Understanding oral health behaviour s and their impact on Oral Health-Related Quality of Life (OHRQoL) may serve as an instrument to articulate the conventional oral health policy framework, thereby ameliorating the overall health of young individuals in the long term.The aim of this study was to investigate the relationship between children’s oral health behaviour s and Oral Health-Related Quality of Life in the capital governorate, Kuwait.12-14) was used to collect the data. Chi-square, t-tests, and ANOVA were used to examine the association between oral behaviour s and children’s OHRQoL.A cross-sectional study involving 607 children aged 12–14 years, randomly selected from schools in Kuwait Capital Region. A validated Oral Health Behaviour s and OHRQoL Child Perception Questionnaires (CPQp > 0.05) but for the overall OHRQoL, frequency of soft drink intake was the only significant predictor associated with 0.2-unit decrease  in total OHRQoL scores.About 52.2% of participants were males and the overall response rate was 93.8%. The mean ± SD for total OHRQoL impact was 3.1 ± 0.58, while the total mean for individual domains- for oral symptoms, functional limitations, emotional well-being and social well-being were 2.89 ± 0.63, 2.89 ± 0.72, 3.1 ± 0.91 and 3.4 ± 0.61, respectively. There was no significant difference in total OHRQoL impact score by frequency of last dental visit, flossing, use of mouth rinse or chewing gum (High frequency of soft drink consumption was related to poorer OHRQoL. Behaviour changing interventions based on OHRQoL inferences coupled with clinical intervention are needed.The online version contains supplementary material available at 10.1186/s12903-023-03454-5. Oral health is defined as: “the state of the mouth, teeth and orofacial structures that enables individuals to perform essential functions such as eating, breathing and speaking, and encompasses psychosocial dimensions such as self-confidence, well-being and the ability to socialize and work without pain, discomfort and embarrassment” . Oral heOral Behaviours are a set of routine practices for keeping the mouth clean and healthy, which substantially prevent oral diseases and maintain overall oral health. These behaviours include regular dental attendance, tooth brushing, regular use of fluoridated toothpaste, flossing, use of mouth rinse and gum chewing. As these behaviours can affect individual’s oral health status, they may play a role in impacting their Oral Health-Related Quality of Life (OHRQoL).Oral Health-Related Quality of Life is defined as: “a subjective evaluation that depicts individual’s comfort while eating, sleeping and engaging in social interaction; his self-esteem; and his satisfaction with respect to their oral health” . OHRQoL 12-14) is a commonly used self-administered Questionnaire that assess children’s OHRQoL [The Children Perception Questionnaire . Parents/Guardians of participating students were informed by a letter which explained the nature of the study and a written consent form to participate in the study prior to data collection via questionnaires. Students who returned the consent form completed and signed were allowed to participate. A copy of the consent was shared with the parents. This process was partially supported by Kuwait’s School Oral Health Program.This study was conducted in the public schools of Capital Education/Health Region in Kuwait during the academic year 2019-2020. A multistage sampling method was used to get a random sample of equal proportions of both genders. The total number of students (both genders) in Grades 7 and 8 in the Capital Governorate as provided by the Ministry of Education was 3643 designated as the population of interest. The public intermediate schools in the capital area were shortlisted from list of all schools registered with the Ministry of Education (22 schools for girls and 20 schools for boys). Later, a random sampling of 16 schools (8 for boys and 8 for girls) was performed. All students from 7th and 8th Grade in those schools were invited to participate.The estimated sample size was calculated by an online calculator of cross-sectional studies by Raosoft. Inc. . The samOral Health (OH) Behaviours Questionnaire: This included 8 questions based on routine behaviours, such as dental attendance, frequency of brushing, use of oral rinses, consumption of sugars, presence of toothache, etc. The format of the questions was adapted from the validated Health Behaviours in School- Aged Children (HBSC) survey [) survey , 13. TheChildren Perception Questionnaire (CPQ12-14): A modified short form of the self-administered Children Perceptions Questionnaire was used to assess children’s OHRQoL [12-14 questionnaire was previously translated and validated in Kuwait [re CPQ12-: A modifn Kuwait .Two self-administered questionnaires with demographic variables were used:12-14 OHRQoL scores for each domain and overall OHRQoL were determined by aggregating all derived outcomes to factors in the corresponding domains or by summation of such outcomes represented as the whole questionnaire. The overall lower score indicates superior OHRQoL and vice-versa. While the Chi-square examined gender difference in oral health between various behaviours, t-test investigated any gender difference in each domain as well as overall OHRQoL scores. ANOVA test assessed difference in domains and overall OHRQoL scores among various behaviours. Post hock and Bonferroni test were applied to find significant difference within the groups.Data was analyzed using SPSS 22.0 software . The Shapiro-Wilks test was used for the determination of continuous variables for normal distribution while frequency and percentage distribution depicted the categorical variables such as oral health behaviour s in terms of score values  for every response. The CPQp≤ 0.05.Further, the linear regression analyses identified the predictors of OHRQoL domains and total scores. Dependent variables included in the regression analysis were children’s comfort while eating, sleeping and engaging in social interaction, while the independent variables were gender , last dental visit (ref. category: less than 1 year), brushing frequency (ref. category: more than once a day), use of mouth rinse (ref. category: more than once a day), flossing frequency (ref. category: more than once a day), gum chewing (ref. category: more than once a day), frequency of sugar intake (ref. category: more than once a day) and frequency of soft drinks consumption (ref. category: more than once a day). The level of significance for all quantitative tests was be set at p<0.05). These behaviour s were dental visits, toothbrushing, gum chewing, sugar intake and frequency of soft drinks consumption , participated in the study with a response rate of 93.8%. About 60% of students had visited the dentist within the previous year and 23.4% between 1-2 years (Table Table p<0.001) with no difference in all other OHRQoL domains or total OHRQoL (Table The mean (SD) for total OHRQoL impact was 3.1 (0.58), while the total mean for the domains were: 2.89 (0.63) for oral symptoms, 2.89 (0.72) for functional limitations, 3.1 (0.91) for emotional well-being, and 3.4 (0.61) for social well-being respectively. Girls had significantly higher mean oral symptoms scores compared to boys . Furthermore, there is a significant difference between mean scores of students who were having soft drinks more than once a day and those who had it once a day (p<0.05).Another set of supplementary tables , functional limitations (p=0.024), emotional well-being (p=0.005) and OHRQoL impact scores (p<0.001) according to sugar consumption frequency. Students who consumed sugars more than once a day had significantly higher oral symptoms, functional limitations, emotional well-being and OHRQoL impact scores as compared to students having lower frequency of sugar consumption. No significant difference was observed between students who rarely consumed sugar and those who had sugar once a day (p>0.05).There was a significant difference in the mean oral symptoms (p<0.05). Furthermore, there is a significant difference between mean scores of students who were having soft drinks more than once a day and those who had it once a day (p<0.05).Students who consume soft drinks once a day or more reported significantly lower scores in all OHRQoL domains as well as the total OHRQoL impact scores than those who rarely/never have soft drinks a day  and nearly 0.3-unit  lower oral symptom scores (worse symptoms) compared to students who brushed more often. The soft drinks intake above once a day limit was associated with a 0.2-unit lower oral symptom score as compared to students who rarely uses soft drinks a day . The frequency of soft drinks consumption was the only significant predictors of functional limitations after adjusting for gender and oral health behaviour s (Table p<0.001). For emotional symptoms, none of the behaviours were significantly correlated with Emotional well-being. The frequency of soft drinks intake was the only significant predictors of social symptoms after adjusting for gender and oral health behaviours . For the overall OHRQoL, frequency of soft drink intake was the only significant predictor (Table p=0.002).The consumption of soft drinks more than once a day was associated with nearly 0.2 unit lower in social symptoms scores compared to students who rarely use soft drinks a day  ensuring participant confidentiality. Although due to the limitations in the present study design, being a cross-sectional one, inferences so obtained cannot impress a direct impact on the OHRQoL, yet the manifestation of high soft drinks consumption by children does offer a reconsideration towards their present understanding of oral health. The findings of the present study were also in agreement with several studies conducted among Kuwaiti school children [In this study, data were gathered through face-to-face interviews to eschew any data loss, minimize information bias, and enhance the accuracy of data . The reschildren –18.It has also been ruled in that since most of the sweet beverages seem to contain acid and regular sugar, the development of both dental erosion and caries are likely to have been positively influenced by high soft drinks drinking . AlthougThe findings of current study bring our attention towards the importance of adopting the practices which can improve consciousness among caretakers and students for maintaining proper oral hygiene from early stage convalescing overall oral health. A more comprehensive model, on the contrary, such as the common risk factor approach may bring about better outcomes in changing the soft drinks consumption behaviour in children than a conventional oral health education approach . This apTo our knowledge, this study is one of the first studies to explore the relationship between oral health behaviour s and OHRQoL among 12-14 years students in Kuwait. Another strength of the study is the randomness of the sample, the high response rate (93.8%), as well as large sample size (607 students). Furthermore, the present study used a strong, validated, and reliable questionnaire that had been previously used with similar populations . All theOut of all examined behaviours, frequency of soft drinks consumption more than once a day was a significant predictor of OHRQoL among 12-14 years old students. Comprehensive health promotion approaches like the common risk factor approach targeting sugar consumption as policy level change can opt to discourage the consumption of sugar; the teachers and dental experts should engage caretakers with regular counselling, parents should be adequately vigilant to fulfil their responsibility for maintaining the overall well-being of their children and religious adoption of pragmatic preventive measures must be encouraged to improve their OHRQoL perpetually.Additional file 1: Table S1. Oral health behaviors/symptoms as a measure of OHRQoL in both genders of 12-14 years aged students (N=607), Kuwait, 2020. Table S2. Functional Limitations as a measure of OHRQoL in both genders of 12-14 years aged students5653405244411500 (N=607), Kuwait, 2020. Table S3. Emotional well-being as a measure of OHRQoL in both genders of 12-14 years aged students (N=607), Kuwait, 2020. Table S4. Social well-being as a measure of OHRQoL in both genders of 12-14 years aged students (N=607), Kuwait, 2020. Table S5. Total OHRQoL scores of all the oral behaviours in both genders of 12-14 years aged students (N=607), Kuwait, 2020.


