arises as a unique subtype.Previous studies have suggested that mutations in s common , 18. Thefindings , 20. Patrouracil . MCs werHER2 mutation. In patients with CRC, nearly half of HER2 alterations are mutation rather than amplification or protein overexpression. Herein, HER2 amplification was not observed in MC. However, a proportion of patients with MC harbored HER2 mutations. Previous animal experiments have reported that the growth of implanted tumors harboring mutant HER2 can be inhibited by HER2 inhibitors, including trastuzumab, lapatinib, and afatinib, alone and in combination with trastuzumab and tyrosine kinase inhibitors  have recently been widely used in solid and hematological malignancies . We defiy in CRC , 27. Pemy in CRC , 29. Recy in CRC . MC is sy in CRC . The proPOLE mutations accompanied by dramatic TMB elevation, owing to the loss of DNA replication fidelity caused by POLE mutations in the exonuclease domain  was 27.9% in MC, suggesting that up to 30% of patients with CRC MC may benefit from immunotherapy. Furthermore, the mutation pattern of POLE differed between MSS and MSI-H tumors and POLE mutations occurring in the exonuclease domain markedly increased the TMB in MSS tumors.Immunotherapy in MSS CRC tumors still lacks efficacy; therefore, there is an urgent need to identify biomarkers for immunotherapy in MSS tumors. Hypermutation in MSS CRCs is often associated with e domain . Wang etreatment . In thisWe acknowledge that the current study had several limitations. First, the retrospective study design could not exclude selection bias. Second, the clinical data of patients treatments and outcomes were not controlled and collected in the current study; therefore, the clinical impacts of our findings need further confirmation. Finally, on the potential benefit of ICI, the effects of tumor infiltrating lymphocytes need to be evaluated along with the mutational profile as well as MSI and POLE statuses; whereas in the current study still lacks the pathological data for tumor immune microenvironment. On the other hand, the somatic mutational landscape is also affected by the host immune environment, hence serve as a proxy to the activities of the immune cells.n = 129) and AMC (n = 100) via a comprehensive targeted NGS panel, our results reveal the molecular landscapes of MC, AMC, and AC, which could lead to tailored treatment for different histological subtypes of CRC. The selection of baseline clinical and pathological characteristics was relatively intact in this study, allowing the analysis of clinical and genomic features. Our findings shed new light on the treatment and management of patients with MC and AMC. Further prospective studies in patients with MC and AMC are warranted to validate our findings, especially regarding the potential use of immunotherapy.In spite of the limitations, using the large cohorts of MC .TL, FW and WW were employed by OrigiMed.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proteobacteria and Bacteroidetes phyla. Functional potential analyses show broadly convergent patterns, contrasting with their great taxonomic variability. As-related metabolism, as well as other functional categories such as those related to the CH4 and S cycles, differs among the five communities. Particularly, we found that the distribution and abundance of As-related genes increase as the As concentration rises. Approximately 75% of the detected genes for As metabolism belong to expulsion mechanisms; arsJ and arsP pumps are related to sites with higher As concentrations and are present almost exclusively in Proteobacteria. Furthermore, taxonomic diversity and functional potential are reflected in the 12 reconstructed high-quality metagenome assembled genomes (MAGs) belonging to the Bacteroidetes (5), Proteobacteria (5), Cyanobacteria (1), and Gemmatimonadetes (1) phyla. We conclude that SH microbial communities are diverse and possess a broad genetic repertoire to thrive under extreme conditions, including increasing concentrations of highly toxic As. Finally, this environment represents a reservoir of unknown and undescribed microorganisms, with great metabolic versatility, which needs further study.Microbial communities inhabiting extreme environments such as Salar de Huasco (SH) in northern Chile are adapted to thrive while exposed to several abiotic pressures and the presence of toxic elements such as arsenic (As). Hence, we aimed to uncover the role of As in shaping bacterial composition, structure, and functional potential in five different sites in this altiplanic wetland using a shotgun metagenomic approach. The sites exhibit wide gradients of As (9 to 321 mg/kg), and our results showed highly diverse communities and a clear dominance exerted by the IMPORTANCE As microbial communities inhabiting extreme environments are fundamental for maintaining ecosystems, many studies concerning composition, functionality, and interactions have been carried out. However, much is still unknown. Here, we sampled microbial communities in the Salar de Huasco, an extreme environment subjected to several abiotic stresses . We found that although microbes are taxonomically diverse, functional potential seems to have an important degree of convergence, suggesting high levels of adaptation. Particularly, arsenic metabolism showed differences associated with increasing concentrations of the metalloid throughout the area, and it effectively exerts a significant pressure over these organisms. Thus, the significance of this research is that we describe highly specialized communities thriving in little-explored environments subjected to several pressures, considered analogous of early Earth and other planets, that have the potential for unraveling technologies to face the repercussions of climate change in many areas of interest. Extreme environments such as high-altitude wetlands select for adaptations in bacterial communities that enable them to thrive. This particular and fragile environment resembles life before the oxygenation of Earth and could serve as a model for studying life on other planets. Moreover, microbial communities are critical for maintaining biogeochemical cycles, particularly in extreme environments where there is little presence of other life forms. The Salar de Huasco (SH), a high-altitude wetland located in the Chilean Altiplano  at 3,800 masl (meters above sea level), is a Ramsar protected site, considered a hot spot for microbial life 38.aioA, arrA, arsM, and arxA are less common in the environment than acr3, arsB, arsC, and arsD , such as As, is a significant selection pressure and thus one of the main drivers of the composition of microbial communities . Thus, m20–In this study, we tested different established sites in the SH regarding their taxonomic and functional heterogeneity. We focused on As-related genetic elements as well as other relevant metabolic functions. Increasingly, such analyses enable the identification of relatively small subsets of markers associated with a particular ecologically important function. Additionally, their identification and the dynamics of bacterial communities are potentially useful bioindicators for monitoring ecosystem health , 24. TheThe Salar de Huasco possesses an important level of variation within a relatively small area (the maximum separation of sampling sites is 5.9 km), including daily oscillations in a wide range of environmental parameters (temperature and humidity) and others which vary in spatial gradients, i.e., salinity and As , 26 Fig; see alsProteobacteria and Bacteroidetes are the most prevalent phyla in the sampled sediments, which are particularly enriched in the H3, H4, and H5 communities, accounting together for >60% of all observed taxa ; it also has the highest abundance of Firmicutes, Patescibacteria, and Spirochaetes phyla as well as significantly lower proportions of Actinobacteria. Moreover, the H1 community profile presents the highest abundance of Chloroflexi, Actinobacteria, Verrucomicrobia, Planctomycetes, and Acidobacteria phyla. On the other hand, H3, H4, and H5 communities are more similar to each other, despite the vast differences in low-abundance taxa, many of which are exclusively present in only one community (see Table S2).The substantial differences between the SH sampling sites are reflected by the taxonomic compositions of bacterial communities, which showed contrasting patterns and variation between sites . Overallved taxa . In turnProteobacteria genera Roseovarius and Desulfotignum in the H3, H4, and H5 communities  detected, many were not classified within any known phylum (6.5%) or genus (58.3%). Nevertheless, Halomonas and Marinobacter genera were detected, both of which are culturable and seem to be recurrent in the SH . Also, most of the highly connected nodes in the network belong to unknown or undescribed taxa.The most abundant bacteria at the lowest (available) taxonomic rank reflect the same pattern, with a great abundance of the n the SH . Of the As a whole, the beta diversity analysis shows that the dispersion patterns between the communities correlate with the composition analysis. Hence, the taxonomic compositions of H3, H4, and H5 metagenomes are more similar between each other and distinctive from those of H0 and H1, producing three very well-defined groups . This coThe SH metagenomes we analyzed represent the compositions in surface sediments of 5 different sites with a great variation in As content (from 9 to 321 mg/kg). The 337.5 million quality-controlled reads were coassembled and yielded a total of 994,545 contigs  corresponding to 2.38 million genes, which were used to generate abundance/distributions and functional profiles of the 5 communities under study . The resCyanobacteria, and the Calvin Benson cycle. However, when assessing the ability of these bacterial communities to carry out necessary reactions to sustain key biogeochemical cycles, we found that S, N, and CH4 showed significant scores  for all five metagenomes, implying that the necessary metabolic pathways and machinery are present and complete in bacteria isolated from all environments . We also found representatives of several pathways, including sulfur reduction and oxidation, nitrate fixation and reduction, methanogenesis, and methane oxidation, and the utilization of a wide range of the available nutrients. Particularly, a significant difference is present in the CH4 cycle, which is much more enriched in the H1 and H0 communities; consequently, we only detected methanogens and methane oxidizer taxa in these sites. Conversely, a wide range of sulfur-associated pathways were detected in all sampled sites , suggesting its importance in environments where