Ichthyosis is a heterogeneous group of Mendelian cornification disorders that includes syndromic and non-syndromic forms. Autosomal Recessive Congenital Ichthyosis (ARCI) and Ichthyosis Linearis Circumflexa (ILC) belong to non-syndromic forms. Syndromic ichthyosis is rather a large group of heterogeneous diseases. Overlapping phenotypes and genotypes between these disorders is a major characteristic. Therefore, determining the specific genetic background for each form would be necessary.A total of 11 Tunisian patients with non-syndromic (8 with ARCI and 2 with ILC) and autosomal syndromic ichthyosis (1 patient) were screened by a custom Agilent HaloPlex multi-gene panel and the segregation of causative mutations were analyzed in available family members.NIPAL4) and 4 already reported mutations . Yellowish severe keratoderma was found to be associated with NIPAL4 variations and brachydactyly to TGM1 mutations. Two novel variations (c.5898G > C and c.2855A > G in ABCA12) seemed to be features of ILC. Delexon13 in CERS3 was reported in a patient with syndromic ichthyosis.Clinical and molecular characterization, leading to genotype–phenotype correlation in 11 Tunisian patients was carried out. Overall, we identified 8 mutations in 5 genes. Thus, in patients with ARCI, we identified a novel (c.118T > C in Our study further extends the spectrum of mutations involved in ichthyosis as well as clinical features that could help directing genetic investigation. Ichthyosis is a heterogeneous group of Mendelian cornification disorders that includes syndromic and non-syndromic forms. Autosomal Recessive Congenital Ichthyosis (ARCI) and Ichthyosis Linearis Circumflexa (ILC) belong to non-syndromic forms. ARCI is characterized by abnormal desquamation over the whole body due to a dysfunctional skin permeability barrier and altered lipid composition. It is a rare skin disease affecting around 1 in 200,000 individuals , AL 607800) , SDR9C7  609769) , PNPLA1  604125) , CYP4F222 MIM: 6195 [14], S3 MIM: 6276 [15], 603741) , LIPN (M 613924) , SLC27A4 604194) , and ST1A major characteristic of ARCI is partially overlapping phenotypes and genotypes between LI and CIE  Table 11. TherefThe current study focused on 11 newly recruited Tunisian patients affected with either non-syndromic ichthyosis forms  or autosomal syndromic ichthyosis (1 patient). We tried to highlight some clinical features related to specific genes that could facilitate genetic diagnosis. We reported three novel missense mutations in two different consanguineous families, as well as 5 known disease-causing variants in 4 different genes in the remaining subjects. In silico analysis was performed in order to predict the effect of novel mutations on each corresponding protein level.PatientsA total of 11 patients with different ichthyosis phenotypes were enrolled. Among them, five belonged to two consanguineous families: A  and F (F1 and F2). The other six cases  were sporadic. Ten out of 11 patients were affected with non-syndromic ichthyosis . Only one patient suffered from autosomal syndromic ichthyosis.2.DNA extractionThe study protocol conforms to the approval of the local ethical committee of CHU Hedi Chaker, Sfax, in compliance with the Declaration of Helsinki. Written informed consent was obtained from all study participants or the parents for minor children.3.Next generation sequencingTotal genomic DNA was extracted from patients and available members’ family using the phenol–chloroform standard procedure . The DNA4.Sanger sequencingNext generation sequencing (NGS) was performed using a custom Agilent HaloPlex multi-gene panel with 32 ichthyosis linked genes comprising a target size of ~ 64 kbp. The average sequencing depth was 420.3x. A rate of 97.6% of the target position had at least 20 × in depth. Sequencing was performed on an Illumina MiSeq sequencer using MiSeq reagent kit v2 (2 × 150 bp). Reads were aligned using bwa (v. 0.7.17)  against PCR amplification was performed using a thermal cycler GeneAmp PCR System 2720 , in a final volume of 15 μl using 100 ng DNA, 10 μM of each primer, 2 mM dNTP, 10 × PCR buffer, 1U of Taq DNA polymerase (Qiagen) and Q-solution. Primers were generated with primer3 and corresponding sequences are mentioned in Table NIPAL4 (ENST00000311946.7) and ABCA12 (ENSG00000141527.18) by BLAST online software.5.Bioinformatic predictionsPCR products were sequenced using Applied biosystem . Sequences were analyzed, using Bio Edit Program and compared with wild type sequences: Protein sequence alignment across species was performed using CLUSTALW and PolyPhen2 software. The potential functional impact of p.(M63T) mutation on NIPA4 and ABCA12 proteins was predicted using PolyPhen2, SIFT, Mutation Taster, Panther and CADD software.The current study included 11 ichthyosis patients with 3 phenotypes. Among them, five belonged to two consanguineous families: A  and F (F1 and F2). The other six cases  were sporadic  displayed at the age of 3 years larger scales on the forehead and limbs, while the scales were fine and brown on the trunk on an erythematous skin. However, their first cousin (A7), who was affected with LI demonstrated darker, thicker and plate-like scales all over the body, without erythema Fig. a. PatienThe F family included 2 patients F1 and F2). The proband F1, aged 35 years, had a similar phenotype to her 4 year-old daughter (F2). Both patients presented multiple polycyclic erythematous and squamous plaques, with brownish fine scales  presented mild erythematous skin. Scales were white and fine on the face, the trunk and the upper members, while they were thicker and larger on the knees and the legs. White scales were also located on the dorsal aspect of the hands and feet and in the folds. Mild palmoplantar keratoderma and brachydactyly were also observed. There was no ectropion and ears were normal. Interestingly, ophthalmological examination revealed bilateral myopia and microspherophakia.NIPAL4, TGM1, CYP4F22, ABCA12 and CERS3)  in exon 1, which substitutes a methionine to a threonine . It was reported in family A affected members , with two ARCI phenotypes (CIE and LI). To confirm the segregation of this mutation and its association with ARCI, Sanger sequencing was performed in all available family members and control population. This mutation was in heterozygous state for parents and absent in unaffected members and control individuals . It was identified in a CIE patient  who presented a mild erythema of the skin, with small white scales sparing the folds.The second disease-causing variant  occurred in exon4 of ABCA12 gene in family F (F1 and F2 patients) with ILC  were identified (data not shown).Two novel missense mutations were detected at the homozygous state: c.5898G > C (ENST00000389661.4)  and c.2855A > G (ENST00000389661.4)  in TGM1 with two different homozygous nonsense germline mutations (Table TGM1 gene was c.1042C > T (ENST00000206765.6) located in exon 7  and detected in patient Y1 aged 19 years, who showed LI phenotype.In three patients with non-syndromic ichthyosis , the involved gene was ns Table . InteresCYP4F22 gene. This variation was also reported only in heterozygous state in gnomAD database.K1 patient with LI had rather a single homozygous missense mutation  in exon 8 which substitutes arginine to tryptophan  in CERS3 gene in a 15-year old patient (E1) affected with syndromic ichthyosis (CIE with ocular defect).We also identified a homozygous deletion in exon 13 of NIPAL4 gene and p.Glu1966Asp, p.Tyr952Cys in ABCA12 gene. We have to mention that all new variants were not reported before in the gnomAD database.As mentioned in Table Moreover, multiple alignments of corresponding proteins showed that these residues were located within a highly conserved region Fig. b, d.NIPAL4, TGM1, CYP4F22, ABCA12 and CERS3). Among these, three were novel . Using bioinformatic tools, these variations were predicted as pathogenic and causing a structural protein modification suggesting their involvement in ichthyosis. Moreover, already described mutations were pathogenic according to ClinVar database, except for c.844 C > T in CYP4F22 gene which was classified as an uncertain significant variant. However, this variant was associated to both LI and CIE phenotypes [We collected detailed phenotypic data from 11 Tunisian patients with different ichthyosis forms. In order to identify causative gene(s) mutation(s), we used a custom multi-gene panel. One of the most important aspects of this panel is its ability to be easily upgradable in view of novel discoveries. Moreover, compared with whole exome sequencing, analysis of the multigene panel is easier and faster. Overall, we identified 8 disease-causing variants in 5 different genes  with two missense mutations (c.534A > C and c.118T > C). This discordance could be explained by the small number of patients investigated in our study.Unlike ARCI series in literature, where ble gene –33, in oABCA12 gene. These variations and clinical related aspects have never been reported before. In fact, this gene is involved in either HI phenotype [SPINK5 gene [SPINK5 gene, neither in genes involved in erythrokeratoderma variabilis (EKV) were found in ILC patients (data not shown), confirming then different etiologies. Recently, Leersum et al. [ABCA12 mosaicism. This particular form of ichthyosis was explained by the combination of a germline mutation and an acquired postzygotic mutation in this gene. ABCA12 gene was reported also in a keratosis pilaris with missense mutation and in Nevus comedonicus with a high protein expression level in the sebaceous gland without any variation in ABCA12 coding region [Phenotype-genotype correlation was one of the major objectives of the current investigation. Besides finding new variations in genes causing ichthyosis, we tried to highlight novel clinical characteristics associated with particular genotypes. In this context, a particular form of non-syndromic ichthyosis (ILC) with multiple localized polycyclic erythematous and squamous plaques and brownish fine scales was found in patients carrying two novel missense mutations in henotype , with hohenotype –37. On tNK5 gene . As cleam et al.  also repg region .NIPAL4 mutations presented with a phenotype of non-syndromic CIE. Concerning patients with TGM1 mutations, 66.67% of them showed a phenotype of LI. These observations confirmed other studies showing that NIPAL4 mutations were usually associated with a moderate phenotype with fine grey/white scales, while TGM1 mutations were associated with a more severe phenotype with darker, adherent and plate-like scales [Otherwise, our investigation showed that 75% of our patients with e scales .TGM1 gene. This phenotype with ‘long palm/short fingers’ appearance was also