P < 0.01) than those fed the high-energy rye diet or the low-energy diet without rye, which was caused by a reduced weight gain during the initial 17 d of the trial. Final BW and overall feed intake were not affected by rye inclusion or NE level. The NE intake was greater (P < 0.05) and feed efficiency (G:F) was reduced (P < 0.05) in pigs fed rye diets compared to those fed diets without rye, whereas there was no effect of NE level on NE intake or G:F. There was no effect of rye inclusion or NE level on lesion scores. In conclusion, pigs can be fed diets including 40% hybrid rye with only minor changes in growth performance. Increasing the NE level of the first phase diet in the grower-finisher barn may be useful to avoid a reduction in growth performance when feeding hybrid rye.Previous research has shown reduced feed intake and growth rate in pigs fed diets with hybrid rye replacing wheat. A reduction in growth rate caused by reduced feed intake will conceivably be counteracted by increasing the dietary energy level. Our objective, therefore, was to determine the effects of 40% hybrid rye inclusion replacing wheat in diets formulated to be either low or high net energy (NE) on growth, feed intake, energy digestibility, and lesion scores in growing-finishing pigs. We hypothesized that pigs fed 40% hybrid rye would perform better on the high than the low energy diets. A total of 160 pigs (body weight [BW] 70.1 kg) housed in 32 pens, 5 pigs per pen, were fed diets with 0% or 40% hybrid rye , either with low  or high  kcal NE per kg of diet over two growth phases . The BW, and feed disappearance were measured on days 0, 8, 17, 28, 42, and 50. Fecal samples obtained in phase 2 (~100 kg BW) were used to calculate apparent total tract digestibility (ATTD) of gross energy (GE). Lesion scores were measured weekly. The ATTD of GE was unaffected by rye inclusion and was reduced in low vs. high NE diets. Overall, (days 0 to 50), pigs fed the low-energy rye diet gained 0.08 kg/d less ( The inclusion of 40% hybrid rye in diets for growing-finishing pigs resulted in only minor changes in growth performance. Increasing the energy level of the first phase diet in the grower-finisher barn may be useful to avoid a reduction in growth performance when feeding hybrid rye. Access to a variety of grains and grain co-products allows the formulation of swine diets at the lowest cost. While it has been known for many years that rye can be used in swine feeds, the low acreages planted and susceptibility of rye to mycotoxins, specifically ergot, have limited its inclusion . HoweverRecent research has demonstrated that the new hybrid rye varieties can replace wheat  or corn Our objective was to determine the effects of 40% hybrid rye inclusion in diets formulated to be either low or high NE on growth, feed intake, energy digestibility, and lesion scores of growing pigs. We hypothesized that growing-finishing pigs fed 40% hybrid rye would perform better on the high than the low-energy diets.Study procedures were reviewed and animal use was approved by the University of Saskatchewan Animal Research Ethics Board and followed principles established by the Canadian Council on Animal Care . The stuA total of 160 pigs  with an average initial body weight (BW) of 70.1 kg, were placed into two rooms, 16 pens per room, and five pigs per pen. Pens measured 2.4 m × 1.7 m. Flooring of each pen was fully slatted concrete, back, and sides of pens were concrete, with a front gate made of polyvinyl chloride planking. Each pen was equipped with a single-space dry feeder. A nipple drinker was located on the back wall of the pen. The room was ventilated using negative pressure and temperature was maintained within the thermo-neutral zone for pigs. Artificial light was provided for 12-h (0700 to 1900 h) followed by 12-h of darkness.The trial utilized a randomized complete block design with a 2 × 2 factorial arrangement of treatments. Within an area block of four pens, barrows, or gilts were randomly allotted to diets with either 0% or 40% hybrid rye replacing wheat , and witExperimental diets were fed to slaughter weight over two growth phases . Diets had similar inclusion of barley and canola meal. Rye NE value was estimated based on previous research conducted at the Prairie Swine Center (unpublished) that provided a value of 3,509 kcal/kg for DE and chemical analysis of the hybrid rye was used to calculate the NE value using equation 1.8 of Pigs were weighed at the initiation of feeding the experimental diets (day 0), and on days 8, 17, 28, 43, 50, and 57. Feed was weighed and delivered to pigs by hand and feed remaining in the pen feeder on weigh days was weighed back. Collected data were used to calculate pen average daily feed intake (ADFI), average daily weight gain (ADG), and feed efficiency expressed as ADG/ADFI (G:F).Fecal samples were obtained by obtaining freshly excreted samples over a 24-h period, a minimum of one sample per pen, when pigs had received the phase 2 celite-containing diets for one week (~100 kg BW). Samples were only collected if defecation was observed and were immediately frozen (−20 °C). Apparent total tract digestibility (ATTD) of energy was calculated based on the indicator method using AIA as a marker and using the following NF is the nutrient concentration in feces; ND is the nutrient concentration in the diet; MD is the marker concentration in the diet; and MF is the marker concentration in feces.Where NE of the diets was estimated according to Where DE is DE, EE is ether extract, crude protein (CP) is CP, and acid detergent fiber is acid detergent fiber.Lesion scores were measured on the face, ear, body, and tail of each pig weekly in the morning and afternoon of the same day to distinguish between old lesions (morning score) and new lesions (afternoon minus morning score). The scores for the different body parts were summed to create one score per animal. The lesion scoring system, adapted from Fecal samples were dried in a forced-air draft oven at 55 °C for about 72 h minimizing N loss . The feeS and R epimers combined.Rye samples were analyzed for moisture (AOAC 930.15), CP (AOAC 990.03[M]), ether extract (AOCS Am 5-04), ash (AOAC 923.03), acid detergent fiber (Ankom method 12[M]), neutral detergent fiber (Ankom method 13[M]), and amino acids  at the Central Testing Laboratories, Winnipeg, MB, Canada. Diets and rye samples were analyzed for mycotoxins and ergot alkaloids (EA) using UHPLC-MS/MS following solvent extraction at Prairie Diagnostic Services . A total of six EA were determined, Pen was the experimental unit for all variables. Because the sampling unit for BW and lesion scores was individual pigs, an average for each pen was calculated prior to statistical analyses.Growth performance and energy digestibility were analyzed using the GLIMMIX procedure of SAS using a normal distribution. Models included the fixed effects of rye level (0% or 40%), energy level (low or high), sex (barrows or gilts), and interactions. Block was considered a random effect in the model. Overall ADG, ADFI, NE intake, and G:F were analyzed using closeout data. BW, ADG, ADFI, NE intake, and G:F were analyzed as repeated measures including growth phase as repeated term. Growth phase was added as a fixed effect and the interactions of growth phase with the other fixed effects were analyzed. An appropriate covariance structure was selected by comparing the goodness-of-fit measures of different structures. The Kenward-Roger correction was used for the denominator degrees of freedom. Since many pigs had already been sent for slaughter by day 57, growth performance data are only reported until day 50 on test.Lesion scores were analyzed using the GLIMMIX procedure of SAS using a Poisson distribution and Log link function. The repeated measures analysis for lesion scores was overparameterized, so the model was simplified. The lesion score data for both old and new lesions was analyzed for each week separately.P < 0.05 was considered significant and P < 0.10 was a trend.To test the hypotheses, The hybrid rye fed in this experiment had relatively low levels of mycotoxins, including EA. Ergocristine  was the most common EA measured, followed by ergotamine 15%, . IncludiP ≤ 0.001) in the low relative to the high-energy diets. The ATTD of GE and the DE content on a dry matter basis were unaffected by rye inclusion; however, DE content on an as fed basis was greater (P < 0.05) in the 40% rye diets compared to the 0% rye diets  between hybrid rye inclusion and NE level for ADG; pigs fed the low-energy rye diet gained 0.08 kg/d less than those fed the high-energy rye diet or the low-energy diet without rye, with those fed the high energy diet without rye being intermediate (P < 0.05) and feed efficiency (G:F) was reduced (P < 0.05) in pigs fed rye diets compared to those fed diets without rye, whereas there was no effect of NE level on NE intake or feed efficiency , there was an interaction (rmediate . For theficiency .P < 0.001) between hybrid rye inclusion and NE level for BW at days 8, 17, and 28. Pigs fed the low-energy rye diets weighed less than those fed high-energy rye diets or diets without rye  between hybrid rye inclusion and NE level for ADG for the days 0 to 8 and days 8 to 17 periods; pigs fed the low-energy rye diets gained less than those fed the high energy rye diet or the diets without rye. For the days 17 to 28 period, the interaction (P < 0.001) between rye inclusion and NE level for ADG was reversed; pigs fed the low-energy rye diet gained the most weight, and those fed the high-energy diet without rye gained the least, with the other two treatments being intermediate (There was an interaction (rmediate . There wP < 0.01), whereas ADFI was not affected by rye inclusion in any of the other growth phases. Feed intake was 0.14 kg/d greater (P < 0.01) during the days 17 to 28 period and tended (P = 0.082) to be 0.13 kg/d greater during the days 28 to 43 period in pigs fed low relative to high NE diets. Energy level did not affect ADFI for the days 0 to 8, 8 to 17, and 43 to 50 periods . There was no effect of NE level on NE intake throughout the trial .P < 0.001) in pigs fed the low-energy rye diet compared to pigs fed the high-energy rye diet or diets without rye. For the days 17 to 28 period, pigs fed rye diets had better G:F than those fed diets without rye (P < 0.001), whereas, for the days 43 to 50 period, pigs fed the low-energy rye diet tended to have lower feed efficiency compared to pigs fed the wheat diets, while the high energy rye diet was intermediate (P = 0.084). For the days 28 to 43 period, pigs fed low NE diets tended to have worse G:F compared to pigs fed high NE diets  was reduced (NE diets .P > 0.38) or NE level (P > 0.37) on lesion scores for old and new lesions throughout the experiment (data not shown).Minimal aggression occurred throughout the trial, resulting in low lesion scores for old lesions  or new lesions . Overall average lesion score for old lesions was 1.05 both for pigs fed diets with or without rye, and 1.04 and 1.06 for pigs fed diets low or high in energy, respectively. Overall average lesion score for new lesions was 0.21 and 0.17 for pigs fed diets without or with rye, respectively, and was 0.23 and 0.16 for pigs fed diets low or high in energy, respectively. There was no effect of rye inclusion , yet the analysis did not find a significant effect of sex. For this reason, a larger sample size may need to be considered in future trials.Due to its lower NE value compared to wheat, when hybrid rye is replacing wheat in the diet the oil inclusion level needs to be increased. As a result, the price of not only wheat and hybrid rye but also oil needs to be considered when determining whether to formulate diets with hybrid rye. The low NE diets were much cheaper than the high NE diets. This large difference in feed cost per tonne translated to lower feed cost per pig and per kg BW gain for pigs fed low vs. high NE diets. This finding is in agreement with our previous research that has consistently shown lower feed cost per pig and per kg BW gain, as well as higher income subtracting feed cost when pigs are fed lower energy diets .In conclusion, feeding 40% hybrid rye replacing wheat to grower-finisher pigs resulted in similar overall feed intake and final BW, slightly lower overall feed efficiency, and lower overall growth rate for pigs fed the low NE but not the high NE rye diets. The lower overall growth rate was caused by reduced BW gain during the initial 17 d of the trial and it may be useful to consider increasing the NE level of the first phase diet in the grower-finisher phase when including hybrid rye to avoid this reduction in growth performance.


