Depression and its increasing prevalence challenge patients, the healthcare system, and the economy. We recently created a mouse model based on the three-hit concept of depression. As genetic predisposition (first hit), we applied pituitary adenylate cyclase-activating polypeptide heterozygous mice on CD1 background. Maternal deprivation modeled the epigenetic factor (second hit), and the chronic variable mild stress was the environmental factor (third hit). Fluoxetine treatment was applied to test the predictive validity of our model. We aimed to examine the dynamics of the epigenetic marker acetyl-lysine 9 H3 histone (H3K9ac) and the neuronal activity marker FOSB in the prefrontal cortex (PFC) and hippocampus. Fluoxetine decreased H3K9ac in PFC in non-deprived animals, but a history of maternal deprivation abolished the effect of stress and SSRI treatment on H3K9ac immunoreactivity. In the hippocampus, stress decreased, while SSRI increased H3K9ac immunosignal, unlike in the deprived mice, where the opposite effect was detected. FOSB in stress was stimulated by fluoxetine in the PFC, while it was inhibited in the hippocampus. The FOSB immunoreactivity was almost completely abolished in the hippocampus of the deprived mice. This study showed that FOSB and H3K9ac were modulated in a territory-specific manner by early life adversities and later life stress interacting with the effect of fluoxetine therapy supporting the reliability of our model. Major depression, with its strikingly increasing prevalence, challenges the affected people, their families, the healthcare system, and even the economy . Basic nIn our model, PACAP HZ mice were exposed to maternal deprivation (second hit) that evokes epigenetic changes ,21 in thThe prefrontal cortex (PFC) is one of the most important brain territories in higher-order cognitive functions and behavior control . The queThe hippocampal formation is probably the most often examined brain territory in depression ,49. It iBased on these, in order to further support the validity of our model and to gain deeper insight into the functional–neuromorphological changes that underlie depression, in this study, we decided to focus on the recruitment of the PFC and the CA1 and CA3, as well as DG of the hippocampus. The altered histone acetylation pattern is a commonly used indicator of long-lasting regulatory changes in epigenetic alterations in depression models ,59,60. FIn both areas of interest in all of our experimental groups, we successfully detected a strong nuclear H3K9ac-ir. We compared the number of immunoreactive cells in order to assess the effect of the quality of maternal care, stress exposure, and SSRI treatment on this epigenetic marker.1,32 = 7.72; p = 0.039) and the interaction of maternal care and treatment  influenced the H3K9ac immunosignal significantly. Post hoc tests showed that the fluoxetine treatment reduced the number of H3K9ac immunopositive cells in the animal facility-reared (AFR) control group (compare p = 0.036). In line with this, in CVMS-exposed mice, fluoxetine treatment was associated with a reduced number of H3K9ac-ir cells (bars “c” and “d” in p = 0.047). Importantly, in the control vehicle-treated mice with MD180 history, a tendentiously lower cell count was detected (compare bars “a” and “e” in p = 0.068), and the fluoxetine treatment did not affect the H3K9ac-ir, in the mice that experienced maternal deprivation . Additionally, a second-order effect of maternal care and stress  and, more importantly, a maternal care × stress × treatment triple interaction was seen . In AFR mice, exposure to CVMS (bars “a” and “c” in p < 0.001) reduced the H3K9ac immunosignal that was reversed by fluoxetine treatment . A history of MD180 was associated with lower H3K9ac-ir in the control mice (see bars “a” and “e” in p < 10−4). Interestingly, in the CA1 region, fluoxetine treatment  and a third-order effect of the maternal care × stress × treatment interaction  influenced the histone acetylation significantly. Again, upon exposure to CVMS (bars “a” and “c” in p = 0.004), AFR mice showed a lower H3K9ac immunosignal, and if they were treated with fluoxetine , the H3K9ac-ir was higher again. MD180 resulted in a relatively low signal in the vehicle-treated control mice (bars “a” and “e” in p < 10−4). Fluoxetine treatment increased the histone acetylation in MD180 control mice (compare bars “e” and “f” in p = 0.001). Exposure to CVMS increased the H3K9ac-ir , but if the fluoxetine treatment was administered in MD180 PACAP HZ mice upon CVMS exposure, which was the case that carried all three “hits”, the histone acetylation appeared to be lower (compare bars “g” and “h” in p = 0.008).In the CA3 area, the main effect of maternal care  and stress  affected H3K9ac. MANOVA found that the interactions of maternal care × stress , stress × treatment , as well as maternal care × stress × treatment  influenced the histone acetylation in the DG. In AFR animals, the