volcanic-derived sulfur oxidation products leach and flow into the salt flats . Particularly, the five SH communities showed specific differences in some categories such as As resistance (SEED subsystems 3), following the same tendency previously mentioned, where H0/H1 metagenomes are more similar than H3/H4/H5, with 0.21% to 0.22% and 0.26% to 0.27% of reads recruited by this category, respectively. Other significant categories in the communities were carbon, nitrogen, and phosphate metabolism, zinc, nickel, cobalt, iron, and manganese transport, osmotic stress, the circadian clock in lt flats . NonethearsR regulator, the arsC reductase and the acr3 pump were the most overrepresented genes, indicating that the As(V) reduction and subsequent As(III) expulsion strategy is the most prevalent one used by bacteria that inhabit the SH. Moreover, arsM was among the most abundant genes, particularly enriched in the H3, H4, and H5 metagenomes. Nevertheless, genes related to oxidation and respiration mechanisms were less abundant yet present an interesting distribution; those related to oxidation  are enriched in H3, H4, and H5, while those related to respiration (arrA and arxR) are enriched in H0 and H1.To gain a broader view on the communities’ functional potential related to As metabolism or resistance/tolerance, we determined the abundance of the genes belonging to the known mechanisms . Again, acr3 , was more abundant in the sites with higher As concentrations. Moreover, the higher acr3 variants and abundance could be because this pump is present in a wider number of bacteria phyla, covering most of the diversity observed  ; the same tendency was observed for the GC content (36.1% to 69.4%). On the other hand, most of the reconstructed MAGS were represented or detected in the H3, H4, and H5 metagenomes, showing differential abundance patterns in each site  to gain insights into nonculturable bacteria. This resulted in 19 metagenome-assembled genomes (MAGs) that met the completion of ≥80% and redundancy of ≤10% criteria ; togethenadetes) . We wereBacteroidetes  clustered together, as well as the Deltaproteobacteria  and Gammaproteobacteria (MAG129 and MAG144). On the other hand, the only Cyanobacteria (MAG29) is close to the Gammaproteobacteria and the only Gemmatimonadetes (MAG3) is close to the Bacteroidetes. Particularly, MAGs displayed a considerable number of genes regarding As metabolism, where the same tendency shown before was observed, with a dominant presence of genes related to As expulsion  throughout all taxa, followed by reduction (arsC), methylation (arsM), and oxidation (arsH) mechanisms , while it can also be associated with calcium carbonate (CaHAsO4) or adsorbed into Fe and Al oxides , whi, whi8), 30–l oxides . The metl oxides . It is hProteobacteria, Bacteroidetes, and Cyanobacteria  have been cultured and isolated under laboratory conditions , which correlates with the findings reported in the Argentinian Altiplano  gene was also consistently enriched. Thus, these markers could be proposed as bioindicators of As contamination, such as acr3 , improving our understanding of microbial communities  reduction and subsequent As(III) expulsion systems are the most common strategy used to detoxify the cell of As. Furthermore, regarding expulsion pumps, the most abundant was Acr3, followed by ArsB; however, in sites with high As concentrations, ArsP becomes enriched. In addition, 12 high-quality nonredundant MAGs were reconstructed from the metagenomes. These represented the dominant diversity detected across the communities as well as the metabolic variability and the presence of marker genes related to the most recurrent As resistance mechanisms . Finally, in order to further elucidate the strategies and relationships between the microbial taxa and among biotic and abiotic components within the ecosystem, further multidisciplinary studies are required as well as the use of the ever-evolving next-generation sequencing (NGS) approaches. Together with more precise database information, we will be able to better understand the evolutionary process of adaptation to the extreme conditions present in these unique ecosystems.The Altiplano array of ecological niches is a reservoir for microbial diversity, showing great richness in adapted organisms capable of facing these challenging conditions. Particularly, the Salar de Huasco is a highly diverse ecosystem, where salinity and As concentrations contribute to shaping the community composition, mainly represented by in situ  lakes with salinity and As gradients from north to south . In June260/280) ratio, and fluorescence using a Qubit 3.0 fluorometer along with the Qubit dsDNA HS assay kit . Next, paired-end (150 bp) libraries were constructed for each sample in duplicates at the Centro de Biotecnología Vegetal, Universidad Andrés Bello  using the TruSeq Nano DNA kit  according to the TruSeq Nano DNA sample preparation guide 15041110 revision D. Libraries were sent for sequencing at Macrogen Inc.  on a HiSeq 4000 platform (Illumina Inc.). Then, raw data were evaluated using FastQC v0.11.8  according to the manufacturer’s instructions. DNA integrity, quality, and quantity were verified through 1% agarose gel electrophoresis, optical density at 260 nm to optical density at 280 nm  a coassembly , (ii) its annotations , and (iii) an alignment per sample , we proceeded to assemble the quality-controlled reads from all samples using MEGAHIT v1.1.3 , with th4, and Fe biogeochemical cycles in each metagenome using the predicted proteins .We followed the anvi’o v7 pipeline . First, To estimate the abundance of genes related to As metabolism, the read counts for each predicted gene of each sample were obtained using the corresponding BAM file and the coassembly annotated GFF file, with HTSeq-Counts v0.13.5 . Also, wThe contigs in the metagenome profile were clustered through anvi’o v7  using thWe define MAGs (metagenome assembles genomes) as bins with a completion of >80% and redundancy of <10%. Then, we used CheckM v1.0.13  for a moAll MAGs were queried against the same previously constructed database (with the As metabolism genes) using CRB-BLAST v0.6.6  E value to connePRJNA573913.The whole raw data sets and the metagenome assembled genomes have been deposited at DDBJ/ENA/GenBank under the BioProject accession number


Background: Medroxyprogesterone and donepezil could be used as respiratory stimulants in ventilated patients. However, no randomized placebo-controlled trial is available to confirm this approach and compare these drugs. The aim of the current study was to evaluate the effects of donepezil or medroxyprogesterone compared to the placebo in improvement in respiratory status and weaning facilitation in critically ill adult patients receiving mechanical ventilation.Material and Methods: This randomized, triple-blind trial was conducted on 78 ventilated patients in intensive care units (ICU). Patients who were intubated due to pulmonary disorders were ruled out. Patients were randomized in a 1:1:1 ratio to receive 5 mg donepezil (n = 23) or 5 mg medroxyprogesterone (n = 26), or placebo (n = 24) twice a day until weaning (maximum 10 days). The primary endpoints were weaning duration, and duration of invasive mechanical ventilation. Secondary endpoints included rate of successful weaning, changes in arterial blood gas (ABG) parameters, GCS and sequential organ failure assessment (SOFA) score, hemoglobin (Hgb), ICU-mortality, and duration of ICU stay, were measured before and after the intervention and if successful weaning was recorded.Results: Of 78 studied patients who were randomized, 59 weaned successfully. 87% patients in donepezil and 88.5% patients in medroxyprogesterone groups were successfully weaned compared to 66.7% patients in the placebo group. However, this difference was not statistically significant . Changes in pH, mean duration of intubation, and weaning duration were statistically different in donepezil compared with the control group . No significant difference in ABG, Hgb, GCS and SOFA score, and duration of intubation were seen in the medroxyprogesterone group, but weaning duration was significantly reduced to 1.429 days compared with the control group .Conclusion: The results of this clinical trial have demonstrated that the administered dose of medroxyprogesterone and donepezil can expedite the weaning process by reducing the weaning duration compared to placebo. Furthermore, the total duration of invasive ventilation was significantly lower in the donepezil group compared to the control group. Future clinical trials with a larger sample size will determine the exact role of medroxyprogesterone and donepezil in mechanically ventilated patients.Clinical Trial Registration:https://irct.ir/IRCT20190810044500N2 . Mechanical Ventilation (MV) with positive pressure was first introduced clinically in the 1950s during the polio epidemic . AlthougAbout 20–30% of ventilated patients are considered difficult to wean from invasive MV . The timIn spite of insufficient data regarding the efficacy of respiratory stimulants such as caffeine and doxapram , researcProgesterone is a respiratory stimulant improving ventilation function, ventilation capacity, and exercise efficiency in normal individuals and obstructive airways diseases. Progesterone could enhance ventilation, breathing overnight and respiratory gas exchanges . The lipThe aim of the current study was to evaluate the effects of donepezil or medroxyprogesterone compared to the placebo in improving the respiratory status and weaning facilitation in critically ill adult patients with non-pulmonary etiologies receiving mechanical ventilation for the first time. In addition to weaning duration, and duration of invasive mechanical ventilation as our primary endpoint, we evaluated secondary endpoints, including successful weaning rate from MV, changes in arterial blood gas (ABG) parameters, GCS and sequential organ failure assessment (SOFA) score, hemoglobin (Hgb), ICU-mortality, and duration of intensive care units (ICUs) stay, were measured before and after the intervention and if successful weaning was recorded.This randomized triple-blind placebo-controlled clinical trial was conducted in Rahnemoon tertiary referral hospital affiliated to Shahid Sadoughi University of Medical Sciences, which is the neurological referral center of Yazd city. Recruitment was conducted from December 20, 2019, through July 22, 2020.2Sat) greater than 90%, Fraction of inspired oxygen (FIO2) less than 50%, and positive end-expiratory pressure (PEEP) less than 8 cm H2O, Hemodynamic stability during the last 12 h, discontinuation of sedatives or reduction of sedation for the