reported in two Tunisian patients with LI carrying TGM1 mutations [TGM1 mutations. To the best of our knowledge, this phenotype, noted in E1 patient also, has never been reported before in ARCI patients with CERS3 mutations. We suggest that this symptom may be caused by the deletion of ADAMTS17 gene [CERS3, complete sequence of non-coding RNA FLJ42289 and the first three exons of ADAMTS17) was reported in three consanguineous Tunisian families affected with ichthyosis associated with ocular, cardiac and skeletal anomalies [Our findings showed that some clinical features were associated with particular genotypes Table . Thus; wutations . Even thS17 gene . Indeed,nomalies . Since Enomalies . In ordeTGM1 (3/7), NIPAL4 (3/7), or CYP4F22 (1/7) genes. In the literature, this clinical feature was significantly associated with TGM1 [NIPAL4 mutations with varying frequencies (28–73%) [ABCA12, CERS3, ALOX12B, ALOXE3, CYP4F22, lipase N, PNPLA1 and SDR9C7 genes [In our cohort, 7 patients were born as collodion babies and were mutated either in ith TGM1 , and NIP(28–73%) , 45. ButC7 genes , 47.TGM1 mutations, and 50% of those with NIPAL4 mutations. This finding was consistent with literature data and confirmed the previous observation that ARCI patients bearing TGM1 mutations were more likely to develop bilateral ectropion [Ectropion was noted in all studied patients with ctropion .NIPAL4 variation and in the patient with CYP4F22 variation. Although reported in the literature [NIPAL4 and CYP4F22 genes.In our study, skin folds were spared in 2 patients with terature  as a preNIPAL4, TGM1, CYP4F22 mutations and CERS3 deletion, but with varying severity. Thus, in patients with p.Met63Thr NIPAL4 variation PPK was either moderate or absent. However, PPK was rather yellowish and severe in patients with p.Glu178 Asp NIPAL4 mutation as reported in the literature. Indeed, yellowish PPK has been reported in NIPAL4 series and, to the best of our knowledge, never associated with other ARCI genes [NIPAL4 mutation in ARCI patients. In addition, patient C1 showed a constricting band around his right fifth finger, suggestive of pseudoainhum  and alopecia in one patient (M1) carrying CI forms . AlopeciTGM1 gene was found in both CIE (M1 patient) and LI (B1 patient), suggesting the effect of a modifier gene as it was already reported [FLG gene coding the filaggrin protein. We identified 44 missense polymorphisms in M1 not present in B1 (data not shown). Prediction effect of these polymorphisms showed that the majority were benign.Nevertheless, we also revealed that diverse phenotypes were caused by the same gene with an identical mutation. Notably, c.788G > A in reported . In an aABCA12 gene responsible for ILC, a rare clinical form of ichthyosis, as well as novel clinical characteristics associated with particular genotypes.To summarize, we report three novel mutations, two of which were located in TGM1 mutations and associated to deletion of ADAMTS17 gene associated to deletion of CERS3 exon 13.Phenotypic-genotypic correlation suggests that brachydactyly could be related to NIPAL4 and ABCA12 genes, we added new insights to the already reported particular phenotypes linked to specific genes. The involvement of such genes in a particular ARCI form remains discussed. In order to better explain its phenotype heterogeneity, investigation of whole genome is necessary to search for responsible modifier genes.Besides the new reported variations in http://bioinfo.ut.ee/primer3-0.4.0/http://www.ncbi.nlm.nih.gov/blast/https://www.genome.jp/tools-bin/clustalwhttp://genetics.bwh.harvard.edu/pph2/https://sift.bii.a-star.edu.sg/http://www.mutationtaster.org/http://www.pantherdb.org/tools/index.jsphttps://cadd.gs.washington.edu/snv


Iodine status, including Iodine Deficiency (ID) of the children aged 12–59 months of Jaffna District, Sri Lanka, have never been studied. This study thus aimed to assess ID among children aged 12–59 months by monitoring the Urinary Iodine Concentrations (UIC), the prevalence of goitre, and the factors causing ID.A cross-sectional study was conducted among 846 children aged 12–59 months in Jaffna District, Sri Lanka. Sociodemographic characteristics and other factors were collected using an interviewer-administered questionnaire. Dietary pattern of children was obtained using semi-quantitative food frequency questionnaire. We performed urinary iodine estimation and physical examinations to detect the goitre, according to the World Health Organization criteria. A multivariate logistic linear regression model was used to identify the factors that causing ID.The median UIC was 146.4 μg/L (interquartile range = 112.6–185.3 μg/L). Based on the UIC (<100 μg/L), 17.8% had ID, of which 15.7% and 2.1% had mild and moderate ID. The mean consumption of iodine from food was 128.7 (±20.2) μg/day. Gender variation had no influence on ID (p>0.05). Median UIC was significantly associated with living area, wealth status, type of drinking water, and method of iodized salt usage. A higher percentage of ID was significantly associated with younger age [AOR 2.32 (95% CI: 1.31–4.10)], urban area [AOR 1.94 (95% CI 1.27–2.96)], inland regions [AOR 3.20 (95% CI 1.85–5.55)], improper method of iodized salt usage [AOR 3.63 (95% CI: 1.38–9.56)], and low consumption of iodine-containing foods. The neck palpation revealed that only three children had goitre (0.4%).This study revealed that high ID among the children in Jaffna children was due to improper usage of iodized salt, even though the iodized salt is freely available in the region, living area, and age, while the prevalence of goitre was not significantly identified as a public health problem. Deficiency of essential micronutrients, including iodine, has substantial impacts on the health and development of growing children –3. IodinID in a geographic area is caused by low iodine content of soil, water or crops . ID, incIodine status, including prevalence of goitre among the children less than five years and the causative factors of ID have still not been reported from Jaffna District, Sri Lanka. Hence, the children from Jaffna District were selected to find the iodine status, including ID and prevalence of goitre, and the factors contributing to ID were studied.All the chemicals used in this research were of analytical grade.2, and a coastline of 160 km  were selected as Secondary Sampling Units (SSU). A total of 10 households were selected randomly from an SSU. If more than a child resides in a house, a child was selected based on his or her recent birthday at the date of data collection.Age, sex, and birth weight of each child were obtained from the growth chart book, Child Health Development Record (CHDR). The weight of the child was measured with lightweight cloth by using an electronic weighing scale (SECA 811) with an accuracy of ±100g. The height of the child was measured with a portable stadiometer (SECA 213) and Infantometer (SECA 417) with an accuracy of ±0.5 cm according to standard World Health Organization (WHO)’s procedures .Information on the socio-demographic and economic characteristics, drinking water source, and information regarding usage of iodized salt of the selected participants were obtained by a structured interviewer-administered questionnaire. The details of households that fall into the category of urban or rural areas based on the administrative divisions of Jaffna District Secretariat and coastal or inland regions based on the households within the coastal divisions of Grama Niladhari divisions were obtained. A spot morning urine sample of 10mL was collected after first voiding between 08:00 and 12:00 using a sterile wide-mouth plastic urine container with a screw cap covered with black paper. The containers were immediately transferred to a cool box and transported to Biochemistry Research Laboratory, Faculty of Medicine, University of Jaffna for iodine estimation. Collected urine samples were kept at 4°C and analysed within 24 hours.Training to data collectors (pre-intern doctors) was given by principal investigator and a community physician. The training mainly focused on the study’s objective, the technique of interview, administration of questionnaires, collection, storage, and transport of urine samples, and maintaining ethical issues. The questionnaire prepared in English was translated into the native language, Tamil and then back-translated to English to ensure consistency of translation. A pilot study was carried out to pre-test the questions. During the pre-test, the questionnaire was evaluated for suitability and applicability during the interview between parents and data collectors. All questionnaires were regularly monitored for completeness and consistency by the research supervisors. The overall research activities were coordinated by principal investigator.The principle of Sandell-Kolthoff reaction was used to determine the iodine concentration in urine samples ,29. BrieClinical examination of neck for goitre was performed for all children described by Smith et al. (1990) . Goitre TM) , food products of rice flour [67.75 (±28.70) g], formula milk [8.58 (±5.21) g], and a variety of legumes daily. They consumed other main foods, including fish , egg , meat , and cow’s milk  per day. The mean consumption of iodine from food was 128.72 (±20.18) μg/day, which was higher than the World Health Organization’s recommendation [0.82 mL] Among the children who consumed iodine-containing nutrient supplements (n = 796), the mean consumption of iodine from nutrient supplements was 10.77 (±7.6) μg/day. The mean consumption of total iodine by the study population was 139.5 (±22.12) μg/day (128.72 μg/day from food and 10.77 μg/day from nutrient supplements). The mean iodine consumption from nutrient supplementation was not significantly different between coastal [9.98 (±5.66) μg/day] and inland areas [11.05 (±8.17) μg/day]. The mean total iodine consumption (from foods and supplements) of coastal children [146.43 (±18.33) μg/day] was significantly higher than the children who live in inland areas of the District [136.93 (±22.71) μg/day] (p≤0.0001). Children who had ID consumed mean of 10.46 (±7.6) μg/day iodine from supplements, whereas mean of 10.83 (±7.6) μg/day iodine by non-iodine deficient children (p>0.05). Mean iodine consumption from both food and nutrient supplements of iodine deficit children [123.56 (±20.47) μg/day] was lower than the non-iodine deficit children [142.95 (±20.79) μg/day] (p≤0.001).Out of 846 children screened for goitre, only 3 (0.4%) had goitre. On the basis of WHO criteria, among those three children  who had goitre, two children had grade 1, and a child had grade 2 goitre . HoweverIn contrast, 12%, 5.6%, and 0.6% of children consumed goitrogenic substances