dynamics of H3K9ac-ir was similar to that we observed in the CA1 and CA3 regions: CVMS exposure reduced the immunosignal (bars “a” and “c” in p < 10−6), which was tendentiously reversed by the fluoxetine administration (see bars “c” and “d” p = 0.079). The control, vehicle-treated mice that experienced MD180 showed a reduced level of H3K9ac-ir (compare bars “a” and “e” in p < 10−6). The effect of fluoxetine treatment on the H3K9ac-ir depended on CVMS exposure in MD180 mice: in the control animals, fluoxetine treatment elevated the level of H3K9ac-ir , while in CVMS-exposed mice, the SSRI reduced the histone acetylation (compare bars “g” and “h” in p < 10−6).In the DG, the main effects of maternal care , and the interaction of maternal care and stress  exerted a significant effect on FOSB-ir. In AFR mice, CVMS exposure caused a significant elevation of the FOSB-ir in both the vehicle-treated  FOSB cell count was detected, compared with the control fluoxetine-treated AFR mice (see bar “b” in p = 0.002) than that observed in the respective AFR groups  and the third-order effect of maternal care × stress × treatment interaction  affected the FOSB-ir. In CVMS-exposed AFR mice, fluoxetine treatment reduced the FOSB-ir to an almost undetectable level (compare p = 0.009). In vehicle-treated, CVMS-exposed MD180 mice, the FOSB signal was also very low , which could not be further reduced by fluoxetine administration (compare bars “g” and “h” in p = 0.51).In the CA1 area, the main effect of maternal care , as well as the stress × treatment  and maternal care × stress × treatment  interactions, on FOSB-ir to be significant. In the control AFR mice, fluoxetine increased the FOSB cell count (compare bars “a” and “b” in p = 0.012). CVMS exposure also increased the FOSB signal in AFR mice (compare bars “a” and “c” in p = 0.008), while fluoxetine reduced this to the basal level (see bars “c” and “d” in p = 0.008 and images p = 0.010) or CVMS exposure (see columns “c” and “g” in p = 0.034). In the CA3 area, ANOVA revealed the main effect of maternal care  and the triple interaction of maternal care, stress, and treatment  influenced the FOSB signal significantly. In AFR mice, stress exposure increased the FOSB cell count (compare bars “a” and “c” in p = 0.042), which was reversed by fluoxetine treatment , which could not be further decreased by fluoxetine administration .In the DG, the main effect of treatment  the behavioral anomalies as assessed in depression and anxiety tests, (b) the functional–neuromorphological alterations in multiple limbic areas, and (c) the altered physical and endocrinological measures of the stress effect ,21. HereHistone acetylation is one of the commonly examined types of epigenetic modifications that do not affect the DNA sequence proper, but contribute to the control of gene expression . The levThe dynamics of the hippocampal H3K9ac-ir differed from that in the PFC. MD180 reduced histone H3 acetylation in all the hippocampal divisions, which is in agreement with the work of Sun et al. . CVMS reFOSB is a commonly used neuronal activity marker in stress research reflecting that the cellular response to a particular stimulus requires a response at the level of gene transcription ,68,69,70The phenomenon that chronic stress exposure increases the FOSB-ir in the PFC is well-known ,71,72,73As the history of MD180 was associated with a very low, almost undetectable FOSB signal in the hippocampus, it has to be stated that there was not much space for the SSRI treatment to reduce the FOSB-ir. One may argue that a technical error may have occurred that prevented the immunolabeling, but because the PFC sections of the same animals in the same staining process gave a reliable signal, we assume that the low hippocampal FOSB-ir in MD180 mice is a true area-specific observation. An alternative interpretation of this phenomenon could be that the effect of the fluoxetine treatment cannot be detected by FOSB labeling upon MD180, but other markers may still mirror the effect of SSRI treatment ,42,43. NTaking the complexity of the model and the capacity limitations of our animal facility into consideration, we had to restrict the study to male animals. Considering that the examined brain areas are estrogen-sensitive ,76, the In this study, we did not examine the histochemical characteristics of the cells that gave a positive signal for H3K9ac and FOSB. Further co-localization studies have to determine if there is a cell-type-specific alteration in the histone acetylation or neuronal activity pattern in the PFC and the hippocampal subdivisions. Considering that for some types of neurons, the expression of the neuronal activity marker immediate early genes is not characteristic ,78, we cSeveral lines of epigenetic markers have been identified in the past ,84, and With respect to the limitations of this study, our three-hit concept-based mouse model for depression in male PACAP HZ mice ,6,21 repThe experimental