past 48 h, neurologic stability with Ramsay score ≤5, and body temperature between 36 and 38 centigrade degrees (°C).Inclusion criteria were age older than 18 years, MV through the endotracheal tube for at least 24 h (hrs) prior to study enrollment, oxygen saturation  > 25 mmol/L, plasma creatinine >180 mmol/L or creatinine clearance <30 ml/min, or more than 25% increase in creatinine over the last 24 h; contrast injection in the last 6 hours; serum sodium level >150 mEq/L, serum potassium level <3.5 mEq/L, metabolic alkalosis with arterial pH > 7.5, GCS = 3, and concomitant administration of potential respiratory stimulants. Had not taken donepezil or medroxyprogesterone during the previous 14 days . We also excluded patients with known underlying lung disease as the severity of lung disease can influence the duration and success rate of the weaning.The anesthesiologist, ward nurses, and the data collector were all blinded to the intervention assignments throughout the study. Patients were randomized in a 1:1:1 ratio to one of three groups including medroxyprogesterone (n = 26) or donepezil (n = 26) or placebo (n = 26) based on a random number table. Before attempting to wean patients from MV, the necessary assessment were performed before conducting a spontaneous breathing trial. For intervention groups, medroxyprogesterone and donepezil were administered 10 mg/day (5 mg/BID) and patients in the control group received the placebo twice daily until weaning and for a maximum of 10 days. Weaning failure was considered as the need for tracheostomy or intervention for more than 10 days.For preparation of placebo tablets, the determined amount of lactose, microcrystalline cellulose, and stearic acid was weighted exactly, mixed with a tumbler mixer, and directly compressed with a single punch tablet press machine. The hardness and thickness of placebo tablets were adjusted similar to donepezil and medroxyprogesterone tablets. Placebo was prepared in the Pharmaceutics Laboratory of Shahid Sadoughi School of Pharmacy, Yazd. Donepezil and medroxyprogesterone tablets were purchased from Sami Saz and Iran Hormone pharmaceutical companies, respectively.2, PaCO2, HCO3, pH, and O2Sat), GCS, Hbg, and SOFA scores were recorded before the intervention, after 24 h, and if successful weaning happened.Demographic information, registry numbers, and dates of reception were recorded in the prepared questionnaire. The following variables including ABG , or invasive ventilation with low pressure support, for some cases supplemental oxygen through a T-piece connection applies, If the patient develops signs of poor tolerance, weaning is considered to have failed and mechanical ventilation is reinstituted .By applying permuted block randomization, we randomly allocated the eligible patients into one of three arms of the study. For this reason, we used blocks of four. The principal investigator, who was unaware of the interventions, gave one digit number to each prepared formulation from 1 to 78. The first eligible person was given to number 1, the second person as number 2, and so on. The anesthesiologist, the ward nurses, and the data collector were all blinded to the intervention assignments throughout the study. In the end, the prepared formulation were decoded and assigned to the proper arm of the study by the main principal.−3 standard deviation in donepezil and medroxyprogesterone groups of 0.3 and 7.4, respectively in order to reach a mean difference  of 4 with the following specifications and using the sample size eq.The sample size was estimated as 26 per group based on earlier experience and HCOα = 0.05; β = 0.2).t-test and Mann-Whitney, respectively. Moreover, paired t-test was used to compare changes of variables at groups over 24 h and repeated measurement to compare the changes of variables over time. Chi-square and Fisher’s exact tests were used to test to compare qualitative variables. Two-way ANOVA and General Liner model were applied to evaluate the simultaneous effects of medications, time and variables changes. All the statistical analysis was conducted by Statistical package for social science (SPSS) software version 23 and two-tailed p-values < 0.05 were considered statistically significant.The quantitative and qualitative variables were reported as mean ± standard deviation (SD) and number (frequency), respectively. The normality of the data was checked by Kolmogorov-Smirnov test. The distributed quantitative variables were compared between groups by using the independent sample p-Value = 0.015).In total, 152 patients were screened for eligibility in three neurological ICUs of our hospital. We enrolled 78 intubated patients without primary pulmonary diseases. The mean age of the participants was 41.23 ± 24.2 years old. The total intention to treat population were 73 patients who randomly assigned to the following groups: 26 patients each assigned to the control, donepezil, or medroxyprogesterone groups. Five patients were withdrawn from the study subsequently. Eventually, 73 patients completed the study for outcome measurement . Patientp-Value = 0.02).The mean (SD) of total duration of invasive ventilation was 142.8 (47.4) hrs in the medroxyprogesterone and 124.8 (51.36) hrs in the donepezil groups vs. 176.4 (64.8) hrs in the control group, for a between-group difference of 33.6 h for medroxyprogesterone group and 51.6 h for donepezil compared to the control group. As presented in p-value = 0.793). The duration of weaning was a mean (SD) of 98.8 (46.8) hrs in the control group vs. 64.5 (42.96) hrs and 56.4 (35.76) hrs for medroxyprogesterone and donepezil respectively, for a between-group difference of 34 and 42.4 h in the donepezil and medroxyprogesterone groups, respectively. This value did significantly in the groups   .p-Value = 0.111).From 73 enrolled patients who were randomized in 1:1:1 ratio to one of three groups, 16 (66.7%) patients in control, 23 (88.5%) patients in medroxyprogesterone, and 20 (87%) patients in the donepezil group experienced successful weaning during the study period. However, the rate of successful weaning were greater in the donepezil and medroxyprogesterone compared to the control group this difference was not statistically significant by Chi-Square test . The rate of ICU mortality was not different between groups, as 4 (25%) patients in control, 5 (21.7%) in medroxyprogesterone, and 3 (15%) patients in the donepezil group expired during study follow-up .The comparison between group differences was done using the ANOVA and Tukey HSD test. In successfully weaned patients, the total duration of ICU stay was the mean (SD) of 453.6 (232.8) hrs in the medroxyprogesterone and 352.8 (180) hrs in the donepezil groups vs. 434.4 (170.4) hrs in the control group, for a between-group difference of −19.68 and 79.2 h for the medroxyprogesterone and donepezil groups respectively. However, this value did not differ significantly between the groups . Despite the upward trend in the profiles of the GCS over time in all three groups, no significant difference was observed between groups   .p-value = 0.453). The rate of ICU mortality in non-successfully weaned patients was 2 (25%) in control, and no patient in other groups .In non-successfully weaned patients, the total duration of ICU stay was on average 559.2 (276) and 758.4 ( 278) hrs in the medroxyprogesterone and donepezil groups in comparison to 532.8 (232.8) hrs in the control group, for a between-group difference of −24.0 h and −223.2 h, respectively. This value did not change significantly in the groups  parameters, O2Sat, Hgb, and SOFA score was done using the general linear model test. The results did not differ significantly after 24 h in non-successfully and overtime in successfully weaned patients. There were no significant differences in mean difference of variables between each group compared to the others  and 4.Although, the effects of hormones such as progesterone on respiratory and ventilatory performance have been evaluated in previous studies, the present study is the first randomized, controlled clinical trial evaluating the effectiveness of medroxyprogesterone in facilitation of weaning in patients receiving invasive mechanical ventilation. The effect of donepezil was previously assessed in a clinical study to facilitate the weaning course. However, in this non-randomized study, there was no control group and the effect of donepezil on ABG was evaluated in only 16 difficult to wean patients. According to the design of this aforementioned study, it had all the limitations of non-clinical trial studies. Therefore, this study is also the first randomized clinical trial of donepezil in expedition the weaning course in mechanically ventilated patients.This triple-blind randomized clinical trial designed to compare the improvement in respiratory status and weaning facilitation in mechanical ventilated critically ill adult patients receiving donepezil or medroxyprogesterone vs. placebo. The mean total duration of invasive ventilation was significantly lower in the donepezil compared to the control group while the duration of weaning did differ significantly between the groups. In addition, the rate of successful weaning were greater in the donepezil and medroxyprogesterone groups compared to the control group, this difference was not statistically significant. Donepezil and medroxyprogesterone had no significant effect on the total duration of ICU stay, rate of ICU mortality, and GCS in successfully weaned patients which was not too far from expected, since there are several factors in the ICU setting influencing these parameters and it is not possible to control for all these confounders. In fact, parameters such as age, chronic use of corticosteroid, atrophy of diaphragmatic, or severe limitation of airway flow are far more vigorous factors affecting the successful rate of weaning from mechanical ventilation or outcomes of critically ill patients . Taken tMechanical ventilation is implemented to improve oxygen delivery and ventilation, and decrease the breathing workload. It is by the physician to and is therefore a means of treating the patient. Physicians adopt MV to stabilize the patient until the diagnosis and therapeutic management of the disease is performed . In otheDespite the salvage of MV in critically ill patients, MV is associated with numerous complications. The most common complications are pneumothorax, broncho pleural fistula, nosocomial pneumonia, and respiratory alkalosis, which can impair cerebral perfusion, predispose patients to cardiac dysrhythmias, and prolong the weaning process . DifficuSome other agents such as methylphenidate, benzquinamide, ethamivan, bemegride, nikethamide, naloxone, almitrin, theophylline, methyl