containing foods, namely manioc (cassava), cabbage, and sweet potato, respectively. The ID was significantly high (28.3%) in children who consumed goitrogenic substances containing foods than the children who consumed non-goitrogenic substances containing foods (15.6%). Children who consumed goitrogenic substances containing foods had the median UIC of 137.9 μg/L than that of children who consumed non-goitrogenic substances containing foods (151.2 μg/L) (p≤0.05). A child with goitre was from coastal areas and consumed seafood predominantly. Only a child affected with goitre consumed cassava, while the other two children did not consume the goitrogenic foods.In the present study, the ID, prevalence of goitre and factors influencing ID, including social determinants and dietary patterns in Jaffna children aged 12 to 59 months, were evaluated. This study demonstrated that the median UIC of children of 12 to 59 months was within the normal range (146.4 μg/L), between 100 μg/L and 200 μg/L ,21. In aWe noticed that 22.5%, 18.8%, 16.7%, and 12.4% of children aged 12–23 months, 24–35 months, 36–47 months, and 48–59 months, respectively, had ID. The increased risk of ID among children of 12–23 months was more when compared with those of 47–59 months. This was supported by a previous study . It was On the other hand, the studies among children aged 6–12 years revealed that the older children had a higher risk of getting ID than those of younger ages ,48. The Females aged 6–12 years had higher median UIC than that of male counterparts . We founThe weight and height of the children were not significantly affected by iodine-deficient children compared to non-iodine deficient children. Morales-Suárez-Varela et al. (2018)  observedThe children from rural areas had a higher median UIC and a lower ID than those of children in urban areas. The present study was consistent with previous studies ,53. FurtThe income and family type of the households were not associated with iodine status. Völzke et al. (2013)  reportedThis study also revealed that utilization of iodized salt to the food preparation by mothers was 100%, which was an ideal value and was higher than the WHO recommendation (>90%) . HoweverEven though the mean consumption of iodine from the food and supplementation was higher than the WHO recommendation of ≥90 μg/day , 14 chilID in a population is a public health problem if the total goitre rate is more than 5% . Further-) and is entirely absorbed via the small intestine. However, two-third of the ingested iodine is excreted by the kidney, and the remaining one-third is taken by the thyroid gland [Three children with goitre had excreted the normal level of iodine in their urine [185.74 μg/L]. The children with goitre might have less iodine uptake or excess iodine intake by the thyroid gland or any defect in the thyroid gland in the synthesis of thyroxin hormone; thus, the absorbed iodine is excreted through urine . In the id gland , where iid gland . The preid gland  that goiid gland  and goitIDDs prevalence is high in the Jaffna District under the present study settings was related to poor dietary intake of iodine-containing food and importer usage of iodized salt. The present study showed that children who consumed the seafood had less ID. Seafood contain a higher amount of iodine than other foods, including crops . Fish coThe present study concluded that the prevalence of ID was high (17.8%). Among the children, 15.7% and 2.1% of children were affected with mild and moderate ID, respectively, while none were affected with severe ID. The mean consumption of iodine from food was (128.7 μg/day) was higher than the World Health Organization’s recommendation. Median UIC was significantly associated with living area, wealth status, type of drinking water, and method of iodized salt usage. Children having ID decreased with age, namely, children aged 12–23 months had a two-fold chance of getting ID than those of 48–59 months. Less ID was observed among the children from rural areas (15.9%) than those from urban areas (23.9%), while children from the inland areas had higher ID (20.2%) than the children from the coastal areas (11.4%). The risk of ID among children in urban areas and inland regions was approximately two- and three-fold higher than those of counterparts, respectively. The increasing educational level of the mothers marginally increased the iodine status of the children. Significantly higher median UIC (159.0 μg/L) was observed in children of mothers who added iodized salt after cooking the foods than those who added iodized salt by other methods. This study also showed that the risk of ID was approximately three times higher in children when the mothers added the iodized salt after washing with water or solubilized with water than that in the children of mothers who added the iodized salt after cooking the foods. The present study also revealed that the consumption of less iodine-rich foods contributed to the ID in Jaffna children. Conversely, ID was not associated with height, weight, and birth weight of the children. Moreover, family income, wealth index, type of drink water, and nutrient supplements were not associated with ID. Consumption of iodine-containing nutrient supplements was insufficient, and it did not improve the iodine status of children. As our study limited to estimate the amount of iodized salt consumed, we recommend incorporating the iodine availability from the table salt. The present study found a low (<5%) prevalence of goitre, and did not have a significant impact on public health.


The recently proposed Cognitive Experiential Leadership Model (CELM) states that leaders’ preference for rational thinking and behavioral coping will be related to their level of transformational leadership. The CELM was based on research that principally used cross-sectional self-report methods. Study 1 compared both self-ratings and follower-ratings of leadership styles with leaders’ self-rated thinking styles in 160 leader-follower dyads. Study 2 compared both self-ratings and coworker-ratings of leadership styles with leaders’ self-rated thinking styles for 74 leaders rated by 607 coworkers. In both Studies, leaders’ rational thinking, imaginative thinking, and behavioral coping correlated positively with their self-rated transformational leadership. However, only behavioral coping, but not rational thinking, was correlated with follower-rated (FR) transformational leadership in Study 1, and thinking styles were unrelated to other-rated transformational leadership in Study 2. These results partly support and partly challenge the CELM. Practically, this study suggests that leadership may be improved by leaders developing their capacity for behavioral coping. Would anyone argue with the proposition that leaders who think better are likely to lead better? In order to be testable, however, this proposition requires a definition of good thinking and good leadership. Recently, transformational leadership , and underlies most behavior. Like other dual-process theories, according to CET rational thinking is slow, conscious, relatively affect-free, and more useful in novel situations. In contrast, the experiential system is defined as a broad intuition-based system that encapsulates emotion, concrete reasoning, generalization, spontaneity, and imagination  and Defensiveness (>1.5 SD above the mean) scales, as specified in the CTI Manual (N = 320).Each participant was issued with a unique code number that they entered when completing measures online. Leader and follower code numbers were retained by the researcher so that paired data could be matched. A total of 245 leaders and 239 followers, among whom there were 191 matched pairs completed online questionnaires. Thirty-one pairs were omitted from the sample because of incomplete responses (>5%) or for falling outside the acceptable bounds of the Constructive Thinking Inventory’s (CTI) Validity  was higher than the average age of their followers . Gender was similar in both the leader  and follower samples . Leaders had an average tenure of 5.75 years (SD = 6.37) in their current position. Followers had an average tenure of 3.19 years (SD = 3.73) in their current position. The participants were from a range of industries with the largest sub-groups being from retail, sales, and marketing (20.0%); healthcare (15.6%); engineering, mining, and construction (14.4%); and education (9.4%).Among the 160 dyads, the average age of leaders  and CTI, and they also completed the Multifactor Leadership Questionnaire ; followers completed the MLQ-5X (other report) and other measures that are not the focus of the present study . The experiential-thinking scale contains 30 items that represent three facets of experiential thinking: imagination, emotionality, and intuition . The imagination subscale measures respondents’ preference for visual stimuli and tendency to think visually . The emotionality subscale represents respondents’ emotional reactivity . The intuition subscale represents respondents’ tendency to base their decisions on how they feel . An overall experiential thinking scale is calculated by combining these subscales. All items were presented in five-point Likert format with response options ranging from 1  to 5 (Definitely True). The REIm’s scales had good Cronbach’s alpha internal consistency reliability coefficients except for emotionality  and behavioral coping . The emotional coping factor consists of four subscales: self-acceptance, absence of negative overgeneralization, non-sensitivity, and absence of dwelling. Behavioral coping consists of three subscales: positive thinking, action orientation, and conscientiousness. Facets of destructive thinking are represented on four scales: personal superstitious thinking , categorical thinking , esoteric thinking , and naïve optimism . The CTI also includes two lie scales: defensiveness  and Validity . As noted, participants scoring outside the acceptable bounds for Validity or Defensiveness were excluded from the sample. Items were in a five-point Likert format with scores ranging from 1  to 5 (Definitely True). The CTI scales had good Cronbach’s alpha internal consistentcy reliabilities , transactional , and passive-avoidant . Transactional leadership has two subscales (contingent-reward and management-by-exception-active). Passive-avoidant leadership has two subscales , we calculated Pearson’s correlations between thinking styles and both self-rated and other-rated leadership styles. In addition, where multiple thinking styles correlated with transformational leadership, we regressed transformational leadership on thinking styles in order to examine the unique contribution of thinking styles to transformational leadership.via a