design corresponds to our most recently published works ,21. BrieIn total, 17 PACAP HZ male mice were identified in the AFR litters, while 25 PACAP HZ male mice were found in the MD180 litters. We did not use the female offspring in this study, and we also excluded the wild-type and PACAP KO mice, based on our earlier findings ,21.Both AFR and MD180 mice were randomly assigned to four subgroups, as shown in Half of the subgroups  were treated with intraperitoneal (ip) fluoxetine  injections. The mice in subgroups a, c, e, and g received physiological saline (0.2 mL vehicle ip) injections. Each group  consisted of 4–6 animals.The mice were kept under standard housing conditions , at the animal facility of the Department of Anatomy, the University of Pécs, in standard (30 cm × 30 cm × 28 cm) polycarbonate cages with 4–5 mice per cage group. The mice had ad libitum access to drinking water and normal standard rodent chow and were placed on fresh nesting material in a clean cage every other day.In this work, the stored brain tissue samples collected in our most recent study  were useMouse Brain Atlas by Paxinos and Franklin [Immunolabeling was performed on manually selected sections based on the Franklin . The braw/v) H2O2. The reaction was controlled by visual observation in a light microscope and stopped in PBS after 7 min. After several PBS washes, the preparations were mounted on gelatinized slides, air-dried, and dehydrated in ethanol solutions . After clearing in xylene (2 × 10 min), the sections were cover-slipped with Depex .The sections were rinsed 4 × 15 min in PBS, permeabilized with 0.5% Triton X-100 for 1 hour, and blocked with 2% normal goat serum (NGS) (Jackson Immunoresearch). Then, the sections were placed into a solution of 2% NGS containing anti-acetyl-lysine 9 H3 histone antibodies  or into a 2% NGS solution of rabbit anti-FOSB antiserum , followed by overnight incubation at 20 °C. Upon washes in PBS, a biotinylated goat anti-rabbit IgG solution was applied for 1 hour . Subsequently, after rinsing in PBS, the preparations were treated for 1 hour with a peroxidase-conjugated avidin–biotin complex (Vectastain ABC Elite Kit). After further PBS washes, the antibody binding was visualized in a Tris buffer (pH 7.4) containing 0.02% 3,3′diaminobenzidine  and 0.03% . The FOSB serum  was generated also in the rabbit, immunized with a C-terminal part of human FOSB protein. This serum was also characterized earlier by our group [The H3K9ac antiserum was raised in the rabbit using a synthetic peptide (range of residues 3-52), including the acetylation site at Lys9 in histone H3. The manufacturer published that the serum is specific to mice . From all mouse brains, five selected coronal sections of the PFC and the hippocampus were imaged. The count of immunoreactive nuclei was determined using the manual cell counting multipoint tool of ImageJ software  in non-edited images. All data were collected by an expert neurohistologist colleague who was not informed about the identity of the preparations.In the case of the PFC, the whole cross-sectional surface area of the PFC in an area of 500 µm × 750 µm C was evaFor publication purposes, the selected representative images were contrasted, cropped, and edited into photo montages using Photoshop software .All data are presented as the mean of the group. The error bars depict the standard error of the mean in all graphs. The statistical evaluation was carried out using Statistica software . Few data points beyond the two sigma range were excluded from the assessment. After testing for normality by Shapiro–Wilk test  and homo


Red raspberry is a kind of fruit with high nutritional values. To evaluate the comprehensive quality of 24 red raspberry varieties in Northeast China, physicochemical properties, bioactive compounds and sensory characteristics were measured, followed by principal component analysis (PCA) and cluster analysis (CA). Altogether, eight important property indexes for processing attributes were selected out using PCA, including titratable acidity (TAC), sugar-acid ratio (SAR), pH, length, diameter, weight, sucrose and citric acid. Six individual sugars, including l-rhamnose monohydrate, fructose, glucose, sucrose, maltose and d-trehalose anhydrous, as well as eight organic acids, including oxalic acid, tartaric acid, malic acid, α-ketoglutaric acid, lactic acid, citric acid, fumaric acid and succinic acid, were identified in red raspberry. The two main clusters according to individual sugar, organic acids and SAR indicated that varieties including ‘European red’, ‘DNS9’, ‘Bulgaskc’, ‘Canby’ and ‘Samodiva’ were suitable for fresh-eating or processing to juice or other products directly because they had suitable SAR; other varieties with relatively low SAR were unsuitable for fresh-eating and need to adjust their excessive sour taste during processing. Rubus idaeus L.) belongs to the Rosaceae family and is widely cultivated in Asia [The red