Aspergillus, Botrytis, Mucor and Penicillium. This set comprised strains affiliated to 32 species distributed among 20 genera. Time-course monitoring of mold growth was performed to assess the inhibitory activity resulting from either diffusible or volatile compounds produced by each yeast strain. All yeasts displayed antagonistic activity against at least one of the mold targets. Mucor was the most affected being strongly inhibited by 68% of the tested strains, followed by Botrytis (20%), Aspergillus (19%) and Penicillium (7%). More notably, the approach used allowed the detection of a wide array of yeast-induced mold response profiles encompassing, besides the decrease of mold growth, the inhibition or delay of spore germination and the complete arrest of mycelial extension, and even its stimulation at different phases. Each factor considered  as well as their interactions significantly affected the antagonistic activity of the yeast isolates. The highest inhibitions were mediated by volatile compounds. Total inhibition of Penicillium was achieved by a strain of Metschnikowia pulcherrima, while the best performing yeasts against Mucor, Aspergillus and Botrytis, belong to Lachancea thermotolerans, Hanseniaspora uvarum and Starmerella bacillaris, respectively. Notwithstanding the wide diversity of yeasts tested, only three strains were found to possess a broad spectrum of antagonistic activity, displaying strong or very strong inhibition against the four fungal targets tested. Our results confirm the potential of wine yeasts as biocontrol agents, while highlighting the need for the establishment of fit-for-purpose selection programs depending on the mold target, the timing, and the mode of application.Grapes’ infection by phytopathogenic fungi may often lead to rot and impair the quality and safety of the final product. Due to the concerns associated with the extensive use of chemicals to control these fungi, including their toxicity for environment and human health, bio-based products are being highly preferred, as eco-friendlier and safer alternatives. Specifically, yeasts have shown to possess antagonistic activity against fungi, being promising for the formulation of new biocontrol products.In this work 397 wine yeasts, isolated from Portuguese wine regions, were studied for their biocontrol potential against common grapes phytopathogenic fungal genera:  The most frequently reported yeasts associated with wine-related ecosystems either belong to oligotrophic and oxidative species affiliated to genera such as amomyces .in vitro screenings using dual-culture assays  added with 20% (v/v) glycerol. The selected isolates were transferred to YPD agar , grown at 28°C and maintained at 4°C.Aspergillus niger strain AN1 (isolated from wine bottle cork), Botrytis cinerea strain BO1 (isolated from table grapes), Mucor sp. strain MU3 (isolated from wine grapes) and Penicillium sp. strain PE3 (isolated from wine grapes). Stocks of mold cultures were prepared in Potato Dextrose Agar (PDA) slants flooded in liquid paraffin and maintained at room temperature. Prior to their use, mold cultures were transferred to PDA plates and grown at 25°C. Spore suspensions of each fungal strain were obtained by harvesting spore mass at the surface of 15 days old colonies with glass beads using PBS buffer 1X with 0.01% Tween 20 and 50% (v/v) glycerol, to assist in spore dispersal and ultra-freezing preservation. After filtration through sterile glass wool, spore concentration was determined using a hemocytometer, adjusted to 1 × 106 or 1 × 104 spores/mL for the ascomycetous phytopathogens and Mucor sp., respectively. Spore suspensions were divided into aliquots and stored at −80°C.Four strains of phytopathogenic fungal genera were used, namely 2.2.Metschnikowia pulcherrima-clade species were affiliated to Met. pulcherrima as proposed by Han. opuntiae and Han. guilliermondii was achieved by 5.8S-ITS-RFLP analysis, in which ITS1/ITS4 amplification products were digested with restriction enzyme DraI, and separated on 2% agarose gel on 1 × TBE buffer as previously described by Genomic DNA extraction of the autochthonous isolates was performed by phenol-chloroform-isoamyl alcohol method from single-strain pure cultures . The ide5 (5′GTGGTGGTGGTGGTG3′), as single primers, following the methodology described previously by 5 and csM13 genomic fingerprinting patterns, were estimated using the Pearson coefficient and clustering was based on the UPGMA method. A cut-off value of 80% similarity was used to define the number of clusters inside each genus.The molecular diversity of yeast isolates belonging to the most prevalent genera was also evaluated through PCR-fingerprinting, using minisatellite csM13 (5′GAGGGTGGCGGTTCT3′) and microsatellite (GTG)2.3.7 cells/mL) onto solid media containing the specific enzyme substrates.The ability of the set of yeast strains to secrete lytic enzymes  eventually associated with the suppression of growth of fungal pathogens was evaluated by spotting 3 μL of freshly grown yeast culture suspensions , 6.7 g/L yeast nitrogen base without ammonium sulfate, 10 g/L glucose, and 20 g/L agar. After incubation at 28°C for 5 days, the plates were stained with 0.1% (w/v) ruthenium red after the colonies being rinsed off with distilled water. Colonies showing a purple halo were considered positive .For glucanase, chitinase and cellulase activities YPD plates containing 0.2% β-D-glucan from barley or 0.2% chitin from shrimp shells or carboxymethyl cellulose (CMC) sodium salt were used, respectively. After incubation, at 28°C for 5 days, the plates were rinsed with distilled water and stained with 0.03% (w/v) Congo red. A clear zone around the colony was indicative of the presence of glucanase or chitinase activity . For the2PO4, 0.5 g/L MgSO4.7H2O, 15 g/L agar and a carbon source of commercial mannan, azo-carob-galactomannan (2 g/L). The pH of the medium was adjusted to 5.5. Positive mannanase producers were detected based on the clear zone formed on the medium around the colony after 5 days of incubation at 28°C  and amylase activity was detected by the presence of a bright lysis zone around the colonies, while the rest of the medium remained violet, as the dye solution stained the non-hydrolyzed starch .2.4.in vitro assays approaches related to distinct modes of action underlying their inhibition. For all the assays, yeast strains were pre-cultivated in YPD medium and incubated overnight at 28°C. Prior to inoculation of each mold target, the suspensions of spores, prepared as described above, were removed from the freezer, and allowed to attain room temperature. The incubation period and the measuring points were previously established by performing mock trials in which the time taken for each mold target to extend from the point of inoculation to the edge of the Petri dish was observed. Based on these experiments it was established, a total incubation period of 40 h and 4, 5, and 7 days for Mucor sp., B. cinerea, A. niger and Penicillium sp., respectively, and daily measurements of radial mycelial extension. Due to the rapid growth of Mucor sp. additional measurements, at 16 h, were performed.The antagonistic activity of the set of 397 yeast strains against four common phytopathogenic fungal agents was evaluated using two 2.4.1.Penicillium sp.) distance from the center of a YPD agar plate (ø = 90 mm), and allowed to grow during 48 h at 25° C. Then, 3 μL of a mold spore suspension were inoculated in the center of each Petri plate and again incubated at 25°C. The radial mycelial extension of the phytopathogens in the direction of each yeast was recorded. For each mold target, and each round of assays, plates without yeast inoculation were used as external controls.A dual screening assay in which the mold target is confronted with each yeast strain (CY assay) was used to investigate the potential inhibitory effect, mainly resulting from diffusible compounds, of the yeast strains against the four phytopathogenic fungi. Briefly, 3 μL of seven fresh yeast cultures were equidistantly positioned at 3 cm or 1.5 cm  with inhibitory activity against the four phytopathogenic fungi. To avoid yeast-mold target contact, Petri dishes with four compartments, containing 4 mL of YPD agar in each sector were used. In two compartments, 50 μL of pre-grown fresh yeast culture were spread plated, while in the other two, 3 μL of a mold spore suspension were inoculated in central corner of the plate. For each mold target, and each round of assays, plates without yeast inoculation were used as external controls. The plates were sealed with Parafilmin vitro antagonistic activity assays is presented in the A schematic overview of the two 2.5.control) and in the presence of a yeast strain (RGtested) at the end of the incubation period, was calculated as described by The inhibition of radial growth-IRG (%), which considers the radial growth-RG (mm)-of the mold target in the absence (RGcontrol) and in the presence of a yeast strain (AUCtested) during the incubation period, calculated as follows:The effect of each yeast strain on mold growth profiles was also quantified considering the daily measurements of radial mycelial extension during the overall established period of incubation. The area under the mycelial extension/time curve  was calculated applying the linear trapezoid rule using GraphPad Prism software 9.3.1 . The obtained data were used to determine an additional parameter to assess yeast antagonistic ability  which relates the AUC (mm.day) of growth of the mold target in the absence (AUCThe reproducibility of antagonistic assays was assessed performing at least 20% of duplicate assays (CY and VOCs) using randomly selected yeast strains against the four targeted strains and evaluating the coefficient of variation (CV%) and respective confidence intervals obtained for the RG (mm) and AUC (mm.day) measurements.Principal-component analysis (PCA) was carried out to represent yeast strains correlated to the inhibition of the four mold targets by diffusible and volatile compounds as response variables using JMP 11.0 software .t-test or Tukey’s procedure at p < 0.05, using JMP 11.0 software .As the data of the inhibitory activities (IRG and IAC) against the four targets by the two modes of action did not meet the assumptions of a normal distribution (Shapiro–Wilk test), prior to ANOVA analysis, data was transformed using the Aligned Rank Tool (ARTool) . Pair-coJ’) evenness index was calculated (n > 20 isolates), as a quantitative estimate of the genomic and antagonistic distribution of the strains based on the number of profiles types of PCR-fingerprinting and of the mold responses. In other words, this evenness index expresses how evenly the strains are distributed among the different profiles obtained for each taxonomic group. If all yeast strains are represented in equal numbers in the obtained profiles, meaning great diversity, then J´ = 1, if one profile strongly dominates, meaning less diversity, J′ is close to zero . The similarity of the 397 yeast-induced mold target responses was calculated using the Pearson coefficient and UPGMA clustering. A cut-off value of 80% was applied for the definition of distinct response profile types. To facilitate comparisons among taxonomic groups (yeast genera and mold target), Pielou’s , so that