logarithm transformation. No other statistical assumption breaches were found, and, for ease of interpretation, descriptive statistics are reported for the untransformed data. Principal components analysis with direct oblimin rotation was used to statistically investigate potential common methods bias for the self-report measures. There was little evidence for common methods bias, with the largest single component only accounting for 21.57% of the variance  versions of the MLQ. For self-rated passive-avoidant leadership, the skew was corrected by removing three outliers, for follower-ratings, the skew was corrected variance . Descripr = 0.26, p = 0.001). However, self-rated and follower-rated transactional  and passive-avoidant leadership were not significantly correlated . Thus, there was a weak alignment between how followers evaluated their leaders’ styles and how leaders evaluated themselves.Pearson’s product movement correlations were calculated between leaders’ self-rated and follower-rated leadership styles in order to assess the level of agreement in their ratings of leadership styles. Self-rated and follower-rated transformational leadership correlated significantly but weakly . These analyses were supplemented with relative importance analysis, with relative weights calculated in order to determine the unique contributions of each thinking style variable as a predictor of leadership style . The resF = 15.48, p < 0.001] and 11.8% of the variance in follower-rated transformational leadership . Looking at the significance of the βs and relative weights in The regression analyses found that the thinking styles accounted for 48.2% of the variance in self-rated transformational leadership  but did not account for significant variance in other-rated transformational leadership . For self-rated transformational leadership, only behavioral coping was a significant predictor in the regression , and no thinking style variables significantly predicted other-rated transformational leadership.The regression analyses found that the thinking styles accounted for 30.2% of the variance in self-rated transformational leadership  but was approaching significance in Study 2 .Theoretically, the CELM expects that rational thinking will be connected with transformational leadership because rational thinking contributes to adaptation by leaders, and the selection of a transformational leadership style is an effective adaptation . The absIt is possible that a relationship exists between leader’s preference for rational thinking and the extent of their transformational leadership that other raters – especially followers – do not detect. Rational thinking, which engages a slower thinking system, may be associated with more deliberative and less action-focused decision-making that followers may perceive as evincing less transformational leadership. This possibility is in contrast to the relationship between leaders’ preference for behavioral coping and transformational leadership. Behavioral coping is a component of how constructively leaders’ experiential thinking is used . ExperieA limitation of the current studies was the low internal consistency reliabilities obtained for some measures that were of focal theoretical interest – emotionality (Study 1) and behavioral coping (Study 2). via coaching (e.g., The studies presented in this paper aimed to provide a more methodologically robust test of the CELM  than hasng e.g., . TherefoThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Murdoch University Human Research Ethics Committee. Written informed consent for participation was obtained for this study in accordance with the national legislation and the institutional requirements.GC conceptualized the studies, collected and analyzed the data, and wrote the manuscript. SW analyzed the multi-level data in Study 2, and reviewed and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hepatocellular carcinoma (HCC) is the most common malignant disease worldwide. Although the diagnosis and treatment of HCC have greatly improved in the recent years, there is still a lack of accurate methods to predict the prognosis of patients. Evidence has shown that Hippo signaling in tissues adjacent to HCC plays a significant role in HCC development. In the present study, we aimed to construct a model based on the expression of Hippo‐related genes (HRGs) in tissues adjacent to HCC to predict the prognosis of HCC patients.Gene expression data of paired normal tissues adjacent to HCC (PNTAH) and clinical information were obtained from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The HRG signature was constructed using four canonical Hippo‐related pathways. Univariate Cox regression analysis was used to screen survival‐related HRGs. LASSO and multivariate Cox regression analyses were used to construct the prognostic model. The true and false positive rates of the model were confirmed using receiver operating characteristic (ROC) analysis.The prognostic model was constructed based on the expression levels of five HRGs  in PNTAH. The mortality rate of HCC patients increased as the risk score determined by the model increased. Furthermore, the risk score was found to be an independent risk factor for the survival of patients. ROC analysis showed that the prognostic model had a better predictive value than the other conventional clinical parameters. Moreover, the reliability of the prognostic model was confirmed in TCGA‐LIHC cohort. A nomogram was generated to predict patient survival. An exploration of the predictive value of the model in HCC tissues indicated that the model is PNTAH‐specific.We developed and validated a prognostic model based on the expression levels of five HRGs in PNTAH, and this model should be helpful in predicting the prognosis of patients with HCC. We built a prognostic model based on the Hippo signaling for the first time, and found that the expression of the Hippo signaling in adjacent tissues can better predict the survival of liver cancer patients. Paired normal tissues adjacent to tumor (PNTAT) are often used as a normal control for cancer research because of the shortage of healthy samples. However, whether PNTAT is truly “normal” is controversial. Recent studies have shown that the transcriptomic profiles of PNTAT are distinct from those of healthy and tumor tissues.Hippo signaling was primarily discovered for its control of organ size in Drosophila and is highly conserved in mammals.GSE14520 using univariate Cox regression analysis. In addition, we screened out five key HRGs  to construct a prognostic model using least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses. We further confirmed that the model is good at predicting prognosis through survival and ROC curve analysis and found that the risk score calculated by the model formula was an independent risk factor through univariate and multivariate Cox regression analyses. Finally, we generated a nomogram to provide clinicians with a quantitative method for predicting survival. Moreover, the reliability of this model was validated by analyzing the PNTAH expression profiles from TCGA‐LIHC. Exploration of the predictive value of these five HRGs and the constructed model in HCC tissues demonstrated that the model is PNTAH‐specific. In summary, the present study may help reveal the underlying role of Hippo signaling in PNTAH and provide a useful prediction tool for HCC survival.In this study, we first constructed a Hippo‐related gene (HRG) signature using four canonical Hippo‐related pathways. We then identified 14 prognostic HRGs based on PNTAH expression profiles from 22.1GSE14520 dataset , GSE102079 dataset , and GSE112790 dataset (15 NL samples) were downloaded from the GEO database (www.pubmed.com/geo). The ComBat function from the sva package in R was used to remove the batch effects of the three datasets. TCGA‐GTEx cohort was downloaded from the UCSC Xena browser (http://xena.ucsc.edu/), among which 110 normal liver samples of GTEx, 50 PNTAH samples, 371 HCC samples, and the corresponding clinical data of TCGA liver cancer hepatocellular carcinoma (TCGA‐LIHC) were selected for the next analysis. To make data from different sources more compatible, the UCSC Xena project recomputed all expression raw data based on a standard pipeline to minimize differences from distinct sources. Principal component analysis (PCA) was applied using the PCA function implemented in the FactoMineR package. The 232 PNTAH samples from GSE14520 were used to construct the prognostic model, and 50 PNTAH samples from TCGA‐LIHC were used for validation.Expression profiles from the 2.2http://software.broadinstitute.org/gsea/msigdb/). After merging the four canonical pathways, a Hippo‐related signature was constructed. The heatmap was plotted using the pheatmap package in R to show the expression levels of the HRG signature in different samples. The protein–protein interaction (PPI) of the HRGs was predicted using STRING (https://string‐db.org/) and visualized using Cytoscape (v3.7.2). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out using the R clusterProfiler package. Enrichment results were visualized using the enrichment plot package. A p‐value <0.05 was set as the cutoff criterion for both GO and KEGG functional analysis.Four Hippo‐related canonical pathways, GO_HIPPO_SIGNALING, KEGG_HIPPO_SIGNALING_PATHWAY, REACTOME_SIGNALING_BY_HIPPO and WP_HIPPOYAP_SIGNALING_PATHWAY, were obtained from the Molecular Signature Database  + (the expression of gene2 in PNTAH × regression coefficient of gene2) + … + (the expression of genen in PNTAH × regression coefficient of genen).To screen out the prognostic HRGs, the association between PNTAH HRG expression and overall survival (OS) was evaluated using univariate Cox regression. Genes with a Based on the median risk score calculated using the PNTAH expression profiles, the patients were divided into low‐ or high‐risk groups. The difference in OS between the two groups was analyzed using the Kaplan–Meier method and log‐rank test. The risk score distribution, number of patients examined, and the heatmap of the prognostic HRGs in different risk groups were displayed. Univariate and multivariate Cox regression analyses were performed to explore whether the risk score could be an independent indicator of OS. The true and false positive rates of the prognostic model were analyzed using the receiver operating characteristic (ROC) and the area under the curve (AUC). A nomogram was constructed to estimate the 1‐, 3‐, and 5‐year survival rates of HCC patients using the rms package in R, and calibration of the nomogram was measured using calibration curves. Moreover, the predictive value of the constructed model was further confirmed using independent data from