xanthine, levosimendan, and acetazolamide have been tried as the respiratory stimulants , but theThe efficacy of acetazolamide in the duration of intubation was evaluated as regards the results did not show reduction in intubation and weaning process .Donepezil and medroxyprogesterone at dose of 10 mg are well tolerated without causing any life-threatening side effect . Mechani2 reduction and PO2 increment  illustrated the effective role of medroxyprogesterone in PaCOncrement . It shou2, PaCO2, HCO3 concentration, and O2Sat parameters while contrary to Abbasi´s study pH changes were significant in this study. Although no significant different in pH changes were seen in the donepezil compared to control group after 24 h, changes in pH were significant overtime indicating that several doses of the donepezil are required for an efficient response. It can be argued that the results of ABG parameters such as pH will often influence the weaning process accompanied with better clinical outcome in such patients  as well as non-randomization limited the establishment of a strong relationship between use of donepezil and facilitation of weaning . Our respatients .We also evaluated the effect of donepezil and medroxyprogesterone on the improvement of the patient’s GCS. In the clinical setting, the improvement in GCS is one of the important criteria for initiation of weaning. In other words, low GCS at the beginning of the weaning process is considered a major reason for prolongation of this process . In this2 are two main indices for calculation of SOFA score. Due to the lack of changes in GCS and PaO2 in both groups compared to placebo, changes in this index were not significant between intervention and placebo groups, so these two drugs are not effective in reducing the SOFA score and mortality rate.SOFA is a valuable score for the management of patients in ICUs which predicts patients’ outcomes . GCS andDuration of weaning was significantly reduced in intervention groups compared to the control group. The underlying mechanisms for donepezil are presumed to be due to the performance of the cholinergic system in improving respiratory coordination, increased neuromuscular transmission, improved upper respiratory muscle function, neuronal control of respiration, and increased salivary secretion . The perThis study has several limitations. First, the main limitation was the small size of the subject groups. Despite more than 7-month study duration and inclusion of consecutive patients, only limited number of patients entered the study according to inclusion and exclusion criteria. Second, while the dose range of medroxyprogesterone was different in previous data, the effective dose for weaning from MV is yet to be determined. Although the interventions performed in this study were safe and no side effects related to these drugs were reported during the study period, but considering the relative short length of the intervention and follow-up period, the necessary precautions should be taken in this regard. Furthermore, the lack of a fixed and predefined protocol for all patients for whom weaning is to be attempted is another limitation which should be taken into consideration in future studies. The last limitation is the presence of factors more strongly influencing the discontinuation of MV or ICU outcomes .Various population pharmacokinetic parameters and contribution of different factors in weaning process might be the reasons for medroxyprogesterone or donepezil treatment to achieve its response goal during the studies. Therefore, these results require further confirmation with larger controlled clinical trials to clarify and establish medroxyprogesterone and donepezil role on weaning process. Moreover, other confounding factors that strongly influencing the patients’ outcome should be considered.Previous investigation found that some respiratory stimulants could facilitate weaning presses. Medroxyprogesterone and donepezil are two safe and successful respiratory stimulants, suggesting that they could be considered as a weaning-facilitating strategy. Our results illustrated that the administered dose of medroxyprogesterone and donepezil can expedite the weaning process presumably by central stimulation of the respiratory system.The results of the current study have demonstrated that administering medroxyprogesterone or donepezil in the weaning process significantly reduced the weaning duration compared to placebo, while the total duration of invasive ventilation was significantly lower in the donepezil group compared to the control group. There were no statistically significant differences in other evaluated outcomes except pH changes. Although the findings of the present study are interesting, care should be taken in implementing these results.


Lactobacillus was enriched in the gut microbiota of individuals with low FCRs (and thus high FE), while Prevotella abundance was significantly higher in individuals with high FCRs (and thus low FE). This may be linked to carbohydrate consumption and incomplete digestion. The activity of pathways involved in the metabolism of cofactors and vitamins was greater in pigs with lower FE. We also identified differences in pyruvate-related metabolism, including phenylalanine and lysine metabolism. This suggests that pyruvate metabolism is closely related to microbial fermentation in the colon, which in turn affects glycolysis. This study deepens our understanding of how gut microbiota are related to pig growth traits, and how regulating microbial composition could aid in improving porcine FE. However, these results need to be validated using a larger pig cohort in the future.To enhance pig production, feed efficiency (FE) should be improved; however, the mechanisms by which gut microbes affect FE in pigs have not been fully elucidated. To investigate the differences between the composition and functionality of the gut microbiota associated with low and high FE, microbial compositions were characterized using 16S rRNA sequencing, functional annotations were performed by shotgun metagenomics, and metabolomic profiles were created by GC-TOF-MS from female Landrace finishing pigs with low and high feed conversion ratios (FCRs).  Feed consumption is the largest variable expense associated with swine production, accounting for 50–85% of the total cost . TherefoTreponema, Methanobrevibacter, and Actinobacillus genera. These bacteria are enriched in the feces , and regulate the energy harvest and carbohydrate metabolism of the host –6. Gut mhe feces , 13, ilehe feces , 14, caehe feces , 16 and nd colon , 17. Somrobiotic . Additioobiotics , 19. Mosobiotics , with thobiotics –23. Hostobiotics , 25.Although many studies have been conducted on growth-related traits and gut microbiota, the results are sometimes conflicting. This may be due to differences in the studies animals and experimental conditions. However, a study that minimized such genetic, nutritional, and management diffienences also found multiple RFI-associated taxonomic differences, none of which were common to all geographic locations or batches within a location .An increased understanding of the community structure and functional capacity of the gut microbiota will help elucidate the interaction between microbial activity and host physiology and metabolism. Therefore, in this study, colonic microbiota sequencing was undertaken to investigate the difference between the colonic digesta microbiota of two groups (high and low FE) of female finishing Landrace pigs, with regards to community structure and composition, functions, and metabolites. Correlations between these bacterial population variables and pig production performance were then identified. We hope that our founding will allow for a greater understanding of the microbial activity and material digestion that occurs in the large intestine, and how this affects the growth traits of pigs.ad libitum from automated individual feeding systems , that recorded the feeding behaviors of each of the pigs, including the daily feed intake and daily body weight gain. All experimental pigs were healthy and antibiotic-free during the study period.In this study, we utilized 120 purebred female Landrace pigs, provided by Tianjin Ninghe Primary Pig Breeding Farm . All experimental pigs were weaned at the age of 28 days and were raised under similar feeding management regimes. When a pig's body weight reached 30 kg, it was transferred to the fattening room, where 10 pigs were housed in each pen. During the study, all experimental pigs were fed the same commercial formula diet and were kept under controlled farm management conditions. The feed was mainly composed of corn, soybean meal, lysine, and calcium hydrogen phosphate. This was available Two groups of 20 pigs were chosen for a further assessment based on feed and weight gain data obtained between 120 and 165 days: the HFCR group (low FCR and thus high FE) and the LFCR group (high FCR and thus low FE). The FCR values for these groups were significantly different . At 166 All experimental procedures described in this study were approved by the Animal Welfare Committee of China Agricultural University (Permit Number: DK996) and conducted under the approved slaughtering guidelines (GB/T 17236–2008) of the Quality Supervision, Inspection, and Quarantine Committee of the People's Republic of China. All efforts were made to minimize animal suffering during the study.A QIAamp DNA Stool Mini Kit  was used to extract the fecal and colonic digesta DNA following the manufacturer's instructions. The V3–V4 region of the 16S rRNA gene was amplified (341F−806R) by polymerase chain reaction (PCR) using universal bacterial 16S rRNA gene PCR amplicon primers , forwardThe 16S rRNA gene sequence analyses were performed using QIIME (version 1.80) . Tags weSRP116179.Clean short reads of the shotgun metagenomics were assembled using SOAPdenovo version 1.05 . Low quaP < 0.05 and adjusted by FDR  with threshold value < 0.05. Z-score of row was calculated to homogenization control a heatmap to demonstrate the relative abundances of antibiotic-resistance genes between the colonic microbiota of the high- and low-FE groups.To identify differences in microbial communities and genes between the two groups, wilcox tests were carried out. The significance level was declared at Each colon content sample (50 mg) was transferred into a 2 mL tube, and 500 μL of a pre-cold extraction mixture of methanol/chloroform  with 10 μL of internal standard  was added. Next, the samples were vortexed for 30 s and homogenized with a ball mill for 4 min at 40 Hz, followed by ultrasonication for 5 