most of them were expected to be originated from this region . Among tMetschnikowia (105 isolates) represented by Met. pulcherrima, Starmerella (84 isolates) represented by Sta. bacillaris (83) and Sta. stellata (1), Hanseniaspora (62 isolates) represented by Han. uvarum (48), Han. guilliermondii (11) and Han. opuntiae (3), Saccharomyces (53 isolates) represented by Sac. cerevisiae, Lachancea (28 isolates) represented by L. thermotolerans and Aureobasidium (21 isolates) represented by Aur. pullulans only. All together these genera represented approximately 89% of the isolates.Thirty-three species belonging to 20 different genera of yeasts were represented in the yeast set . The sixPichia (9 isolates), Candida, Meyerozyma, Torulaspora and Zygosaccharomyces with 5 isolates each, Rhodotorula (4 isolates) Naganishia (3 isolates) and Wickerhamomyces (2 isolates). The genera Filobasidium, Hyphopichia, Nakazawaea, Saccharomycodes, Yamadazyma and Zygoascus were represented by a single isolate.In lower numbers, we found the genera The sequence data of D1/D2 domains of all strains used in this study have been deposited in GenBank under the accession numbers MG832576 to MG832583, MG877743 and MG87744  and OQ303.2.The production of fungal cell wall-degrading enzymes is considered to be one of the important characteristics associated with the activity of biocontrol agents . Thus, hAur. pullulans strains, being the only species displaying mannanase and pectinolytic activities. In contrast, none of the Aur. pullulans strains exhibited glucanase activity while, notably, 21 out of 28 L. thermotolerans strains produced this enzyme. Protease release was found in a higher number of isolates (37), while cellulase activity was found to be the most common hydrolytic activity detected among different yeast species (7).Only 12 out of the 20 genera included strains displaying at least one enzymatic activity, and any isolate exhibited all the enzymatic activities . Besides3.3.3.3.1.All 397 yeast strains were screened for their ability to inhibit four important fungal pathogens using two dual-culture growth inhibition tests. A very good intra-plate reproducibility of the assays (CY and VOCs) was found, with the average coefficient of variation for RG (mm) measurements at the end of incubation period being minor, homogeneous and similar in both types of assays .Pic. membranifaciens and R. nothofagi exhibited the worst overall performance, not reaching 25% inhibition against any of the targets either in the CY or VOCs assays. Considering all the assays performed against all targets, the IRG (%) induced by yeasts volatile compounds production ranged from −19%, consistent with a stimulant effect by an Aur. pullulans strain, to 100%, corresponding to the total inhibition of Penicillium sp. PE3 by a Met. pulcherrima strain. The greatest inhibition activity against A. niger AN1 (81%), B. cinerea BO1 (94%) and Mucor sp. MU3 (94%) were also attained in the VOCs assays by a Han. uvarum, a Sta. bacillaris and a L. thermotolerans strain, respectively.In general, all yeasts exhibited an antagonistic phenotype, with an IRG > 5%, against at least one of the mold targets tested, through the production of diffusible (CY) and/or volatile compounds (VOCs) . The strB. cinerea BO1 and Mucor sp. MU3. On the other hand, the maximum inhibition levels against all targets in this assay were lower than those induced by volatile compounds: two Han. uvarum strains were the most effective in inhibiting the growth of A. niger AN1 (56%) and Mucor sp. MU3 (49%) while strains of Sta. bacillaris and Sac. cerevisiae were able to reduce 60 and 66% of the growth of B. cinerea BO1 and Penicillium sp. PE3, respectively.A stimulant effect of yeasts on mold growth was also observed in the CY assays, but only against p < 0.05) on their taxonomic affiliation, the mode of action, the mold target and the interactions between all factors , could be noticed, with the majority of the strains grouping together away from the remaining genera according to PC1 and being separated in the first and second quadrant, respectively, according to PC2.In general, the IRG (%) values obtained in the different assays for the isolates belonging to the same genus were broadly distributed and showed several outliers, as this behavior was highly variable with the target and/or the mode of action . Indeed, factors . To eval factors . The res3.3.2.A. niger AN1, B. cinerea BO1, Mucor sp. MU3 and Penicillium sp. PE3, obtained in the control and in the presence of each yeast strain were analyzed by hierarchical clustering with Pearson correlation coefficient and UPGMA  evenness index of diversity, calculated for the most representative genera/species (n > 20 isolates), using a cut-off value of 80% similarity for each dendrogram (5 and csM13 genomic fingerprinting patterns (L. thermotolerans (J’ = 0.37), the 28 isolates induced a wide diversity of responses on A. niger AN1 (J’ = 0.97) and Mucor sp. MU3 (J’ = 0.83) in the VOCs and CY assays, respectively. Conversely, notwithstanding the high genotypic diversity found for Aur. pullulans (J’ = 0.86), the volatile compounds produced by the 21 isolates induced the same response profile on A. niger AN1 and highly similar ones on Penicillium sp. PE3 (J’ = 0.28).The high diversity of mold response profiles induced by the different yeast strains was further highlighted by the analysis of Pielou’s , with a higher discriminating power, for the categorization of yeasts based on the determination of the area under the curves  of mold growth in the absence and in the presence of the different yeast strains, during the incubation period . As seene assays ,B.Principal component analysis was also performed to assess the distribution of the 397 yeast strains based on IAC (%) . Althoug3.3.3.pulcherrima strains were considered for all the six classes, from S through 4, according to their VOCs-induced effect on Penicillium sp. while the three strains of Han. opuntiae tested were placed in classes 1,2, and 3, based to their VOCs-induced antagonistic activity  against B. cinerea BO1.For a biological interpretation of the data, the yeast strains were classified in different categories based on their inhibitory activity using the IAC (%) metric: class 0 (no effect: -5 < IAC < 5%), class 1 (weak effect: 5% ≤ IAC < 25%), class 2 (moderate effect: 25% ≤ IAC < 50%), class 3 (strong effect: 50% ≤ IAC < 75%) and class 4 (very strong effect: IAC ≥ 75%). Strains with stimulatory activity, identified by a negative IAC (< −5%) effect of mold growth were included in class S. Mucor sp. MU3 in the CY assay and thus a less stringent criterion was applied, and isolates included in class 2 for this target were considered instead in the following analysis.In order to identify the most promising candidates to be used as biocontrol agents we restrained our subsequent analysis to those isolates exhibiting a more prominent inhibitory activity by selecting strains only included in classes 3–4 by both type of assays (CY and VOCs). Interestingly, no isolate was found in those classes for Mucor sp. MU3 (68%) followed by B. cinerea BO1 (20%), A. niger AN1(19%) and Penicillium sp. PE3 (7%).In a first approach we aimed to identify the most effective strains against each mold target, irrespective of the mode of action. The Venn diagrams presented in A. niger AN1 and comprise strains of Sta. bacillaris (1), Han. uvarum (9) and Han. opuntiae (2) (Han. uvarum that was assigned to the pattern B3 (Han. uvarum strains.Twelve isolates were found to be highly effective by both modes of action against tiae (2) . These yttern B3 . The aveHan. uvarum, one of Met. pulcherrima and the only representative of Zygoascus meyerae showed a strong inhibitory activity against B. cinerea BO1 by both modes of action  in both assays ranging from 39.1% by one Sac. cerevisiae strain, assigned to patterns D1 and B2  and Mey. guilliermondii (2), which inhibited Penicillium sp. PE3 growth since day 3 until de end of the experiment , Can. glabrata, Han. uvarum, Hyp. burtoni, L. thermotolerans, which effectively led to an early arrest of A. niger AN1 growth  values of 73, 67, and 68%, respectively.We then aimed to identify strains with broad spectrum of antagonistic activity –F. No ye4.Vitis vinifera L. grapevine is one of the most cultivated (in an estimated surface area of 7.3 million hectares) and valued fruit crops, given the many applications of grape production, from its fresh consumption (table grapes and raisins) to its use in the production of juices and wines  and Shemer® (Met. fructicola NRRL Y-27328), both targeting only B. cinerea. Thus, the quest for new yeasts with reliable biocontrol prospects against grape phytopathogenic fungi continues.Presently, there is an increasing demand for more sustainable and eco-friendly practices in the agricultural ecosystems aiming the replacement, or at least the reduction, of the chemical products that are currently being used . A consiA. niger, B. cinerea, Mucor sp. and Penicillium sp. This set of yeast strains, isolated from grapes/grape-juices and spontaneous grape-juice fermentations derived from different Portuguese wine-regions, was found to be highly diverse in their taxonomic groups, encompasses mostly non-Saccharomyces species (approximately 87%) and 53 Sac. cerevisiae strains and thus represents the broad biodiversity common in the targeted niche  against all the phytopathogenic fungi tested were found. Overall, the strains affiliated to species of  cinerea  and of Llus spp. . Althoug cinerea , our repminiatus . Strainsexpansum  the biocee beans  or grapeee beans .Biocontrol activity of yeasts by direct mold antagonism is mainly mediated by the competition for nutrients and space, secretion of non-volatile compounds  and production of volatile compounds . In thisIn addition, the original time-course mold growth monitoring approach, performed in the present study, allowed the detection of a high diversity of mold growth effects induced by yeasts. Indeed, it has been demonstrated that the comparison based on time series analysis enables a more thorough and objective comparison of the influence of environmental conditions on mold growth dynamics . All theHerein we uncover a remarkable intra and interspecific diversity in the yeast inhibition levels against mold targets, which is in line with previous works . BesidesThe diversity of yeast-mediated mold growth responses led us to propose another parameter, IAC (%), based on the area under the mycelial extension/time curve , which integrates the different effects over time. The AUC measure is also preferred in other fields of study such as pharmacology where, for example, it is used to accurately estimate the extent of a body’s exposure to a particular drug over time . The useAur. pullulans strains exhibiting the highest diversity of lytic enzymes released, in line with the recognized ability of this species , or competition for nutrients, or the secretion of extracellular enzymes as the underlying mechanism of inhibition . However species . On the  species , althoug species . NeverthMet. pulcherrima and Sta. bacillaris was detected against the most phylogenetically closer targets, A. niger and Penicillium sp. Also, a delay in spore germination of these mold targets by strains of Hanseniaspora spp. and Sta. bacillaris was found. These observations suggest the involvement of diffusible compounds on mold inhibition, with the distinct effects being dependent on their toxicity, and rates of production and diffusion  surpassing 90%. Alcohols and their respective esters, have been associated with the antagonistic activity of yeasts against phytopathogenic fungi . The mosA. niger, Mucor sp. and Penicillium sp. These compounds are known to be used by fungi as chemical signals to control physiological processes such as nutrient acquisition, sporulation, sexual development and spore germination  can be found in the article/AM-F, FC, and MT were responsible for funding acquisition, AM-F and RT conceived and designed the experiments. ME, PL, and JS performed the experiments. ME prepared the original draft. ME, PL, FC, RT, and AM-F organized and analyzed the data. ME, RT, and AM-F wrote the manuscript. AM-F supervised the study. All authors contributed to the article and approved the submitted version.This work was financially supported by ABCyeasts project no. NORTE-01-0247-FEDER-039793, co-financed by FEDER through NORTE 2020. ME is a recipient of a PhD grant with the reference PD/BD/150587/2020 from the FCT Doctoral Program in Applied and Environmental Microbiology (DP_AEM).FC and MT was employed by Proenol.