TCGA‐LIHC.2.4GSE14520. HCC patients were divided into high‐ or low‐expression groups based on the median expression of the key HRGs in HCC tissues. Moreover, the risk score was calculated with the established formula for PNTAH, using the HCC tissue expression profiles, and the patients were divided into high‐ or low‐risk groups based on the median risk score. The survival curve was drawn using the Kaplan–Meier method, and the difference in the survival rate between different groups was verified by the log‐rank test. Similarly, the prognostic value of the key HRGs and the constructed model for the HCC tissues of TCGA‐LIHC cohort were also analyzed.To investigate whether the prognostic model built using PNTAH also had prognostic value in HCC tissues, the expression profiles of HCC tissues were extracted from 2.5https://www.r‐project.org). Univariate Cox regression analysis was conducted to estimate prognosis‐related HRGs. The Kruskal–Wallis rank sum test was used to determine whether the HRGs were differentially expressed among NL, PNTAH, and HCC. LASSO regression analysis was used to prevent overfitting. Multivariate Cox regression analysis was performed to construct a prognostic model. An independent t‐test was performed to analyze the association between the risk score and conventional clinical characteristics. A nomogram was created using the rms package in R. ROC analysis was performed to test the true and false positive rates of the model. The survival curve was plotted using the survival and survminer package of R. Forest maps were plotted using the forsetplot package of R. The survivalROC package was used to generate the ROC curves, and the AUC values were calculated according to the ROC curves. All tests were two‐tailed and considered significant when p < 0.05.Statistical analysis was carried out using R 4.0.1 , suggesting that the expression of NF2 in PNTAH was of great significance in HCC patients. However, since the HR of NF2 was much higher than that of other genes, it was temporarily excluded from the forest map of the univariate regression analysis. In addition, these 14 genes were found to be differentially expressed among NL, PNTAH, and HCC  had a lower survival rate than the low‐risk group (n = 105)  Figure . The risn Figure . The res3.5p = 2.949e‐02)  Figure . The ris) Figure .3.6p < 0.001) and tumor stage  remained as independent prognostic factors for patients , advanced stage (p = 0.005), and more recurrence (p = 4.009e‐06). To show the relationship between individual predictors and survival rate, a nomogram model was developed based on the data of the GEO cohort and converted to scale within a certain range  to construct the prognostic model. High expression levels of BIRC3, CSNK1E, MYC, and NF2 in PNTAH were associated with poor prognosis in HCC patients, while MINK1 expression was associated with a good prognosis. We divided patients into high‐ or low‐risk groups based on the median risk score calculated by the model formula and found that the high‐risk group had a lower survival rate. The AUC calculated by the ROC curve was 0.750, indicating that the model could predict the survival of HCC patients. We then confirmed that the risk score was an independent prognostic indicator after adjusting for other clinical parameters. ROC curve analysis demonstrated that the risk score had a better predictive value than other clinical parameters. Finally, we established a nomogram that predicted the survival of HCC patients well. The use of this tool could help clinicians dynamically assess a patient's prognosis based on different levels of clinical parameters and implement more targeted interventions accordingly. Furthermore, we also observed a similar trend in the survival analysis and ROC curve analysis of an independent dataset from TCGA, which further confirmed the reliability of this prognostic model. Given that PNTAH might be a precancer state, the five HRGs and the constructed model could have better prognostic performance in HCC tissues. We investigated the prognostic ability of the five HRGs and constructed a model using the expression profiles of HCC tissues from GEO and TCGA‐LIHC. Interestingly, the results showed that neither the five HRGs alone nor the constructed model had prognostic value in HCC tissues. Thus, we concluded that the prognostic model based on HRGs had a prognostic value specific for PNTAH.We further explored five selected HRGs. NF2 is a well‐established tumor suppressor and an essential upstream regulator of Hippo signaling.Some limitations of this study should also be considered. First, while the expression profiles were downloaded from the GEO and TCGA databases, the sample size was not large enough. Second, we only focused on the mRNA levels of these genes, and the five genes at the protein level should be further investigated. Third, the results of our study are descriptive, and the potential molecular mechanisms of these five genes warrant additional functional experiments. In addition, it has not been determined whether the median risk score we chose in the GEO and TCGA databases can be used as the threshold in real‐world clinical practice to identify high‐risk and low‐risk patients.5In the present study, we constructed and validated a prognostic model using PNTAH expression profiles, and this model could predict the survival of patients with HCC. The differentially expressed HRGs may provide a new perspective for the elucidation of the molecular mechanisms of HCC.The authors have no conflicts of interest to declare.(I) Conception and design: Y Zeng; (II) Administrative support: H Ren; (III) Provision of study materials or patients: QB Pan; (IV) Collection and assembly of data: BN He, N Yang, YT Zhang, HY Yuan; (V) Data analysis and interpretation: QB Pan, FB Qin; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All data were publicly available and downloaded from online databases; therefore, this study did not require additional ethical approval.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.


GRM8, SPC25, FSD1L, SLC386A, FAM72A and SLC39A10) were screened via LASSO Cox regression, which provided the basis for developing a novel prognostic risk model. Based on the risk model, HCC patients were subdivided into high-risk and low-risk groups. Kaplan-Meier curve indicated that patients in the high-risk group have a lower survival rate compared with those in the low-risk group. The prognostic model showed good predictive efficacy, with AUCs reaching 0.802 at 1 year, 0.694 at 2 years, and 0.672 at 3 years. Univariate and multivariate cox regression analysis demonstrated that the risk score had significant prognostic value and was an independent prognostic factor for HCC. Moreover, this model also showed a good diagnostic positive rate in the ICGC-LIRI-JP and GSE144269. Finally, we demonstrated the efficacy of the AF-risk model in HCC patients following sorafenib adjuvant chemotherapy. And revealed the underlying molecular features involving tumor stemness, immune regulation, and genomic alterations associated with the risk score. Based on a large population, we established a novel prognostic model based on 6 AFs to help identify HCC patients with a greater risk of death. The model may provide a reference for better clinical management of HCC patients in the era of cancer precision medicine.Hepatocellular carcinoma (HCC) is the most common primary liver cancer with poor prognosis. An optimized stratification of HCC patients to discriminate clinical benefit regarding different degrees of malignancy is urgently needed because of no effective and reliable prognostic biomarkers currently. HCC is typically characterized by rich vascular. The dysregulated vascular endothelial growth factor was proved a pivotal regulator of the development of HCC. Therefore, we investigated the capability of angiogenic factors (AFs) in stratifying patients and constructed a prognostic risk model. A total of 6 prognostic correlated AFs ( Hepatocellular carcinoma (HCC), as the most common primary liver cancer, is amalignant tumor with poor prognosis . HCC is Tumor’s access to the blood system is mainly accomplished by sprouting angiogenesis . AngiogeFor example, WNT2 has been confirmed to correlate with prognosis and considered to be an angiogenic growth factor that promotes liver regeneration . ExpressIn this study, we constructed and verified an effective prognostic risk model based on the expression of informative AFs. The investigation of the risk score deepened further understanding of the divergence of molecular features underlying different risk groups. This model was also proved to be suitable for patients following sorafenib adjuvant chemotherapy and we created the predictive nomogram. As a whole, this prognostic model might help guide the prognostic status of patients with HCC.http://xena.ucsc.edu/). We also obtained two independent validation cohorts of HCC patients from the ICGC portal  and the GEO , with transcriptomic and clinical data available.The raw counts of RNA-Seq data and corresponding clinical information of TCGA-LIHC patients were collected as the training cohorts. Data were downloaded from UCSC Xena (http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). The final AFs gene set studied here was the combination of genes from the two resources  and FDR of each gene was analyzed by DESeq2 (version 1.34.0) package . The twop-value < 0.01 and HR > 1 or HR < 0.5. Kaplan-Meier analysis was also performed to screen the prognostic candidate AF genes using R package survival (version 3.2-13) (Univariate Cox regression was performed for each AF genes to obtain the prognostic genes with  3.2-13) . MultivaThe prognostic candidate AF genes were screened by univariate cox regression and log-rank test. Tumor samples of the TCGA-LIHC were used as the training cohort to establish the LASSO model. A lasso penalty was used to find the best gene model utilizing an R package glmnet. The risk score for each sample can be calculated with the final LASSO model coefficient as follows:The median risk score was used as cutoff for high-risk group (with higher risk score) and low-risk group (with lower risk score).CIBERSORT algorithm was used to evaluate the infiltration of 22 immune cell types . The immMutation comment file (MAF) of TCGA-LIHC cohort was downloaded from the GDC client. Differential analysis and visualization of somatic mutations were performed using maftools package. The Fisher’s exact test was used on all genes between two groups to detect differentially mutated genes. Segment file of the TCGA-LIHC cohort was downloaded from FIREHOSE and analyzed using the GISTIC 2.0 pipeline .AFs-derived risk scores, TNM stages, clinical stage, gender, age and grade were used as independent prognostic