min in ice water. This was repeated three times. After centrifugation  for 15 min, 200 μL of the supernatant was transferred to a fresh tube. After evaporation in a vacuum concentrator, 50 μL of methoxyamination hydrochloride (20 mg/mL in pyridine) was added. The samples were then incubated at 80°C for 30 min, and then derivatized by 70 μL of BSTFA reagent  at 70°C for 1.5 h. Gas chromatography coupled with a time-of-flight mass spectrometry analysis of the colonic contents was performed by Beijing Biomarker Technologies Co., Ltd.  on an Agilent 7,890 gas chromatograph . The raw data analysis, including peak extraction, baseline adjustment, deconvolution, alignment, and integration, was completed using Chroma TOF 4.3X software . The LECO-Fiehn Rtx5 database was used for metabolite identification by matching the mass spectrum and retention index .P < 0.05; After quality control and demultiplexing, the number of available sequences for the 40 fecal samples ranged from 26,158 to 42,174. With a 97% identity cut-off as one OTU, the number of OTUs ranged from 1,658 to 3,030 . The bacde novo sequencing, this was a similar finding to that of the amplification sequencing . Eight genera were significantly enriched in the LFCR group, including Prevotella, while 17 genera were more abundant in the HFCR group, particularly Treponema and Lactobacillus . A totalDifferentially expressed colonic genes were annotated in the KEGG database . SignifiThe CAZy database was used to align and categorize differentially abundant genes into seven CAZy types. In total, 776 and 1,541 genes from the Hco and Lco group, respectively, were mapped to the database. The proportions of CAZy types in the two groups were then compared . GlycosiP < 0.05, Based on statistical analysis of the differential KOs, the differential pathways were investigated, and 22 pathways were found to be significantly different  was performed using the R (version 3.3.2) package ropls. The values of R2Y and Q2Y were 0.998 and 0.571, respectively, indicating that the model built by the OPLS-DA method could distinguish the correct sample group by metabolic expression; it could therefore be used to screen for differential metabolites . At the s and FE . Among ts and FE , palmitoRecently, with the widespread application of next-generation sequencing, the understanding of the mechanisms by which microbes affect their hosts has considerably increased. Studies have found that approximately one-tenth of the host transcriptome is regulated by microorganisms . Any micPrevotella, Treponema, and Lactobacillus were also found in other studies, and our metagenomic results found that the two most significantly different species in terms of their abundance in each group were Prevotella sp. CAG_604 and Lactobacillus reuteri. Gene annotation showed that many genes in both groups belonged to the Oscillospira genus, the growth of which is probably induced by potentially pathogenic bacteria and can be considered a sign of a healthy gut  under the Sequence Read Archive (SRA) Accession Number The animal study was reviewed and all experimental procedures described in this study were approved by the Animal Welfare Committee of China Agricultural University (Permit Number: DK996) and con-ducted under the approved slaughtering guidelines (GB/T 17236–2008) from the Quality Supervision, Inspection, and Quarantine Committee of the People's Republic of China.ZT and CW: conceptualization and funding acquisition. ZW, KX, and ZT: methodology and software. ZW, YH, and HA: resources. ZW, YH, KX, and ZT: data curation. ZW and ZT: writing-original draft preparation and visualization. ZT and KX: writing-review and editing. KX and CW: project administration. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study assesses the attitudes (willingness) and preparedness of non-frontline physicians across different specialties in the Kingdom of Saudi Arabia (KSA) toward the management of hospitalized coronavirus disease (COVID-19) patients.This cross-sectional study conducted between April 15, 2020, and May 5, 2020, included 6209 physicians working in KSA. An electronic questionnaire was designed and validated for the assessment of 3 categorical outcome variables, namely, attitudes, confidence, and knowledge levels. Pearson’s chi-square test was used for comparing the distribution of the proportions of these 3 categorical variables.P < 0.0001). Lower confidence levels were found for airway management skills (38.1%), particularly among dermatologists and radiologists.Most participants (63.2%) were willing and prepared to treat COVID-19 patients. A significantly large proportion of participants specializing in anesthesiology (78.2%) had higher knowledge levels, followed by those from plastic surgery (71.1%), pediatrics (69.7%), and obstetrics and gynecology (69.1%)  use and confidence in airway management skills were proportionally related to the level of willingness to participate in COVID-19 patient management. There is an urgent need to train doctors from certain specialties on PPE use and airway management to enable their frontline support of severely ill COVID-19 patients. Home management is deemed appropriate for patients with non-severe symptoms , who can be adequately isolated in an outpatient setting.4 Suggested or confirmed COVID-19 patients accompanied by severe symptoms warrant hospitalization. The management of patients with severe disease comprises appropriate infection control and supportive care, including oxygenation support. In such patients, the disease is treated and managed by frontline physicians. In Saudi hospitals, these physicians include emergency physicians, intensivists, pulmonologists, and infectious disease physicians. However, there is an urgent need for assistance from health care professionals across other specialties, owing to the high risk of exposure. For example, during the early stages of the SARS epidemic in Toronto, 60% of the infected health care providers comprised critical care staff.5 The WHO recommends multidisciplinary collaboration for disaster mitigation during epidemics. However, several previous studies have found that physicians from different specialties worldwide are unprepared to respond to disasters, owing to a lack of appropriate education, training, or experience.7 To date, no published study has investigated the knowledge levels, skills, or preparedness of non-frontline physicians in the Kingdom of Saudi Arabia (KSA) in the context of surge management.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus belonging to the coronavirus family, was first identified on December 31, 2019, in Wuhan City, Hubei, People’s Republic of China.Accordingly, this work aimed to study the attitudes (willingness), levels of knowledge on personal protective equipment (PPE) use, and levels of confidence in airway management skills, associated with the COVID-19 pandemic, among non-frontline physicians across the KSA. We believe that this study has the potential to provide basic data for facilitating strategic planning during the pandemic, especially if the current situation worsens, as well as to enable preparedness for future outbreaks.In this cross-sectional study conducted between April 15, 2020, and May 5, 2020, non-frontline physicians were enrolled from across the KSA. Physicians who currently reside in the KSA were included, regardless of whether they were currently practicing or retired. Physicians across all clinical specialties were included, except frontline physicians who, by definition, are likelier to directly deal with suggested or confirmed severe COVID-19 cases in Saudi hospitals . The sample size was estimated by considering the outcome variables  as a single composite outcome variable.Data were obtained using a self-administered online questionnaire, and participation was voluntary. Participants were recruited through the Saudi Commission for Health Specialties (SCFHS), which is responsible for the supervision and evaluation of training programs, as well as setting of controls and specification of standards for practicing health professionals (licensure). The SCFHS maintains a database of all physicians who live and work in the KSA and facilitates research by the distribution of surveys to health care workers (HCWs). Following the receipt of ethical approval for this study, the Data and Business Department at SCFHS sent out e-mails to all the targeted registered physicians (91 364), followed by a reminder e-mail 2 weeks later. A total of 7696 responses were received; of them, 127 did not agree to participate in the study while 6209 completed the survey.9 in which surveys were used for assessing the preparedness of physicians toward the SARS and Avian influenza outbreaks, as well as the opinions of 2 national experts. The questionnaire was modified to suit the ongoing COVID-19 pandemic, with the incorporation of additional questions.A questionnaire was developed based on information obtained from previous studies,The questionnaire was examined further for inadequate expressions or concepts. Additionally, the content of the questionnaire was validated for clarity and adequacy by 2 experts specializing in disaster preparedness and emergency preparedness. Finally, a pilot survey was performed, and the questionnaire was distributed to 27 candidates who represented the study population. The reliability of the questionnaire was assessed using Cronbach’s alpha for the 3 domains, which resulted in attitude, 0.98; confidence, 0.97; and knowledge, 0.96, indicating a high level of internal consistency.The final version of the questionnaire comprised 5 sections: (1) demographic characteristics, (2) preparedness and willingness to manage patients with COVID-19, (3) confidence in airway management skills, (4) knowledge on PPE use, and (5) willingness to participate in COVID-19 patient management. The section on demographic characteristics included items on sex, age, marital status, and years of experience, among others. In sections 2–4, a linear numeric response format was used. The last section pertained to the physicians’ willingness to participate in patient care, their area of interest, and reasons for their unwillingness to participate.Twelve items for the evaluation of physicians’ attitudes, 7 for measuring confidence level, and 15 for measuring the knowledge level were employed. In the attitude sections, participants were asked several questions about their involvement in specific training for COVID-19 preparation and their willingness to receive such training. In the confidence of airway management skills section, they were asked about how well they are trained in airway management skills and whether they can participate in