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of the study was to evaluate the value of lung ultrasound (LUS) in patients with cardiogenic shock treated by venoarterial extracorporeal membrane oxygenation (VA-ECMO).A retrospective study was conducted in Xuzhou Central Hospital from September 2015 to April 2022. Patients with cardiogenic shock who received VA-ECMO treatment were enrolled in this study. The LUS score was obtained at the different time points of ECMO.2/FiO2 and LUS scores and pulmonary dynamic compliance(Cdyn) after 72 h of ECMO treatment (P < 0.001). ROC curve analysis showed that the area under the ROC curve (AUC) of T72-LUS was 0.964 .Twenty-two patients were divided into a survival group (n = 16) and a nonsurvival group (n = 6). The intensive care unit (ICU) mortality was 27.3% (6/22). The LUS scores in the nonsurvival group were significantly higher than those in the survival group after 72 h (P < 0.05). There was a significant negative correlation between LUS scores and PaOLUS is a promising tool for evaluating pulmonary changes in patients with cardiogenic shock undergoing VA-ECMO.The study had been registered in the Chinese Clinical Trial Registry(NO.ChiCTR2200062130 and 24/07/2022). Even with important advances in revascularization strategies and heart failure pharmacotherapies, venoarterial extracorporeal membrane oxygenation (VA-ECMO) has been widely used to provide life support in patients with refractory cardiogenic shock and has significantly improved their survival rate , 2. AlthLung computed tomography (CT) is regarded as the gold standard for noninvasive evaluation of pulmonary oedema, but it is not convenient for critically ill patients with cardiogenic shock undergoing VA-ECMO due to the exposure to ionizing radiation and the need for transportation, which limits its application . At presHowever, the application of bedside LUS in patients with cardiogenic shock undergoing VA-ECMO support has rarely been reported and has not been systematically studied. In this study, patients with cardiogenic shock who received VA-ECMO were reprospectively enrolled, and the LUS score was obtained before and during VA-ECMO support. The aim of this study was to assess the potential utility of the LUS score in patients receiving VA-ECMO support.A retrospective study was conducted in a tertiary-level teaching hospital . The study protocol was reviewed and approved by the Ethics Committee of our hospital and conforms to the Declaration of Helsinki.Between September 2015 and April 2022, twenty-two patients who underwent VA-ECMO support and LUS measurement and with cardiogenic shock were enrolled in the study. Eleven patients had acute myocardial infarction, 8 had fulminant myocarditis, and 3 had heart failure after cardiac surgery. The patients were divided into survivors (n = 16) and nonsurvivors (n = 6).In our centre, when the following criteria are met, VA-ECMO treatment is required for patients with refractory cardiogenic shock : (1) PerDemographic and clinical data were collected from the hospital records and our organization’s proprietary database. The demographic data included age, sex, and body mass index (BMI). The clinical data included the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, comorbidities, ICU mortality, length of ICU stay, duration of ECMO and duration of ventilation.The ECMO setup involved femoral vein catheterization, femoral arterial catheterization and superficial femoral artery catheterization . The femThe adult-related indications for ECMO weaning were used as a reference in our centre, which included the following : stable 2O, the positive end expiratory pressure (PEEP) between 10 ~ 15 cmH2O, and the oxygen intake concentration between 30 − 50%, and the setting of respiratory rate refers to the changes of tidal volume and ECMO airflow to match. The pH value and arterial blood carbon dioxide partial pressure measured by arterial blood gas analysis were maintained within the normal range.A protective mechanical ventilation strategy was implemented during ECMO for all the patients, and standardized nursing work was performed under the guidance of doctors. The protective mechanical ventilation strategy included –15 selecBased on the scheme proposed by Bouhemad et al , the pat0, T24, T48, T72, T96, TW and TR. According to the above periods, the corresponding ventilator parameters and the blood gas analysis results of the right upper limb artery blood sample were also recorded, including PaO2/FiO2, PaCO2, and pulmonary dynamic compliance (Cdyn).The lung ultrasound scores of the patients were obtained at different periods, including at the initiation of ECMO, each morning in the following four days and at the termination of ECMO and ventilation as T2/FiO2. ROC curves analysis was used to determine the diagnostic value of the significantly changed variables during ECMO support for the ICU survival status of the patients. A p value < 0.05 was considered statistically significant.Statistical analysis was performed with IBM SPSS Statistics 26 . Normal distribution was formally tested with the Shapiro–Wilk test. Continuous data are presented as the mean with standard deviation. ANOVA was used to compare repeated measurement data at different time points, and a T test was used for comparisons between two groups. Categorical data are presented as frequencies and percentages. Categorical variables were compared using the chi-square test. The Pearson method was used to compare the correlation between the LUS score and PaOTwenty-two patients with refractory cardiogenic shock who received VA-ECMO support in our centre were included in the study, including 13 males and 9 females aged 14 to 77 years. There were 11 cases of acute myocardial infarction, 8 cases of fulminant myocarditis and 3 cases of cardiac postoperative assistance. All patients received endotracheal intubation and ventilator-assisted breathing.P > 0.05). However, the length of ICU stay in the survival group was longer than that in the nonsurvivor group (P < 0.05). Detailed baseline characteristics for the study population are shown in Table According to discharge survival status, the patients were divided into a survival group n = 16) and a nonsurvivor group (n = 6). There were no significant differences with regard to age, sex, basic diseases, APACHE II, ECMO running time, or mechanical ventilation support time between the two groups . The LUS scores at T72, T96, TW and TR in the nonsurvivor group were higher than those in the survival group, and the differences were statistically significant (P < 0.05). Repeated measures analysis of variance was calculated as follows: FTime = 165.7 (P < 0.001); FGroup = 142.6 (P < 0.001); FTime × grouping = 54.30 (P < 0.001) . PaO2/FiO2 in the nonsurvivor group was lower than that at T0 at T48, T72, T96, TW and TR (P < 0.05). PaO2/FiO2 at T72, T96, TW and TR in the nonsurvivor group was lower than that in the survival group, and the difference was statistically significant (P < 0.05). Repeated measures analysis of variance was as follows: FTime = 30.96 (P < 0.001); FGroup = 262.9 (P < 0.001); FTime × grouping = 28.92 (P < 0.001) . Repeated measures analysis of variance was as follows: FTime = 2.464 (P < 0.05); FGroup = 0.025 (P > 0.05); FTime × grouping =1.286 (P > 0.05) . Cdyn in the nonsurvivor group was lower than that at T0 at T24, T48, T72, T96, TW and TR (P < 0.05). Cdyn at T72, T96, TW and TR in the nonsurvivor group was lower than that in the survival group, and the difference was statistically significant (P < 0.05). Repeated measures analysis of variance was as follows: FTime = 3.000 (P < 0.05); FGroup = 94.13 (P < 0.001); FTime × grouping = 39.40 (P < 0.001)  .  afterload due to reverse ECMO flow in the aorta , and pat2/FiO2 ratio of VA-ECMO-assisted patients always depends on the ratio between the patient’s spontaneous cardiac output and the ECMO flow and FiO2 in the ECMO oxygenator and ventilation setting, oxygenation parameters cannot more accurately indicate the severity of lung function injury in patients during pulmonary oedema episodes . These findings indicated that lower LUS scores might be linked with better patient survival. Nonetheless, future regression analysis is needed to explore whether LUS scores can function as an independent risk factor or predictor of the survival of patients with cardiogenic shock in the ICU receiving VA-ECMO support. Given the small sample size of the current study, the analysis was not conducted.An increased incidence of pulmonary oedema during VA-ECMO treatment is closely associated with mortality. Moreover, the LUS score was used to predict the prognosis of ARDS caused by COVID-19 and paediatric diseases, and indicated that the LUS score was associated with a higher risk of PICU mortality and longer PICU stay days after 72 h of VV-ECMO support . SimilarThere are several limitations of our study. First, it is important to note the retrospective design and the single-centre setting as limitations and that the small sample size may have led to selection bias. For further research, the sample size should be expanded for a stratified analysis. Second, the evaluation of back partitioning requires the patient to be moved, which increases the difficulty of nursing and has the risk of catheter displacement, and multiple measurements are required to ensure the accuracy of the data.Third, left heart decompression procedures have been implemented by numerous medical centers to improve pulmonary edema and lung ultrasound scores. However, it should be noted that our study did not include patients undergoing left heart decompression, therefore, our findings cannot be generalized to this population.In conclusion, LUS can be an important means for the dynamic and continuous assessment of lung function in patients with ECMO and can accurately assess the degree of lung lesions in critically ill patients, and the LUS score is expected to be a valuable reference index for predicting ICU prognosis in patients receiving VA-ECMO treatment for cardiogenic shock.