factors through univariate cox regression and AFs-derived prognosis risk model. Nomogram was finished based on the results of multivariate cox regression analysis. The calibration curves of the nomogram were constructed to test consistency between 1-, 3- and 5-years survival probability prediction and actual observation. The performance of the nomogram was evaluated using the concordance index (C-index) and time-dependent receiver operating characteristic (ROC) curves. Nomograms analysis and visualization were performed using R packages rms (version 6.2-0)  and survFirst, the large population of liver hepatocellular carcinoma (LIHC) patients from the TCGA database was used as the training cohort. We downloaded the transcriptomic and clinical data for 371 tumor samples and 50 adjacent normal samples. A total of 8,250 differentially expressed genes were found between tumor and normal samples . We then systematically collected AF genes from NCBI and MSigDB (see Methods) and noticed 1,038 AFs were differentially expressed  > 1 or HR < 0.5, p-value < 0.05, EGF, GRM8, TRPM6, SLC38A6, BLM, BARD1, CLSPN, PRIM2, MSH2, FAM72A, SPC25, IGF2BP3, CENPP, GTF2IRD1, TMC7, FSD1L, and SLC39A10. The distribution of expression levels and Kaplan-Meier curves of the four genes with top significance were displayed  and low-risk group (n = 159) according to the median cut-off value of risk score. The overall survival (OS) time of patients in the high-risk group was remarkably decreased  and GSE144269 (N = 68) were collected. Patients were all separated into high- or low-risk groups according to the risk score. The high-risk group of the ICGC-LIRI-JP validation cohort also showed significantly lower survival rate than the low-risk group . The predictive capacity was proved as AUC reaching 0.602 at 1-year, 0.632 at 2-years, and 0.709 at 3-years in ICGC-LIRI-JP cohort , tumor stage, virus infection status, etc. . Chronicetc. . We obsep-value < 2.2e-16, Next, we investigated the associations between the AFs risk score and the immune response in tumors. The mutation burden (TMB) was not differed between high- and low-risk groups , while tp-value <0.05). Among them, the mutation frequency of HCC driver gene TP53 was enriched in the high-risk group , this observation suggested the classic role of TP53 in cell-cycle regulation and guarding genome stability might also contribute to the malignant progression of HCC . Previously, multi-omics integration analysis revealed three HCC subtypes, one of which exhibited few CTNNB1 mutations companied by poor prognosis  was analyzed through maftools and GISTIC 2.0. As shown in n of HCC . While trognosis . This wap-value < 0.0001, Accumulating evidence supported that sorafenib was effective in extending the time of progression in HCC . In ordep-value < 0.05, TIDE is the tumor immune dysfunction and rejection score, depicting the primary mechanisms of tumor immune evasion. It was proved to predict the clinical response and outcomes of patients following immunotherapy . We usedFinally, a graphic prognostic nomogram based on the 6-AFs genes was developed for 1-, 3-, 5- and 10-years prediction of OS for HCC patients from TCGA. The tumor stage, grade, age, and gender were also included . MeanwhiIn the present study, we established an important prognostic model based on 6 DE-AFs genes significantly related to the prognosis of HCC, and further verified it in two independent validation datasets. The patients in the high-risk group showed poor prognosis, which was consistent in the three cohorts. Through univariate and multivariate Cox regression analysis, the risk score had significant prognostic value and was an independent prognostic factor of HCC. The model suggested that high risk may cause the regulation of immune mechanism, and these 6 gene signatures in the model could be used as potential prognostic molecular markers of AFs in HCC.Several prognostic staging systems have been built for liver cancers, such as the Japan Integrated Staging score , the CanGRM8, SPC25, and FAM72A was negatively correlated with favorable outcomes and also observed in other cancer types such as lung cancer (GRM8) was elucidated to promote the survival of squamous cell lung carcinoma (LUSC) tumor cell through inhibiting cAMP pathway and activating MAPK pathway and the transcription level of GRM8 was reversely correlated with the prognosis of LUSC cases  is a complex ecosystem consisting of various types of cells and the extracellular matrix with obvious heterogeneity . Tumor cSorafenib is an oral multikinase inhibitor, its action mechanism includes inhibition of both MAPK/ERK-mediated cell proliferation and angiogenesis driven by VEGF signalling . SorafenNomograms have been widely used as prognostic devices in oncology and medicine . ConstruIn summary, we constructed and validated a novel risk model consisting of 6 prognostic-associated AFs genes. This risk model showed effective and independent prognostic power, thereby providing important insight into the survival prediction of HCC. To our knowledge, this is the first study to predict prognosis of HCC patients based on the expression levels of AFs. Therefore, our study provided novel insights into the relationship between the regulation of AFs and development of HCC. In addition, we also revealed the underlying molecular features involving tumor stemness, immune regulation and genomic alterations between high/low-risk groups in this model. We expected further verification and mechanism exploration by the accumulated datasets in the future.


Galinsoga quadriradiata, across populations at different elevations in the Qinling and Bashan Mountains in central China. Seed mass–area ratio (MAR), an important seed dispersal-related trait, of 45 populations from along an elevational gradient was measured, and genetic variation of 23 populations was quantified using inter-simple sequence repeat (ISSR) markers. Individuals from four populations were then planted in a greenhouse to compare their performance under shared conditions. Changing patterns of seed dispersal-related traits and populations genetic diversity along elevation were tested using linear regression. Mass–area ratio of G. quadriradiata increased, while genetic diversity decreased with elevation in the field survey. In the greenhouse, populations of G. quadriradiata sourced from different elevations showed a difference response of MAR. These results suggest that although rapid evolution may contribute to the range expansion of G. quadriradiata in mountain ranges, dispersal-related traits will also likely be affected by phenotypic plasticity. This challenges the common argument that dispersal ability of invasive plants increases along dispersal routes. Furthermore, our results suggest that high-altitude populations would be more effective at seed dispersal once they continue to expand their range downslope on the other side. Our experiment provides novel evidence that the spread of these high-altitude populations may be more likely than previously theorized and that they should thus be cautiously monitored.Detecting shifts in trait values among populations of an invasive plant is important for assessing invasion risks and predicting future spread. Although a growing number of studies suggest that the dispersal propensity of invasive plants increases during range expansion, there has been relatively little attention paid to dispersal patterns along elevational gradients. In this study, we tested the differentiation of dispersal-related traits in an invasive plant,  Galinsoga quadriradiata across populations at different elevations in Qinling and Bashan Mountains, China. Our results suggest that the dispersal ability of G. quadriradiata decreases along elevational dispersal routes as a result of genetically based rapid evolution and phenotypic plasticity, challenging the common argument that dispersal ability of invasive plants increases along dispersal routes. Our findings provide new insight into how the dispersal ability of invasive plants may change along the elevational gradient. This is important for revealing the upward expansion dynamic of invasive plants in high mountains.In this study, we tested the differentiation of dispersal-related traits in an invasive plant  Invasive plants, which tend to spread uncontrollably and cause environmental or economic damage , land-usPlants have been shown to do this via two main strategies: phenotypic plasticity and genetic adaptation . Other eSolidago canadensis, for which genetic diversity decreased significantly with elevation  How do population-level traits of G. quadriradiata related to dispersal vary across an elevational gradient? (ii) Are these changes attributable to phenotypic plasticity, genetic adaptation or a combination of both? (iii) What do these changes suggest about the future expansion of G. quadriradiata in the mountains of central China?In this study, we investigated how dispersal-related traits of invasive Galinsoga quadriradiata  is an annual herbaceous plant originating in Central and South America. It is a harmful agricultural invasive weed, mainly established in moist, warm temperate and subtropical zones around the world on abandoned land or farmland  are characterized by complex topography and distinct environmental conditions between the northern and southern regions . To calculate HSW, we randomly selected 100 ripe seeds from the collected seeds of each population and weighted them. At least five replicates were made for each population. We used WinSEEDLE Pro  to measure seed length, pappus length and pappus width. Each time, we randomly selected 30 ripe seeds from an elevational population and scanned and analysed them. We repeated this 10 times for each population.G. quadriradiata, is typically approximated by morphological characteristics . Seven primers  were used to amplify the 233 samples from 23 of the 45 populations. The polymerase chain reaction (PCR) system for ISSR analysis was as follows: total volume 25 µL; 12.5 μL 2 × Es Taq MasterMix (Dye), 1 μL 10 μmol L−1 primer, 1 μL 50 ng μL−1 DNA, 10.5 μL ddH2O. All reactions were performed on a gradient PCR instrument (Agilent SureCycler 8800). The PCR procedure was as follows: pre-degeneration at 94 °C for 2 min, degeneration at 94 °C for 30 s, annealing at 50.5–55 °C for 30 s , extension at 72 °C for 1 min, 6 cycles; degeneration at 94 °C for 30 s, annealing at 50.5–55 °C for 30 s , extension at 72 °C for 1 min, 32 cycles; final extension at 72 °C for 3 min and preservation at 4 °C. The amplification products were separated in 2 % agarose gel (containing 0.5 mg mL−1 ethidium bromide) in 1 TBE (Tris-Borate-EDTA), and the separated bands were visualized under UV light by using an Electrophoresis Documentation