airway management teams. In knowledge of PPE use, they were asked about the usefulness of certain measures in the prevention of acquiring COVID-19 .All responses were rated on a linear numeric response format. The total attitude, confidence level, and knowledge level scores were in the ranges of 12–60, 7–35, and 15–75, respectively. The total scores were categorized as low, medium, and high based on the percentiles of scores , with appropriate cutoffs for attitude levels , confidence levels , and knowledge levels .P-value ≤ 0.05 was considered statistically significant.Data were analyzed using SPSS version 26.0 statistical software . A descriptive statistical analysis was performed. Frequencies and percentages were used for the quantification of the categorical study and outcome variables. Pearson’s chi-square test was used to compare the distribution of the proportions of the 3 categorical outcome variables  in relation to the categorical study variables. A Ethical approval was obtained from the Institutional Review Board of the Princess Nourah Bint Abdulrahman University (IRB log number 20-0136). Only physicians who agreed to participate were directed to questions in the survey tool.P < 0.0001). Participants with a current “married, with children” (65.0%) relationship status, those who were staying alone (67.5%), and physicians who were still practicing (63.6%) had significantly higher attitude scores than those with other relationship statuses, physicians staying with family members, and retired physicians, respectively (P < 0.0001). In terms of the physicians’ specialties, a significantly large proportion of physicians who specialized in anesthesiology (74.8%) had higher attitude score levels, followed by those specializing in internal medicine (65.3%), pediatrics (65.5%), and plastic surgery (65.2%) (P < 0.0001). A significant increasing trend was observed in the attitude scores in relation to the designations of the participants; physicians with designations of assistant consultant, associate consultant, and consultant showed higher attitude scores than those with a designation of a fellow or resident (P < 0.0001). A similar increasing trend was observed in the attitude scores in relation to the categories of 5 experience levels (P < 0.0001). Physicians working at the secondary care level (67.7%), tertiary care level (69.5%), and at more than 1 care level (69.0%) showed significant higher, positive attitudes than those working at the primary care level (P < 0.0001).P < 0.0001). Participants who were “married, with children” (39.3%), staying alone (44.4%), and still in practice (37.0%), had significantly higher confidence levels than those with other relationship statuses, staying with family members, and retired . A significantly larger proportion of participants with a specialization in anesthesiology (78.4%) had higher confidence levels, followed by those specializing in pediatrics (56.5%) and plastic surgery (40.3%) (P < 0.0001). A significant trend was observed between the physicians’ confidence levels and their designations, with larger proportions of assistant consultants (41.9%), consultants (39.1%), and associate consultants (38.2%) showing higher confidence levels than fellows and residents (P = 0.001). An increasing trend was observed between the 5 categories of experience level and levels of confidence in airway management skills pertaining to COVID-19 patients, with a larger proportion of participants with greater experience levels showing higher confidence levels (P < 0.0001) as well. Participants working in private hospitals (42.4%) had higher confidence levels than those working in other types of hospitals (P < 0.0001). Significantly larger proportions of participants working at the secondary care level, tertiary care level, and at more than 1 care level  had higher confidence levels than participants working at the primary care level (P < 0.0001).P < 0.0001). Participants with a current “married, with children” (69.8%) relationship status and who were still practicing (68.7%) had significantly higher knowledge levels than those with other relationship statuses and those who were retired (P < 0.0001). In terms of specialization, a significantly larger proportion of participants with a specialization in anesthesiology (78.2%) had high PPE knowledge levels, followed by those specializing in plastic surgery (71.1%), pediatrics (69.7%), and obstetrics and gynecology (69.1%) (P < 0.0001). A significant increasing trend was observed between PPE knowledge levels and participants’ clinical designations, with larger proportions of assistant consultants, associate consultants, and consultants showing greater PPE knowledge levels than fellows and residents (P < 0.0001). A similar increasing trend was observed between the categories of the 5 experience levels, with higher proportions of participants who had 11–15, 16–20, and > 20 years of total experience , showing higher knowledge levels than those who had < 5 and 6–10 years of total experience (P < 0.0001). Moreover, larger proportions of participants working at the secondary care level, tertiary care level, and at more than 1 care level  had significantly higher knowledge levels than those working at the primary care level (P < 0.0001).This study has certain limitations that must be considered in the interpretation of its results. First, the study sample was not evenly distributed across the specialties. Second, the knowledge levels were assessed based on personal impressions rather than accurate measurements. Further studies assessing the actual knowledge levels and comparing them with HCWs’ willingness to deal with infected patients during epidemics and pandemics should be undertaken. Third, the results have limited generalizability to other populations as this study included HCWs in Saudi Arabia only.In this study, we aimed to measure the willingness of non-frontline physicians to provide patient care for those with COVID-19, in addition to assessing their knowledge on both airway management skills and proper use of PPE, which are the 2 fundamental components in dealing with COVID-19 cases. This study is the first to investigate the knowledge levels, skills, and preparedness of non-frontline physicians in the KSA, in the context of management of the COVID-19 surge. To the best of our knowledge, this is the first study conducted on non-frontline physicians in Saudi Arabia for this pandemic.10 A similarly lower proportion was reported in another study, in which only 48% of New York City HCWs were willing to work during the SARS outbreak.11 Additionally, weather-related disasters and mass casualty events have been shown to be associated with greater levels of willingness to provide care to victims among HCWs than radiological, nuclear, biological, or chemical disasters. The strongest barriers have been identified in association with biological outbreaks.12Most of the participants were willing and prepared to participate in COVID-19 patient management (63.2%); this value was higher than that reported in previous studies. One previous study showed that only 53.8% of public health department employees were willing to work during the influenza pandemic.15 Shapira et al. found that female respondents were less willing to work than the male respondents,14 whereas living alone was associated with a higher willingness, as the risk of infection transmission to family members was low.12 Based on these findings, efforts should be made for the provision of training female physicians in dealing with pandemics. Additionally, there is a need for in-depth investigations of the barriers that affect more than 40% of the physician pool, represented by women, for mitigating the related challenges and enhancing the levels of willingness.In the present study, the male sex and living alone were associated with higher willingness levels , consistent with previous findings. Women, in general, showed lower levels of willingness to participate in care provision during disasters .17 Therefore, it is recommended that annual training and re-training programs be conducted for HCWs, with a focus on PPE use . Moreover, all health care facilities should have a stockpile of PPE and ensure open communication channels with the infection control department.The majority of this study’s participants were familiar with the use of PPE (68.3%); this may be correlated with a higher level of willingness to work during the COVID-19 outbreak, as well as trust in work safety, which may have a strong impact on the willingness to participate in disaster management, in general.19 Interestingly, physicians who were not usually accustomed to dealing with purely medical cases, such as plastic surgeons (65.2%), showed higher willingness levels than family physicians (59.3%).The levels of willingness were higher in the 45–64 years age group, as well as among participants with a consultant designation , and those with > 16 years of experience (68.0%). These factors are correlated with higher levels of knowledge and confidence, which may aid HCWs in dealing with a new pandemic. HCWs with greater levels of experience and self-efficacy demonstrated a higher level of willingness to work during influenza pandemics.The participants showed lower levels of confidence in airway management (38.1%), with female physicians and those from certain specialties (dermatology and radiology) showing both lower confidence levels in airway management and lower willingness to manage COVID-19 patients. Since participants with higher scores in the 3 categories displayed more willingness to participate in frontline care of COVID-19 patients, willingness (induced) could be a secondary trait to higher levels of knowledge and confidence. As these scores were highly linked to the physicians’ specialties, those physicians also did not receive urgent training because of their willingness. Therefore, an establishment of a hands-on training program for HCWs in basic and advanced airway management skills, in addition to use of PPE, is recommended from lead agencies.Higher levels of knowledge and confidence in airway management are proportionally related to a greater level of willingness to participate in COVID-19 patient management. There is an urgent need for training programs focusing on PPE use and airway management skills for HCWs. Physicians across specialties who are associated with lower levels of contact with critically ill patients, such as those working in the departments of radiology, dermatology, and family medicine, must undergo special training programs to gain knowledge on COVID-19 patient management. Studying the barriers of willingness is a recommended area for future research.