In patients with heart failure, left atrial remodeling often occurs to varying degrees. Left atrial enlargement has been proved to be an important predictor of cardiovascular‐related adverse events. However, the relationship between left atrial diameter (LAD) with heart failure (HF) with preserved ejection fraction (HFpEF), reduced ejection fraction (HFrEF) and mid‐range ejection fraction (HFmrEF) remains unclear.We want to explore the correlation between left atrial diameter and HFpEF, HFmrEF, and HFrEF.A total of 210 patients with heart failure who underwent hospitalization in Ningbo Medical Center Lihuili Hospital, Zhejiang, China, from January 1, 2020, to June 31, 2021, were reviewed. The basic demographic characteristics, blood test, and the related indexes of echocardiography of the subjects were collected and analyzed.p = .007), and LAD is negatively correlated with left ventricular ejection fraction (LVEF) .There is a significant difference between HFpEF and HFrEF group in LAD . And LAD is negatively correlated with LVEF , which may predict the prevalence of HFrEF.We studied the data of 210 individuals with heart failure. There is a significant difference between HFpEF and HFrEF groups in LAD ( The results showed that the LAD of HFmrEF was smaller than that of HFrEF and HFpEF.2This research was conducted following the Declaration of Helsinki. This retrospective study was approved by the Institutional Review Board of Ningbo Medical Center Lihuili Hospital (No. LY2023YJZ059) and waived the individual consent for this analysis.2.1A total of 210 consecutive subjects with HF referred to Ningbo Medical Center Lihuili Hospital from January 1, 2020, to June 31, 2021, were reviewed. According to the LVEF, there were 70 patients  in HFrEF group (LVEF < 40%), 70 patients  in HFmrEF group (LVEF = 40%–49%) and 70 patients  in HFpEF group (LVEF ≥ 50%). Inclusion criteria: (1) Patients with complete medical records during hospitalization; (2) Patients met the diagnostic criteria of HF according to ESC 2016: Guidelines for Diagnosis and Treatment of Acute and Chronic Heart Failure.2.2In the study, the basic demographic characteristics of the subjects were collected and analyzed, including gender, age, smoking, and drinking; Previous disease history: hypertension and diabetes.Blood test results during hospitalization included B‐type brain natriuretic peptide (BNP), creatinine (Cr), albumin, triglyceride (TG), total cholesterol (TC), low‐density lipoprotein, high‐density lipoprotein, and homocysteine (HCY).The related indexes of echocardiography include left atrial diameter, left ventricular end‐diastolic diameter (LVEDd), left ventricular end‐systolic diameter (LVEDs), interventricular septum thickness at end‐diastolic (IVSd), left ventricular ejection fraction (LVEF) and left ventricular posterior wall diastolic thickness (LVPWD).The medication of each person was counted, which was limited to diuretics, spironolactone, digoxin, anticoagulants, statins, β receptor blockers, angiotensin‐converting enzyme inhibitor (ACEI)/angiotensin‐receptor blocker (ARB).2.3SPSS23.0 statistical software was used to test the distribution characteristics of samples with Shapiro–Wilk. The measurement data of normal distribution was expressed by mean ± standard deviation, and one‐way analysis of variance was used for comparison between groups. The measurement data of nonnormal distribution are expressed by median and quarterback difference, and independent sample median test and Bonferroni correction are adopted. The counting data were expressed as percentage, and the comparison between groups was carried out by two tests. Spearman's correlation coefficient was used to evaluate the correlation between left atrial diameter and HFpEF, HFmrEF, and HFrEF.p < .05 was considered statistically significant.A receiver operating characteristic curve (ROC) was generated to assess the ability of the LAD to predict the prevalence of HFrEF and atrial fibrillation, and area under the curve (AUC) was calculated. The optimal cutoff value for the LAD was determined with the highest Youden index. A 33.1p < .001). There was no significant difference in age, gender, smoking history, drinking history, diabetes history and atrial fibrillation among three groups . There was no significant difference in BNP in HFpEF group was lower than that in HFrEF ; BNP in HFpEF group was lower than that in HFmrEF group ; There was no significant difference in BNP between HFrEF group and HFmrEF group .A total of 210 patients were included in the study, including HFpEF group, HFmrEF group, and HFrEF group, with 70 cases each. The proportion of basic diseases in HFpEF group was the highest (74.3%), followed by HFmrEF group (57.1%) and HFrEF group (35.7%) (p < .001). Similarly, LVEDs in HFpEF group were the smallest , followed by HFmrEF group  and HFrEF group  (p < .001). IVSd in HFpEF group was thicker than that in HFrEF group , and IVSd in HFmrEF group was thicker than that in HFrEF group . There was no significant difference in IVSd in HFpEF group and HFrEF group . There was no significant difference in LVPWd among three groups .LVEDd was the smallest in HFpEF group , the second in HFmrEF group  and the largest in HFrEF group  (p = 1.0), albumin , TG , TC , HCY  among HFpEF, HFmrEF, and HFrEF.In terms of biochemical indexes, there were no significant differences in Cr , beta blockers (60 (85.7) vs. 58 (82.9) vs. 43 (61.4); p = .001), ACEI/ARB (50 (71.4) vs. 54 (78.3) vs. 40 (57.1); p = .05) than HFpEF groups. There were no significant differences in the use of spironolactone (56 (80.0) vs. 61 (87.1) vs. 64 (91.4); p = .141), digoxin (6 (8.6) vs. 11 (15.7) vs. 11 (15.7); p = .357), anticoagulants (32 (57.5) vs. 27 (38.6) vs. 34 (48.6); p = .471), and statins (54 (77.1) vs. 47 (67.1) vs. 47 (68.1); p = .358) among HFpEF, HFmrEF, and HFrEF groups.In terms of drug treatment, HFmrEF and HFrEF groups used more diuretics (65 (92.9) vs. 67 (95.7) vs. 59 (84.3); The summary related to the clinical characteristics of the three groups is shown in Table 3.2p < .001; Figure ROC analysis revealed that the optimal cutoff value of the LAD for predicting the prevalence of atrial fibrillation in patients with heart failure was 0.286 , a positive correlation between LAD and LVEDd , and no correlation between LAD and BNP and NYHA grades (p = .308 and p = .272).There was a negative correlation between LAD and LVEF , there was no significant difference in LAD between HFrEF group and HFmrEF group , and there was no significant difference in LAD between HFpEF group and HFmrEF group .3.5p = .047; Figure ROC analysis revealed that the optimal cutoff value of the LAD for predicting the prevalence of HFrEF was 0.17 .In this retrospective study, a total of 210 patients with heart failure were included. According to the new diagnostic criteriap < .001). Hypertension plays an important role in pathological left ventricular hypertrophy, which inducing process of heart failure.The comparison of basic diseases showed that HFrEF had the lowest proportion of hypertension patients (35.7%) and HFpEF had the highest proportion of hypertension patients (74.3%), with statistical difference . Previous studies suggest that there may not be a clear BNP threshold that can effectively distinguish HFpEF from HFrEF.From the perspective of left ventricular structure and function, LVEDd and LVEDs in HFrEF group is the largest, and IVSd in HFpEF group is the thickest, suggesting that HFrEF is characterized by left ventricular enlargement and systolic dysfunction, while HFpEF is mainly characterized by left ventricular concentric remodeling/hypertrophy and diastolic dysfunction, which is similar to previous studies.p = .007), and LAD is negatively correlated with LVEF . Therefore, it is speculated that LAD may be used to predict the occurrence of HFrEF.This study shows that with the decrease of systolic function, the left atrium gradually expands, namely the lower LVEF is, the larger LAD is. There is a significant difference between HFpEF and HFrEF group in LAD (It is found that LAD has high sensitivity (sensitivity for 80.0%) in predicting HFrEF, because left atrial enlargement is an important pathogenic mechanism of left atrial remodeling. Because left atrial enlargement reflects chronic left ventricular end‐diastolic pressure increase, patients with heart failure with left atrial enlargement tend to have longer course of disease, worse cardiac function and more serious cardiac remodeling.This study is a single‐center retrospective study, which has the inherent limitations of retrospective study and a small sample size. Second, all patients excluded AF/AFL through symptoms, signs, past history, physical examination, past and postadmission ordinary and 48 h ambulatory electrocardiogram, which may ignore the possibility of occult AF, and the results may be biased. In the future, a larger and multicenter cohort study is needed to verify and support it. In addition, we failed to obtain the data of left atrial volume in this study. Although LAD has a strong correlation with left atrial volume,5LAD is negatively correlated with LVEF, which may predict the occurrence of prevalent HFrEF.The authors declare no conflict of interest.This research was conducted following the Declaration of Helsinki. This retrospective study was approved by the Institutional Review Board of Ningbo medical center lihuili hospital and waived the individual consent for this analysis.