and Analysis System 120 (Eastman Kodak Company). DL2000 ladder (Dongsheng Biotech Ltd) was used as DNA molecular weight markers.We used the inter-simple sequence repeat (ISSR) markers to analyse the genetic diversity of B method , and dil−1, respectively. Eight replicate plants from each population were grown, yielding a total of 32 pots. Pots were randomly arranged in an 80-m2 greenhouse, and the positions of the pots were changed at random every 2 weeks. Seedlings were watered daily and were replaced if mortality occurred within 1 week of the start of the experiment. The plants began to bloom in early June and the seeds began to disperse in late June. We collected mature seeds every 2 days until all plants no longer yielded seeds. All seeds were stored in envelopes and air-dried under laboratory conditions until measurement. We then analysed seed dispersal-related traits using the same method as described above for the field survey.To explore the influence of genetic differentiation on dispersal ability of different populations in a common environment on the basis of field survey, we randomly selected populations from four elevations. In late April 2016, seeds from the four populations (BS1 (223 m asl), QL5 (680 m asl), QL22 (1307 m asl) and BS15 (1756 m asl); We constructed linear mixed-effects models to evaluate the effects of elevation on diaspore-related traits , using the packages ‘lme4’ and ‘lmerTest’ in R-3.5.3 . ElevatiI) is usually used as an evaluation index, with higher values reflecting higher genetic diversity. Expected heterozygosity (He) is often used to measure the genetic diversity of a population, with higher values indicating richer the genetic diversity.Only distinct, reproducible and well-resolved PCR fragments were included in the statistical analysis for genetic differentiation. Inter-simple sequence repeat bands were scored as presence (1) or absence (0) characters, to construct the binary matrix. To compare the amount of total genetic variation partitioned within and among populations, three methods were employed, namely the hierarchical analysis of molecular variances (AMOVA), the analysis of Shannon’s diversity  and Nei’Linear mixed-effect models were then constructed to test for associations between genetic diversity and elevation (fixed factor) for the field seedlings, using mountain (Qinling and Bashan) as a random factor. We then constructed the phylogenetic tree of UPGMA based on Nei’s genetic distance using MEGA-X . We alsoseeThe results of the linear mixed-effect models revealed that elevation has significant effects on HSW, NSC, seed length, pappus length, pappus width and MAR see, NSC Fisee, seed leI and He were significantly negatively correlated with population elevation . Furthermore, these trends were associated with reductions in genetic diversity at high elevations, suggesting that adaptation is largely responsible for the observed patterns. However, some of contradicted results from our greenhouse study showed greater dispersal-related traits of higher-elevation population in unstressed environment, suggesting that trait plasticity could still play an important role in the range expansion of invasive species in mountain ranges.Elevational variation associated with seed dispersal-related traits of invasive species has not yet been fully explored, creating a knowledge gap of how phenotypic plasticity and genetic diversity affect the invasive success of plants at high altitudes. In this study, we examined the phenotypic plasticity and genetic diversity of dispersal-related traits in invasive Spatial selection theory hypothesizes that dispersal phenotypes are spatially separated and that only the best dispersers will accumulate towards the range front . This thFor wind-dispersed Asteraceae seeds, dispersal ability is often approximated by measuring seed MAR, which is correlated with terminal seed velocity . Higher G. quadriradiata seeds exhibiting positive correlations between MAR and elevation  and the rate of gene flow (m), are strongly influenced by the demography and spatial distribution of populations, with optimal parameter values in central populations and less optimal values in marginal populations  and other dispersal-related traits has also been detected by others (G. quadriradiata in central China. This differentiation was also associated with dispersal-related traits of this species, suggesting that dispersal ability may be genetically based. Moreover, differentiation in MAR between elevational populations in the greenhouse experiment (seeG. quadriradiata in mountain ranges. Although our results support the idea that mountain ranges can act as natural barriers to plant invasions, the plasticity demonstrated in the greenhouse experiment implies that once acclimated to high elevations, acclimating back to low elevations on the other side will occur quickly and therefore should be cautiously monitored. Further research is needed to link the traits from this study directly to dispersal ability as well as to investigate these patterns across broader geographic gradients.Trait variation among populations has been associated with changes in natural selection pressure along elevation . Long-teiment see. Our resThe following additional information is available in the online version of this article—I and He.plab008_suppl_Supplementary_MaterialsClick here for additional data file.plab008_suppl_Supplementary_DataClick here for additional data file.


In the past decade the treatment of patients with heart failure with reduced ejection fraction has quickly gained momentum. Numerous randomised controlled trials (RCTs) have proved the benefit of angiotensin receptor-neprilysin inhibitors (ARNIs) and sodium-glucose transport protein 2 inhibitors (SGLT2i) in addition to the current ‘guideline-directed medical therapy’ (GDMT) –3.The novel European Society of Cardiology (ESC) guideline on heart failure (HF) that will be published this year will undoubtedly recommend a substantial change in GDMT, presumably in accordance with the previously published ESC position papers and the American College of Cardiology consensus document , 5. UntiNetherlands Heart Journal.Despite the significant improvement in pharmacotherapy for HF, previous studies have demonstrated that real-life patients do not reach the target doses that lead to the beneficial results observed in RCTs, on which the guidelines base their recommendation , 7. It iThe authors retrospectively analysed the effectiveness of GDMT in terms of improvement of left ventricular function and prognosis after a mean follow-up of 3.3 years in a cohort of 378 HF patients with reduced left ventricular ejection fraction (LVEF) between 2012 and 2018. The maximal tolerated dose of each drug type was also recorded, including the reason for intolerance. All patients were newly diagnosed with HF and GDMT was initiated and/or up-titrated according to the ESC guideline , in an oThe mean age of the patients was 65.5 years and just under 35% were women. After up-titration 85% of patients used a beta-blocker, 81% a renin-angiotensin-system (RAS) inhibitor and only 53% a mineralocorticoid receptor antagonist (MRA). Of the patients on medication 73% reached > 50% of the target dose of beta-blockers, for RAS inhibitors 73% and for MRA 77%. The most important reasons reported for not reaching the target dose were bradycardia, hypotension and renal dysfunction.p = 0.002) for mortality and 0.85  for the combined endpoint of mortality and/or HF hospitalisation.After an arbitrarily chosen 9‑month follow-up period, LVEF on average improved from 28% to 39%. In patients with a non-ischaemic cardiomyopathy, higher plasma levels of N‑terminal pro-B-type natriuretic peptide or older age, the LVEF was more likely to improve. The 1‑year mortality rate was 10% and a 33% mortality rate was observed after a median follow-up of 3.3 years. Each 5% increase in LVEF was associated with a hazard ratio of 0.84  or revascularisation remains unclear in this cohort. Potential survivorship bias exists, since 35 patients died before the actual follow-up at 9 months and these patients were not included in the overall analysis. Lastly, the change in functional  class was not described. It was mentioned that the most important reason for not reaching the recommended dose of MRA was that there was no indication at the time of the follow-up visit because patients’ functional status had improved to NYHA functional class I. However, no percentages are provided.Nederlandse Hart Registratie (NHR) will provide more insight into this matter in the future.Despite these limitations, the article by Nauta et al. gives the reader a clear overview of the up-titration of GDMT, LVEF improvement and prognosis of real-life HF patients, albeit the relatively young age and the fact that the treatment is performed in a tertiary care centre suggest that the results could differ from an outpatient population in secondary care. The heart failure registry of the Ultimately this study underlines that it is essential to verify the feasibility of implementing findings from large RCTs in daily clinical care. It shows that the majority of real-life HF patients are able to tolerate GDMT and, more importantly, seem to benefit from this regimen.


Amyloidosis is a disease of deposits classified into systemic and localizedBecause of the severity of the systemic form of the disease and its association with plasmocytoma and multiple myeloma, it is important to distinguish these manifestationsWe present here a case of a 42-year-old patient with ear amyloidosis.® and codeine. Upon the exam, there were hyperemia and edema of the outer ear and pinna , once a week, for 4 weeks, evolving with symptom improvements.Today, the patient is being observed.Amyloidosis is a rare disease, with deposits of protein fibrils. Such protein build up in the tissues may compromise the function of organs, such as the heart,In head and neck amyloidosis, systemic involvement must be ruled out by laboratory tests, such as protein electrophoresis, renal function test, electrocardiogram and abdominal ultrasoundIn this paper, we report partial and temporary stenosis of the external auditory canal, affecting the pinna - a rare manifestation of amyloidosis. In the few reports present in the literature, fullness of the ear and hearing loss are constant findings, which did not happen with pain - which was not reported by other papers.In most cases, amyloidosis is systemic and follows multiple myelomaThere are but a few studies on amyloidosis. Among the localized forms, the ear form is rare. The definition of clinical and laboratory characteristics is important for its diagnosis. It is important to investigate multiple myeloma in the systemic form of the disease.