Hepatocellular carcinoma (HCC) is a disease with a high incidence and a poor prognosis. Growing amounts of evidence have shown that the immune system plays a critical role in the biological processes of HCC such as progression, recurrence, and metastasis, and some have discussed using it as a weapon against a variety of cancers. However, the impact of immune-related genes (IRGs) on the prognosis of HCC remains unclear.Based on The Cancer Gene Atlas (TCGA) and Immunology Database and Analysis Portal (ImmPort) datasets, we integrated the ribonucleic acid (RNA) sequencing profiles of 424 HCC patients with IRGs to calculate immune-related differentially expressed genes (DEGs). Survival analysis was used to establish a prognostic model of survival- and immune-related DEGs. Based on genomic and clinicopathological data, we constructed a nomogram to predict the prognosis of HCC patients. Gene set enrichment analysis further clarified the signalling pathways of the high-risk and low-risk groups constructed based on the IRGs in HCC. Next, we evaluated the correlation between the risk score and the infiltration of immune cells, and finally, we validated the prognostic performance of this model in the GSE14520 dataset.FABP6), Microtubule-Associated Protein Tau (MAPT), Baculoviral IAP Repeat Containing 5 (BIRC5), Plexin-A1 (PLXNA1), Secreted Phosphoprotein 1 (SPP1), Stanniocalcin 2 (STC2) and Chondroitin Sulfate Proteoglycan 5 (CSPG5)), which showed better prognostic performance than the tumour/node/metastasis (TNM) staging system. Moreover, we constructed a regulatory network related to transcription factors (TFs) that further unravelled the regulatory mechanisms of these genes. According to the median value of the risk score, the entire TCGA cohort was divided into high-risk and low-risk groups, and the low-risk group had a better overall survival (OS) rate. To predict the OS rate of HCC, we established a gene- and clinical factor-related nomogram. The receiver operating characteristic (ROC) curve, concordance index (C-index) and calibration curve showed that this model had moderate accuracy. The correlation analysis between the risk score and the infiltration of six common types of immune cells showed that the model could reflect the state of the immune microenvironment in HCC tumours.A total of 100 immune-related DEGs were significantly associated with the clinical outcomes of patients with HCC. We performed univariate and multivariate least absolute shrinkage and selection operator (Lasso) regression analyses on these genes to construct a prognostic model of seven IRGs (Fatty Acid Binding Protein 6 (Our IRG prognostic model was shown to have value in the monitoring, treatment, and prognostic assessment of HCC patients and could be used as a survival prediction tool in the near future. Ranking sixth in worldwide incidence, primary liver cancer (PLC) is the fourth-leading cause of cancer-related mortality . HepatocTP53 status in order to improve the prognoses of HCC patients  and 0.62, 95% , respectively. The above results indicated that the seven signatures performed well in predicting the OS of HCC, and we obtained the same results in testing set, too , and they were related to OS in HCC patients. Then, we established a regulatory network based on these 31 TFs and 9 IRGs that had proven significant in univariate analysis. Correlation coefficients > 0.4 and P-values ≤0.001 were set as screening criteria. The TF-based regulatory diagram in Fig. To explore the clinical significance of pivotal IRGs and the corresponding underlying molecular mechanisms, we examined the expression profiles of 318 TFs and found that 31 genes were differentially expressed between HCC and non-tumour HCC samples . Based on univariate-analysis results with P < 0.669, we further included these parameters in multivariate Cox regression analysis for analysis. Multivariate analysis showed that risk score (P < 0.001) was an independent risk factor , main tumour size (>/<=5 cm), multinodular cirrhosis, TNM staging, BCLC staging, CLIP staging and AFP (>/<=300 ng/ml) were included in the analysis. Univariate analysis showed that risk score, main tumour size, cirrhosis, TNM staging, BCLC staging, CLIP staging and AFP were related to OS; while multinodular, cirrhosis, BCLC staging, CLIP staging and risk score were independent prognostic risk factors in multivariate analysis Fig. c, d.We used a stepwise Cox regression model to establish a prognostic nomogram based on the 219 eligible HCC patients with complete clinical information in the TCGA–LIHC dataset for predicting survival at 1, 3 and 5 years. Risk score, age, sex, TNM stage, T stage, N stage, and M stage were all nomogram parameters. The AUCs of OS at 1, 3 and 5 years were 0.791, 0.760 and 0.793, respectively. The C-index was values were 0.78  and 0.73 and  in the training and testing groups, respectively. The results of the clinical factors showed that the AUC values of T stage, TNM stage, and risk score were the highest at 0.757, 0.750, and 0.791, respectively, which suggested that the IPM had moderate prognostic performance Fig. .Fig. 9TWe performed GSEA in the training group to identify the differences between the high-risk and low-risk groups. Among them, the high-risk group had 41 significantly enriched pathways, and the low-risk group had 12 significantly enriched pathways. The enrichment pathways in high-risk groups are mostly related to tumours  or tumour-related pathways  -like receptor signaling pathway”, “Notch signaling pathway”, “VEGF signaling pathway” and “Pathways in cancer”), and metabolic or metabolic disease-related pathways ; the enrichment pathways in the low-risk group are mostly related to metabolism , complement and coagulation cascades and the PPAR string pathway Fig. .Fig. 11BICR5 gene expression and risk score. Female has higher expression level of PLXNA1 than male, while G3/G4 and stage III/IV have higher expression level of PLXNA1 gene. Those of patients in Stage III and IV were higher in SPP1 expression than those of patients in Stage I and II, a difference that was statistically significant. Patients in stage N0 had higher expression of FABP6 and MAPT than patients in stage N1, probably due to the large difference in the number of samples between the two groups. In terms of survival time, patients with HCC in T1/T2 and Stage I/II were significantly higher than those with the disease in T3/T4 and stage III/IV.We analysed the relationship between genetic characteristics and clinical parameters Table  in train+ T cells and CD8+ T cells and risk scores is less than 0.2, but it is relatively close to 0.2. The results showed that all immune cells were positively correlated with risk score to a statistically significant degree , which implying that the higher the degree of immune infiltration, the worse the prognosis of the patient.To further study whether risk score in this IPM could affect the abundance of immune cells in the TME, we performed a correlation analysis between six types of common immune cells and risk score. In Fig. FABP6, MAPT, BIRC5, PLXNA1, CSPG5, SPP1 and STC2. Of the 349 patients included in the cBioPortal, 120 (34.38%) showed genetic changes in these seven genes. With mRNA high (21.49%) was the most common genetic variation, amplification (6.5%) and missense mutation (1.69%) being the next most common , and the same conclusion was reached in the testing set . The patient’s risk score for HCC progressively increases as the expression levels of the genes in the risk signature increase, and the prognosis of HCC worsens as the risk score increases. More importantly, we constructed a nomogram based on these seven IRGs to quantitatively analyse the prognosis of HCC patients. The AUCs for 1-, 3-, and 5-year OS were 0.780, 0.699 and 0.685, respectively, and the C-index was 0.72 (95% CI: 0.68–0.77). HCC is a highly heterogeneous disease, and its prognosis is affected by many factors. We only included and analysed genes related to immunity and ignored the influence of other factors on HCC. Hence, our model does not show a high prognostic performance in predicting the long-term survival rate of patients, which is also one of the inherent defects of the model. We further analysed the risk score and clinically related factors in univariate and multivariate analyses and found that the risk score was associated with higher grade (P = 0.003) and stage III/IV disease (P = 0.004), which indicated that our prognostic model was more significant in advanced HCC patients. We believe that genetic detection