The morbidity of cancer keeps growing worldwide, and among that, the colorectal cancer (CRC) has jumped to third. Existing early screening tests for CRC are limited. The aim of this study was to develop a diagnostic strategy for CRC by plasma metabolomics.A targeted amino acids metabolomics method was developed to quantify 32 plasma amino acids in 130 CRC patients and 216 healthy volunteers, to identify potential biomarkers for CRC, and an independent sample cohort comprising 116 CRC subjects, 33 precancerosiss patients and 195 healthy volunteers was further used to validate the diagnostic model. Amino acids-related genes were retrieved from Gene Expression Omnibus and Molecular Signatures Database and analyzed.Three were chosen out of the 32 plasma amino acids examined. The tryptophan / sarcosine / glutamic acid -based receiver operating characteristic (ROC) curve showed the area under the curve (AUC) of 0.955 (specificity 83.3% and sensitivity 96.8%) for all participants, and the logistic regression model were used to distinguish between early stage (I and II) of CRC and precancerosiss patients, which showed superiority to the commonly used carcinoembryonic antigen. The GO and KEGG enrichment analysis proved many alterations in amino acids metabolic pathways in tumorigenesis.This altered plasma amino acid profile could effectively distinguish CRC patients from precancerosiss patients and healthy volunteers with high accuracy. Prognostic tests based on the tryptophan/sarcosine/glutamic acid biomarkers in the large population could assess the clinical significance of CRC early detection and intervention.The online version contains supplementary material available at 10.1186/s12967-023-04604-7. The targeted amino acids metabolomics was applied to profile the alterations of amino acids in colorectal cancer.Plasma amino acids profile shows capability of differentiating the colorectal cancer from the precancerosiss patients and healthy volunteersA diagnostic model Y = 0.001×Trp +0.029×Sar-0.002×Glu-9.427 (unit: ng/mL) was developed and validated for colorectal cancer diagnosis.Amino acids-related differentially expressed genes analysis supported the alterations of amino acids metabolic profile in tumorigenesis.The online version contains supplementary material available at 10.1186/s12967-023-04604-7. The colorectal cancer (CRC) is one of the leading causes for cancer deaths worldwide, with more than 1.8 million people in 2018 . PatientRecently, the burgeoning omics field provides diverse high-throughput approaches for CRC blood sample-based biomarker discovery . As the In this study, we used a targeted amino acids metabolomics method to profile 32 plasma amino acids in CRC patients and precancerosiss (PC) patients as well as healthy volunteers (HV) from three independent centers. A total of 690 plasma samples were analyzed, and differential amino acids were selected by means of orthogonal partial least squares-discriminant analysis (OPLS-DA) and logistic regression, and further validated, to identify the plasma amino acids as the potential diagnostic biomarkers for CRC. Besides, this study took advantage of transcriptomic data from GEO(Gene Expression Omnibus) database to depict the alterations of the amino acids between CRC tissue and adjacent normal tissue samples, which could consolidate the results of targeted amino acids metabolomics.This is a multi-center, two sets and case–control study. The patients were enrolled in the Second Affiliated Hospital of Naval Medical University , the Affiliated Huaihai Hospital of Xuzhou Medical University  and Taixing People’s Hospital  between 2017 and 2018. The training set included 130 CRC patients and 216 HV, and the validation set was consisted of 116 CRC patients, 33 PC patients and 195 HV. The inclusion and exclusion criterions for CRC patients are as follows: (1) the patients with age ≥ 18 years old and clinically and histologically diagnosed with CRC were enrolled. (2) The patients who were diagnosed severe metabolic diseases, pregnancy, lactation, severe infection or other cancers were excluded. The protocol of this study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Naval Medical University, the Affiliated Huaihai Hospital of Xuzhou Medical University and Taixing People’s Hospital. All study participants signed the informed consent according to the institutional guidelines. The study was carried out in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.After the enrollment, the demographic characteristic data of all participants were documented, and the peripheral blood samples were collected in an EDTA-3 K tube from CRC patients in the morning on the day of surgery(food fasting overnight), and for HV and PC patients, the peripheral sample collection was completed after a food fasting overnight. All the blood samples were mixed gently and then subjected to a 2000 × g, 15 min centrifuge at 4 °C. The supernatant was aliquoted and transferred to CyroMax tubes, and all the plasma samples were stored in −80 °C until retrieval. The sample collection and process were accomplished within 1 h.The plasma sample pretreatment was developed based on protein precipitation. The three internal standards (IS) L-alanine-d4 (Ala-d4), L-methionine-d3 (Met-d3), and L-phenylalanine-d5 (Phe-d5) (400 ng/mL for each IS) were prepared freshly in acetonitrile, and a 50 μL aliquot of plasma sample was drawn and 150 μL acetonitrile (containing three IS) was added to precipitate the protein prior to a 3 min vortex-mixing and then the mixture was centrifuged at 19,060 × g, 4 °C for 15 min. The supernatant was injected directly to Agilent 1290-6460A ultra-high performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) system for analysis.The UHPLC-MS/MS quantitative analysis methods were developed in our group , 25. On P-value < 0.05). The genes related to amino acids metabolism were downloaded from Molecular Signatures Database (AMINO_ACID_AND_DERIVATIVE_METABOLIC_PROCESS). All the analysis were completed using the OECloud tools based on R programming language (https://cloud.oebiotech.com). The enrichment analysis was carried out using Metascape V3.5.Two transcriptomic data sets(GSE164541 and GSE138202) from GEO database were retrieved. The counts of genes were normalized based on BaseMean to obtain the fold change and the negative binomial distribution test was carried out to dig out the differential genes (absolute fold change ≥ 2 and t test was performed to compare the amino acids contents between different groups (IBM SPSS Statistics 21.0), and P < 0.05 was considered to be statistically significant. The multivariate statistical analysis was carried out using Umetrics Simca-p (version 14.1), and principal component analysis (PCA), OPLS-DA models, etc. were built to find the amino acids that contributed most to the model construction, and the amino acids with variable importance for the projection (VIP) value > 1 and false discovery rate (FDR) value < 0.05 would be selected for further diagnosis model construction. The model construction was developed in SPSS and the binary logistic regression was carried out to assess the biomarkers in differentiating the CRC.The metabolomics data were acquired and preprocessed using Agilent MassHunter workstation (version B.07.00). The student’s-P = 0.188, student’s-t test; gender, P = 0.713, χ2 test), and no differences were found in the smoking status and alcohol consumption. The CEA and α-fetoprotein (AFP) values were retrieved as auxiliary diagnosis for all the patients, and in the training set, the CEA of CRC patients was higher than the HV , while the AFP was lower in CRC patients , and in the validation set, the CEA  and AFP  were higher in the CRC patients compared with the HV. In the validation set, 33 PC patients including 20 polyps, 7 inflammatory colitis, 1 intestinal obstruction and 5 mixed hemorrhoids were recruited because higher risk for CRC.The demographic data and clinical factors of the study cohort were summarized in Table P < 0.05, student’s-t test), while Gln, Ser, Glu, Asn, Opr, Orn, Lys, Asp, Cys, Amp, and SDMA obviously increased in CRC , and four amino acids Ile, Thr, Cit, and ADMA didn’t show any differences between CRC patients and HV .The logistics regression model parameters were shown in Additional file Furthermore, We assumed the stage I and stage II CRC as early stage, and stage III and stage IV as advanced stage, and the stage-related amino acids profile was analyzed by a new PCA model. The CRC samples distributed evenly in the PCA model and no stage-related clusters were found   including 5 and 8 paired tumor and adjacent normal tissue samples, respectively, were analyzed and in the GSE 164541, 2172 and 2046 significantly upregulated and downregulated genes were found in tumorigenesis, respectively, and in GSE138202, 4477 and 3791 significantly upregulated and downregulated genes were dug out in tumorigenesis with the threshold q-value < 0.05 and − 1 > log FC > 1, respectively and specificity (0.944), and then validated in an independent group which was consisted of 116 CRC patients, 33 PC patients and 195 HV. The results of validation process presented a good sensitivity (70.0%) and specificity (98.7%), and a better sensitivity (83.3%) and specificity (96.8%) were gained when the training set and validation set were combined. Besides, this model has an obvious superiority over CEA in CRC early diagnosis (stage I and stage II CRC). Although the amino acids profile could not distinguish the early stage and advanced stage of CRC, Opr, Kyn and Trp showed significant increase trend along with the CRC progression. A study reported that the concentrations of some sphingomyelins, phosphatidylcholines and two amino acids Cit and His in plasma were related to CRC progression . These dGenerally, the diagnostic model was generated by a combination of three metabolites (tryptophan / sarcosine / glutamic acid), capable of differentiating between CRC patients and HV. Our research found a significant decrease of Trp in CRC compared with HV, and this result was supported by the gene expression change  and Q2(cum) of components for PCA model of amino acids metabolic profiles in plasma samples from training set. Figure S3B. Polt cumulative R2X(cum) and Q2(cum) of components for OPLS-DA model of amino acids metabolic profiles in CRC and HV plasma samples from training set. Figure S4. Loading scatter plot of amino acid metabolic profiles in CRC and HV plasma samples from training set. Figure S5A. Score scatter plot for PCA model of amino acids profiles in early and advanced stage CRC patients’ plasma samples. Figure S5B. Score scatter plot for OPLS-DA model of amino acids profiles in early and advanced stage CRC patients’ plasma samples. Figure S6A. Volcano plot of differentially expressed genes in dataset GSE164541. Figure S6B. Volcano plot of differentially expressed genes in dataset GSE 138202. Figure S7. Summarized pathway changes in tumorigenesis based on The KEGG pathway analysis. A: pathway enrichment based on GSE164541; B pathway enrichment based on GSE138202. Table S1. Logistics regression model parameters. Table S2. Areas under the curve of diagnostic factors. Table S3. The value of joint factor diagnostic model in the diagnosis of CRC. Table S4. The value of CEA in the diagnosis of CRC.