In the present scenario, the challenge of emerging antimicrobial resistance is affecting human health globally. The increasing incidences of multidrug-resistant infections have become harder to treat, causing high morbidity, and mortality, and are posing extensive financial loss. Limited discovery of new antibiotic molecules has further complicated the situation and has forced researchers to think and explore alternatives to antibiotics. This has led to the resurgence of the bacteriophages as an effective alternative as they have a proven history in the Eastern world where lytic bacteriophages have been used since their first implementation over a century ago. To help researchers and clinicians towards strengthening bacteriophages as a more effective, safe, and economical therapeutic alternative, the present review provides an elaborate narrative about the important aspects of bacteriophages. It abridges the prerequisite essential requirements of phage therapy, the role of phage biobank, and the details of immune responses reported while using bacteriophages in the clinical trials/compassionate grounds by examining the up-to-date case reports and their effects on the human gut microbiome. This review also discusses the potential of bacteriophages as a biocontrol agent against food-borne diseases in the food industry and aquaculture, in addition to clinical therapy. It finishes with a discussion of the major challenges, as well as phage therapy and phage-mediated biocontrols future prospects. Globally, throughout the ages, the primary factor that leads to mortality is infectious diseases. Although infectious disease incidences have been reduced with the advancement in sanitary and hygienic conditions, diseases continued to be a significant threat until the discovery of antibiotics in 1928. The commercialization of antibiotics is regarded as a medical miracle leading to a rise in average life expectancy up to 78.8 years in the United States , and in Bacteriophages are believably one of the most ancient and ubiquitously existing biological entities (viruses) that are capable of infecting and replicating within bacteria and therefore play an important role in sustaining the equilibrium of an ecosystem where bacteria reside. After injecting their DNA, phages use the host machinery to replicate and translate the necessary information required to produce viral progeny (in the case of lytic phages) and enzymes such as spannin, holin, and endolysin. In the case of gram-negative hosts, caudovirales use holin-endolysin or pinholin SAR (Single arrest release) mechanisms for breaking the peptidoglycan bond of the cell wall, resulting in host lysis and the release of viral progeny during lytic cycle . While iE.coli and K. pneumoniae, cystic fibrosis with chronic Pseudomonas aeruginosa infections, and prosthetic joint and osteomyelitis infections due to Staphylococcus aureus . The Shanghai Institute of Phages in China has also started providing clinical treatment with phages to patients suffering from multidrug-resistant infections /lysins are used in the therapy and biocontrol. GMO phages are considered Advanced Therapy Medicinal Product (ATMP) by the European Medicines Agency. For their access to the European market, they must comply with Directive 2001/83/EC, Directive 2001/18/EC (Article 12.2), and Regulation (EC) 726/2004 (Articles 6.2 and 6.3) under current good manufacturing practices (cGMP) . HoweverSeveral studies have indicated the successful treatment of bacterial infections with phage therapy . HoweverThe main rule for finding a bacteriophage for a specific host is to use an environmental sample where the host is located. For example, phages for fish pathogens are generally isolated from coastal or fish farm waters . SimilarPhage characterization generally includes the determination of the plaque morphology, host range and the multiplicity of infection. Structural properties of phages are determined by transmission electron microscopy, thermal and pH stability for therapeutic purpose, and genome/proteome analysis are carried out for checking their suitability for therapy. A plaque assay is performed to assess the lytic efficiency of a bacteriophage where phages producing clear plaques are generally considered lytic/virulent while those forming turbid plaques are considered lysogenic/temperate. Phages can be additionally screened for the presence of integration/recombination/excision/toxin genes that are associated with the temperate life style and have the potential for transduction, which can be tested either by nucleic acid hybridization/PCR or by whole phage genome sequencing . Whole-g−1 h−1  for their use in human, veterinary, aquaculture, and processed foods has increased, but only a limited number of phage products are developed according to guidelines under Good Manufacturing Practices (GMP). To prepare a phage for its use in therapy, it needs to be free of bacterial endotoxins and other gross impurities while concurrently maintaining the phage efficacy. Endotoxins and protein toxins are the main impurities present in phage lysate that need to be removed from therapeutic preparations. Chromogenic Limulus Amebocyte Lysate (LAL) assay  is used −1 h−1 . The tra−1 h−1 . Therefo−1 h−1 , EndoTra−1 h−1 , and LPS−1 h−1 . Dependi−1 h−1 . For int−1 h−1 . For the−1 h−1 .A phage susceptibility test is carried out to simultaneously check hundreds of phage candidates selected from different phage banks and phage laboratories against the bacteria isolated from a patient. A phage susceptibility test can be performed by conventional agar overlay assay, or by direct spot of liquid phage suspension on the targeted bacterial lawn (Direct spot assay). Further, the productivity of phage infection can be evaluated by the Efficiency of Plating (EOP) assay, by studying time kill growth kinetics in for planktonic cultures and the biofilm inhibition/eradication assay . A phageCaudovirales, S. aureus, including MRSA strains and it was found that trehalose and sucrose (0.5 M) were the best additives for protecting the phage particles   and accoterature . Later tterature . Bangladterature . Similarterature . A similterature . Throughterature  it was dterature . Similarfections . Also, tfections . DetaileS. aureus, P. aeruginosa, and E. coli, and to a lesser extent Enterococcus spp. induced infections of different body organs. However, there is a need to develop a relatively standardized protocol to drive the phage therapy beyond its compassionate use to a more accessible frontline therapy.Compassionate therapy refers to using non-standard medicines to treat a patient for which authorized medicines have run out. Article 37 of the “Helsinki Declaration” summarizes the doctrine of the compassionate use of phage therapy and emphasizes the physicians consent to use best practice to treat the patient along with the patients or guardians consent . The regvia pattern recognition receptors  restored healthy gut function and suggested fecal filtrate transfer (FFT) as an alternative and effective approach in comparison to fecal microbiota transplantation (FMT). Similarly, C. difficile infection (RCDI) eliminated the symptoms of infection and restored a healthy gut microbiome.In a recent report by in vivo. So far, most of the phage therapy studies are based on single phage-host pairs or a cocktail of a limited number of phages against the host in vitro. Therefore, understanding the phage interactions with their host and immune response in vivo lays the first stepping stone for the oral therapeutic application of phages in humans.Similarly, E. coli, Campylobacter spp., non-typhoidal Salmonella enterica, Shigella spp., Vibrio cholera, and Listeria monocytogenes were responsible for 96% of the food-borne illnesses. The food industry routinely utilizes several antimicrobial interventions such as chemicals, physical disruption techniques, and irradiation to eradicate the pathogens of the contaminated foods . Similarly, in vitro experiment of phages against A. salmonicida, which showed significant lysis at low MOI in comparison with high MOI , a very limited number of phages  have been characterized completely , and Enterococcus. Bacteriophage or lysins induced cell lysis in the case of gram-negative bacteria may lead to the release of endotoxins which are responsible for inflammatory response and, in severe cases, may cause septic shock. While the use of another phage-derived enzyme, polysaccharide depolymerase helps to overcome this problem because, as such, they do not lyse the bacterial cell but only remove the surrounding polysaccharide layer, thus exposing the bacteria to immune cells  and immune reaction studies should be carried out extensively. The regulations involved in phage preparation and the legal framework should be set up decisively. Also, there is a need to formulate universal and favorable regulations to promote phage or phage-based products for human benefits. In addition, popularizing phage therapy and its benefits over other therapeutic agents will help in the acceptance of phage therapy by medical practitioners. Awareness should be generated among clinicians to offer phage therapy as a treatment option for patients where antibiotics have failed altogether. The areas that need to be strengthened, of course, include expanding phage biobanks/repositories for the timely offering of specific and usable phages, improvements in phage production protocols to provide stable and safe phage preparations, and to relax the regulatory protocols for phage therapy. Looking at the extensive damage to the environment, animal, and human health that antibiotics have posed since their discovery, and simultaneously the benefits that the naturally occurring phages (the living drugs) have offered, it does not seem relevant to completely reject phages.The inclusion of bacteriophages in the treatment of human diseases and food biocontrol has witnessed a significant surge in the last few decades. A large number of studies employing single phages, cocktails, phages in combination with antibiotics, and simultaneous improvements in phage production protocols and the ease of genetic manipulation technologies, have broadened the versatility of phage application. However, there is a requirement for the adoption of favorable regulations to promote phage or phage-based products for human/livestock benefits. The phage therapy provides hope against ever growing menace of antimicrobial resistance however, a major boost is required for widening its application through the involvement of researchers, clinicians, industry, and policy makers. Use of phage therapy also aligns with the goal of one health approach to sustainably benefit the environment with simultaneous improvement in the treatment strategies. Incorporation of phage therapy in medical education and willingness among physicians to consider and apply phages will help accelerate its acceptance. Further, making phage therapy cost-effective by supporting medical tourism and relaxing the stringent regulatory guidelines associated with its compassionate use will help in improving the accessibility of phage therapy as a frontline medical intervention to treat resistant bacterial infections.AJ and TA: conceptualization. AJ: methodology and software. AJ, MV, TA, and RV: validation. NV, BB, RV, and BT: formal analysis. TA: resources and funding acquisition. AJ and MV: data curation. AJ and RV: writing - original draft. MV, TA, NV, and BB: writing - review and editing. AJ, MV, and RV: visualization. TA and BT: supervision. TA and RV: project administration. All authors have agreed to the published version of the manuscript.This work was supported by the National Agricultural Science Fund, Indian Council of Agricultural Research, New Delhi, India and Council of Scientific and Industrial Research, New Delhi, India.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.