should not be considered independently of individual characteristics. Therefore, we also constructed a nomogram combining the risk score and clinical factors, which can easily predict the 1-year, 3-year and 5-year OS of patients. It should be noted that the AUC values were all higher than 0.7. Compared with other clinical factors, the AUC value of the nomogram corresponding to risk score was the highest (AUC = 0.791), and the C-index was 0.78 (95% CI: 0.72–0.84). In addition, when we analysed the risk score combined with clinical factors, the C-index of the test dataset was 0.73 (95% CI: 0.67–0.78), indicating that our IPM has a modest prognostic performance in the test dataset. In the GSE14520 dataset, a series of test results were basically consistent with those in the TCGA dataset. Although the AUC values reached above 0.5  is expressed in DC and participates in the interaction between T cells and DC, and may be involved in regulating the rearrangement of the cytoskeleton during the interaction between T cells and DC [CSPG5 is only expressed in the human brain, and a study showed that it has a new function that binds to ERBB3 tyrosine kinase [ERBB3 somatic mutation is a potential tumour driver [SPP1 can activate the CSF1-CSF1R pathway in tumour-associated TAMs and promote the expression of PD-L1 in HCC, and there is a positive correlation between SPP1 and PD-L1, TAM expression. On the other hand, SPP1 can induce endothelial cells and upregulate VEGF-induced migration of endothelial cells, having a synergistic effect with VEGF in tumour angiogenesis [MAPT is mainly expressed in nerve cells, and more commonly studied in geriatric diseases such as various neurodegenerative diseases including Alzheimer’s disease [MAPT is overexpressed in certain cancers, and participate in the resistance of various tumours to taxane drugs [FABP6 is involved in the bile acid metabolic process and is related to the bile acid intestinal circulation [STC plays a vital role in tumour growth, invasion, apoptosis and metastasis, and promotes local angiogenesis through the VEGF/VEGFR2 signalling pathway [STC2 is related to the poor prognosis of HCC [FABP6. Also, the results of immunohistochemistry in the HPA database showed that except FABP6, the protein levels of other IRGs were significantly different between HCC tissues and normal liver tissues, and they were highly expressed in HCC, which was consistent with our conclusion. At present, FABP6 has not been reported in immunotherapy of HCC, which may be potential therapeutic targets for HCC. We then used the seven IRGs from the cBioPortal to obtain information about genetic mutations. The mutation rates of these seven IRGs are more than 4%, which may be useful for clinical research in the future. Collectively, these findings indicated that the seven IRGs have the potential to predict the prognosis of HCC.The signatures in this IPM have good prognosis performance, which could be potential prognosis and therapeutic targets for HCC. poptosis . Experim stromal . PLXNA1 s and DC . CSPG5 ie kinase , and ther driver . Howeverogenesis . MAPT is disease . Previoune drugs , and itsculation . STC pla pathway . Hongweis of HCC . To furtBIRC5, PLXNA1 and CSPG5 were the core IRGs in this network; all three were positively regulated by 13 core TFs, of which EZH2 could positively regulate the expression of BIRC5, PLXNA1 and CSPG5. EZH2, the hub TF in our PPI analysis . STG and SPP1 are associated with the VEGF signalling pathway, PLXNA1 and SPP1 are associated with DCs or TAMs; CSPG5 is associated with common somatic mutation sites. The application values of MAPT and FABP6 in HCC need further experimental confirmation. In this case, we boldly speculate that EZH2 may mediate the angiogenesis of the VEGF signalling pathway through regulating the expression of the seven IRGs, which may be the possible mechanism of this predictive model related to immune infiltration in high-risk patients. In low-risk patients, we found that the mechanism of these seven IRGs related to the immune infiltration of HCC is related to metabolism. However, the specific mechanism remains to be further explored. The combination of antiangiogenic drugs and tumour immunotherapy will show great prospects in the near future. However, further insights by validation with immunohistochemistry analysis are needed to understand whether the VEGF signaling pathway is linked to high-risk groups.To explore the potential molecular mechanisms associated with these IRGs, we constructed a TF-mediated regulatory network to screen out important TFs that might regulate identified the hub IRGs. sis Fig. a, is shogenetics , immunitgenetics , metastagenetics , angiogegenetics  and apopgenetics . In addigenetics . At presy of HCC  and may pathways . NOD-lik pathway . The inhreatment . Of part+ T cells, CD8+ T cells, neutrophils, TAMs, and DCs. The results showed that for these six cell types, the degree of immune infiltration was positively correlated with the risk score, and the correlations between all immune cells and the risk score were statistically significant (P < 0.05). These results indicated that these cells have a high level of immune infiltration in high-risk patients. TAMs are phagocytes, which are the body’s first line of defence against external threats; they can produce proinflammatory responses to pathogens and repair damaged tissues. However, cytokines and chemokines expressed by TAMs can inhibit antitumour immunity and promote tumour progression [SPP1 and PD-L1 and the infiltration of TAMs in HCC tissues, which played an important role in the immune microenvironment of HCC [+ type 1 T regulatory (Tr1) cells [+ T cells and CD8+ T cells and the risk score was less than 0.4. In addition, studies have shown that CD4+ T cells and CD8+ T cells can suppress the occurrence and proliferation of HCC due to their antitumour immune response [+ T cells in the area surrounding HCC tumour tissue increases significantly and could promote the progression of HCC [+T cells is associated with longer OS [To further assess the immune microenvironment of HCC, we also analysed the correlation between risk score and the following six types of immune cells: B cells, CD4gression . The expt of HCC . All thet of HCC  illustrat of HCC . Wei Y et of HCC . Severalt of HCC , 85. Rest of HCC  illustra1) cells , which iresponse ; howevern of HCC . Shinji onger OS . AlthougWe systematically and comprehensively analysed the application value of our IPM for HCC, which could provide new insights for the treatment of this disease. However, the current study still has certain limitations. The IPM constructed based on the TCGA database has the best predictive performance in the training set. In the testing set, the risk score is not the best indicator to predict the OS of HCC. Limited by clinical factors in the testing group, our model still needs to be further validated in other datasets. Although we increased the reliability of our conclusions by combining multiple datasets, evaluating various aspects of the IPM, and verifying our results using the GSE14520 database. Most of the studies are plagued by a lack of validation in vitro and in vivo validation experiments, and further evidence provided by a well-designed clinical study is needed. It is also essential to use relevant basic experiments to further explain the mechanism of HCC immunotherapy. On the other hand, we have studied six major immune cells, the correlation between immune cells and the risk score was weak, and the association between more immune cell subtypes and HCC is still unclear. Therefore, we suggest that further individual studies and discussions should be conducted in the future. We believe that the era of HCC immunotherapy will soon be realized, and we look forward to it.To sum up, we constructed an IPM with seven prognostic IRGs by combining different data types from multiple databases. Individuals with HCC were automatically classified into high-risk and low-risk groups based on their risk scores, with gene expression as an independent variable. In addition, we established gene-related and clinical factor-related nomograms to facilitate more-comprehensive prognostic assessments of HCC patients. Finally, the results of the association between infiltration abundance of common immune cells in the TME and risk score showed that our IPM could predict the TME to a certain extent. This model will be a reliable tool for predicting prognosis in HCC by combining genomic characteristics, immune infiltration abundance, and clinical factors.