To evaluate the association between OCT-angiography (OCTA) and photopic negative response (PhNR) in open angle glaucoma (OAG) patients and assess the diagnostic accuracy of these parameters in early detection of glaucoma.A total of 152 eyes were enrolled in this study, 28 eyes with mild POAG (group I), 44 eyes with moderate-severe POAG (group 2) & 80 eyes of healthy subjects (control group). Full ophthalmological examination, OCTA and PhNR measurements were underwent for all participants. RNFL, GCC thicknesses, PhNR (implicit time and amplitude) were recorded. The superficial and deep capillary plexus vessel density  were measured by using 6 × 6 mm macula OCTA scans. The peripapillary vessel density (RPC-VD %) were measured by using 4.5 × 4.5 mm optic disk head OCTA scans.There were reduction of the median Interquartile range (IQR) thickness of the GCC and RNFL in OAG eyes versus normal (P < 0.001). RPC-VD%, SCP-VD % and DCP-VD% were significantly reduced in OAG eyes versus normal (P < 0.001). Increased OAG severity was associated with more reduction in PhNR amplitude and increased implicit time. Reduced PhNR amplitude and prolonged latency were significantly correlated with reduced vascular parameters. The RCP-VD and PhNR amplitude demonstrated higher diagnostic accuracy (98.7) with the largest AUC and higher sensitivity and specificity , followed by the PhNR implicit time with (AUC = 0.995) with a diagnostic accuracy 98.7. The SCP-VD, RNFL and GCC thickness had a diagnostic accuracy of , respectively (P < 0.001).OCTA vascular parameters displayed significant positive correlation with PhNR amplitude and significant negative correlation with PhNR implicit time. OCTA and PhNR parameters showed a high diagnostic accuracy for detection of glaucoma, and both may provide promising insight in early detection of glaucoma.This study was retrospectively registered on ClinicalTrials.gov . Open angle glaucoma (OAG) is considered the second leading cause of irreversible vision loss globally . It is aThe pathophysiology and factors contributing to glaucoma progression have not been fully understood . SeveralOptical Coherence Tomography Angiography (OCTA) and adaptive optics-OCT are promising current tools for diagnosing and management of glaucomatous patients. OCTA can measure the blood flow within the retina and the optic nerve, and thus can assess the degree of glaucomatous damage and evaluate the health of the ganglion cells and their axons –8.Recent publications using OCTA had mainly focused on evaluation of the microcirculation inside the optic disk and in the peripapillary area and displayed reduction of the peripapillary microcirculation –11. ReceThe photopic negative response (PhNR) is an objective, minimally-invasive test that reflect the function of RGC and their axons. It can be reduced in glaucoma and disorders that affect the inner-most retina , 17.Structural OCT measurements have limited diagnostic ability in identifying early stages of OAG, especially in patients with atypical optic nerves as in high myopia or uveitis and have moderate correlation with visual field testing , 18–20.Controversy regarding the associations between the vascular, functional and structural parameters in OAG is still present and the sensitivity of these parameters varies greatly. Some studies have found stronger associations between vascular and functional parameters rather than structural ones , 20, 21 The aim of the present study was to evaluate the correlations between vascular, functional and structural changes of the peripapillary RNFL and macular/ganglion cell complex in OAG patients and assess the diagnostic accuracy of OCTA and PhNR parameters in early detection of glaucoma changes.This cross-sectional comparative study included one hundred fifty-two eyes of 76 participants. All participants were recruited from the outpatient clinics of Benha University Hospital. 72 eyes of 36 patients with primary open angle glaucoma (glaucoma group) with a mean age of (40.2 ± 4.39) and 80 eyes of 40 healthy subjects (control group) with a mean age of (40.5 ± 4.02).This study was approved by the local ethics committee (Benha Faculty of Medicine Research Ethics Committee) and written informed consent, which were in compliance with the requirements of the Declaration of Helsinki, was obtained from all participants and the study was retrospectively registered on ClinicalTrials.gov .The clinical diagnosis of POAG was made after the demonstration of changes in the optic disk on clinical examination, e.g., optic nerve rim defect , presence of visual field defects and open angle on gonioscopy. The pre-perimetric glaucoma eyes had characteristic glaucomatous optic disk changes, without presence of visual field defects.The severity of POAG was determined according to visual field testing, patients with a global mean deviation (MD) less than − 6 dB were defined as "mild" POAG and patients with a global MD more than − 6 dB were defined as "moderate-severe" POAG.Our inclusion criteria for both control and glaucoma groups were: age 30 years and older, spherical equivalent (SE) between − 2 and + 2 D, with no history of trauma or any previous ocular surgery, or systemic diseases. Normal healthy participants had normal ophthalmological examination, with non-glaucomatous optic disks, IOP less than 20 mm Hg and best corrected visual acuity (BCVA) of 6/9 or better.Our exclusion criteria were as follows: SE more than + / − 2 diopters (D), history of any ocular surgeries, ocular trauma, optic nerve / macular or other retinal diseases, media opacity , and unreliable visual field results .2 circle centered over the optic disk and the macular map scan, respectively. The superficial and deep capillary plexus vessel density  were measured by using of 6 mm × 6 mm macula OCTA scans {400 × 400 pixels (two repeats/B-scan), scan time 3 s, axial resolution 5 µm and transversal resolution 15 µm}. The peripapillary vessel density (RPC-VD%) was measured by using of 4.5 mm × 4.5 mm optic disk head OCTA scans. The new AngioVue software , incorporating the automated algorithms for mapping capillary density were applied to automatically measure the vessel density in the peripapillary area, superficial and deep capillary plexus. The vessel density (VD) was represented as a percentage by taking the ratio of the total vessel area to the total area of analyzed region. OCTA images with a signal strength index (SSI) more than 6 were included, any images with observable motion or defocus artifacts were excluded.A complete clinical examination  measurement by Goldmann applanation tonometry, refraction, BCVA (converted to LogMAR), and fundus examination) were underwent for all participants. Central corneal thickness (CCT) was measured by the LenStar LS900. OCTA was done using the RTVue XR OCT Avanti System AngioVue Version 201.2.0.93 . Peripapillary RNFL and GCC thickness were recorded from a 3.4 mmPhNR measurements were done “using RETI-port/scan 21,” PhNR implicit time and amplitude were recorded. The PhNR implicit time was calculated as the time interval from the onset of the stimulus to the peak of the negative wave. There are many techniques for PhNR amplitude measurements  , but we 2 (625 nm), on a blue background 25 cds/m2 (455 nm), with Inter-flash interval: 1 s.PhNR was recorded using HK Loop electrodes which was installed into the lower fornix after applying topical anesthetic eye drops, ground gold skin electrodes attached on the central part of the forehead and the reference electrodes attached on the outer canthus skin of each eye. Ganzfeld settings were: red stimulus 0.4 cds/mP ≤ 0.05.Software  was used for the univariate, bivariate, and stratified analyses of the data. Qualitative variables were analyzed by constructing contingency tables with Pearson × 2 test. Analysis of variance (ANOVA) (with LSD as a post hoc test) and kruskal wallis test (KW) were used for multiple comparisons of quantitative variables for more than two groups. The Student t test and Mann–Whitney U test  were applied for the comparison of quantitative variables after establishing their normal distribution by means of the Shapiro–Wilk test and Levene test for equality of variance. Correlations among variables were studied by using the spearman coefficient for the association between variables. ROC (receiver operating characteristics) curve was performed to predict cutoff points for different variables. Differences were considered significant at P < 0.001; 90 µm vs. 108.5, P < 0.001, respectively). In the glaucoma patients the superior and inferior quadrants of RNFL were significantly thinner. More advanced POAG severity were associated with higher magnitudes of MD, increased cup-disk ratio and decreased both RNFL and GCC thicknesses , they were divided according to the mean deviation (MD) into two sub-groups 28 eyes with mild POAG {9 eyes were pre-perimetric} (group I), 44 eyes with moderate-severe POAG (group 2). The healthy control group included 80 eyes. There were no statistically significant differences between the glaucoma and control groups regarding age, gender, refraction or CCT Table . There wes Table .Table 1DP < 0.001, 47.85% vs. 54.8%, P < 0.001; 47.57% vs. 57.25%, P < 0.001; respectively), with a trend of worsening with increased POAG severity, , increased POAG severity was associated with more reduction in PhNR amplitude and prolonged latency, , there were also significant negative correlation with PhNR implicit time ; however, the correlation remained insignificant with GCC thickness . In group (1) the SCP-VD demonstrated significant correlations with RNFL thickness, PhNR implicit time ; however, the correlation remained insignificant with MD, PhNR amplitude and GCC thickness . In group (2) both RCP-VD and SCP-VD demonstrated significant associations with both structural and functional parameters, Table There were statistically significant differences of PhNR implicit time and amplitude between OAG patients versus normal . In patients with moderate /severe POAG, both PhNR amplitude and implicit time demonstrated significant associations with the vascular and structural parameters, which indicates that the function of the inner retina reduced proportionately with the neural loss, Table Although the PhNR amplitude was not significantly correlated with the MD in mild POAG, there were significant correlations between it and RCP-VD, RNFL and GCC thicknesses (P < 0.001), the sensitivity and specificity for a cutoff point of 57.55 were 100% and 97.5%, respectively, with a diagnostic accuracy 98.7. The PhNR amplitude had the ROC curve with the AUC value , the sensitivity and specificity for a cutoff point of 15.45 were 100% and 97.5%, respectively, with a diagnostic accuracy 98.7, followed by the PhNR implicit time with (AUC = 0.995) with a diagnostic accuracy 98.7.Table P < 0.001).Diagnostic accuracy was  for SCP-VD, RNFL and GCC thickness, respectively  Fig. .Fig. 5ROImaging plays a very important role in early detection and management of glaucoma , 4. It iIn the current study we evaluated the vascular, functional and structural changes in the peripapillary and macular area in patients with POAG.There were significant differences in RNFL, GCC thicknesses and visual field MD between the healthy and POAG eyes. Glaucomatous eyes had significant reduction in the inner plexiform layer of macular ganglion cells (GCIPL) and peripapillary RNFL microcirculation. These results are in agreement with previous studies , 9, 18.Regarding PhNR parameters, eyes with POAG had marked reduced amplitude and increased implicit time (IT) in comparison to normal eyes, these results are in accordance with a study by Banerjee et al.  who compp = 0.003, 0.020, 0.026, respectively). The linear relationship between the PhNR and the vascular parameters indicates that inner retinal function declines proportionately with changes in microvasculature in eyes with OAG. We assumed that in the early OAG, the decrease in peripapillary and macular vessel density is significantly associated with the severity of functional loss, regardless of structural loss. Previous studies on RNFL-GC-IPL perfusion also reported that OCTA -VD in the macular area was better associated with functional visual field parameters than structural GCC thickness [We also assessed the strength of association of each vascular perfusion parameters with structural (RNFL & GCC thicknesses) and functional (MD & PhNR) measures in different POAG groups. In patients with mild POAG, both RCP-VD & SCP-VD demonstrated a significant correlation with RNFL thickness, the amplitude of PhNR and IT; however, the correlation remained insignificant with the thickness of the GCC. The PhNR amplitude was not significantly correlated with MD in mild POAG; however, there was a significant correlation between it and RCP-VD, RNFL and GCC thicknesses . These results reflect a better diagnostic performance of the peripapillary vascular parameters than the macular vascular parameters for the diagnosis of glaucoma which are consistent with previous studies [In the current study, we also evaluated the diagnostic accuracy of different OCTA and PhNR parameters for the detection of glaucoma. The RCP-VD and PhNR amplitude demonstrated a higher diagnostic ability (98.7) with the highest AUC and higher sensitivity and specificity , followed by the PhNR implicit time (AUC = 0.995) with a diagnostic accuracy of 98.7. The SCP-VD, RNFL and GCC thicknesses had a diagnostic accuracy of , respectively  can supplement the structural OCT measurement if it has limited diagnostic value, as in high myopia, for the diagnosis and monitoring of POAG.