raspberry  and cluster analysis (CA) were used to access their comprehensive quality. Results of this study could not only provide references for geneticists to develop varieties with higher nutritional value and consumer acceptability, but also help growers choose the best germplasm resources for fresh-eating and processing fruits with improved quality.All the standards for high performance liquid chromatography (HPLC), including l-rhamnose monohydrate, fructose, glucose, sucrose, maltose, d-trehalose anhydrous, oxalic acid, tartaric acid, malic acid, α-ketoglutaric acid, lactic acid, citric acid, fumaric acid, succinic acid, ascorbic acid (HPLC grade) were obtained from Shanghai YuanYe Biotechnology . Other chemicals were obtained from Tianjin Guangfu Chemical Reagent . All reagents were either analytical grade or HPLC grade.The red raspberry was obtained from Heilongjiang Academy of Agricultural Sciences  in July 2021. The 24 red raspberry varieties were introduced from various regions and countries, planted and cultivated in Heilongjiang Academy of Agricultural Sciences. Detailed information of the samples was presented in Red raspberry fruits (500 g) were squeezed after natural thawing, filtered using an 8-layer gauze, then the gauze was washed twice with distilled water. The washing solution and red raspberry juice were mixed and the mixture was brought to 500 mL using distilled water. The mixture (15 mL) was centrifuged at 4000 r/min, 4 °C for 15 min , then filtered through a 0.45 μm membrane to obtain a supernatant for further determination of individual sugar, organic acids, ascorbic acid, et al.n = 50) were measured by a vernier caliper. The weight of red raspberry fruits (n = 50) was measured by an electronic balance  [The length and diameter of red raspberry fruits  .Moisture, titratable acidity (TAC), soluble solids contents (SSC) and pH values were analyzed following the method of the association of official analytical chemists (AOAC) . Total s2-MS column . The mobile phase was consisted of acetonitrile and deionized water . The flow rate for mobile phase was 1.0 mL/min. The injection volume was 20 μL. The column oven temperature was maintained at 37 °C. The elution time was 30 min. Identification of the sugar was carried out by comparison of their retention time with those of the standards. Standard curves were obtained using 0–20 mg /mL sugar standards. Individual sugar contents in samples were calculated by regression equation and expressed as g/100 g dry weight (DW).Individual sugar, including l-rhamnose monohydrate, fructose, glucose, sucrose, maltose and d-trehalose anhydrous, was measured by the HPLC method according to Tilahun et al. , with sl2PO4 , methanol (eluent B). The elution condition was isostatic elution with A:B of 97:3 (v/v). The flow rate for mobile phase was 0.7 mL/min, and the injection volume was 10 μL. The column oven temperature was maintained at 35 °C. Wavelength for the detection was 210 nm. Organic acid contents in samples were calculated by regression equation and expressed as g/100 g DW.Organic acids, including oxalic acid, tartaric acid, malic acid, α-ketoglutaric acid, lactic acid, citric acid, fumaric acid and succinic acid, were measured by the HPLC method according to He et al. . The sysSensory characteristics evaluation of red raspberry was conducted using a 9-point hedonic scale , with slv/v), and filtered through a 0.45 µm durapore membrane. The mobile phase flow rate was 1 mL/min. Volume injection was 10 µL and the column temperature was 37 °C. The detection wavelength was 260 nm. The elution time was 10 min. The identification of the ascorbic acid was accomplished by comparing retention time of the standard. Ascorbic acid contents in samples were calculated by regression equation and expressed as g/100 g DW.Ascorbic acid was measured by the HPLC method according to Pantelidis et al. , with slv/v). Diluted samples (1 mL) were mixed with 4 mL of sodium carbonate solution  and 5 mL of 10% (v/v) Folin-Ciocalteu reagent and were allowed to stand at room temperature for 1 h. The absorbance of mixtures was measured at 765 nm. A calibration curve was obtained using 0–100 µg gallic acid (GAE)/mL and was used to calculate the total phenol contents of samples. Total phenol contents in samples were expressed as g (GAE)/100 g DW.Total phenol contents of samples were determined using Folin-Ciocalteu method according to Rambaran et al. , with mip < 0.05) among accessions. Principal component analysis (PCA) and cluster analysis (CA) were performed by using OriginPro 2021b Statistics .All biochemical determinations were repeated three times, and they were expressed as the mean ± standard deviation (SD). Duncan’s multiple-comparison test, one-way analysis of variance (ANOVA) was performed by using SPSS 20.0 Statistics  to identify significant differences . Therefore, ‘DNS9’, ‘Canby’, ‘European red’ and ‘Samodiva’ might have relatively pleasant sweetness than other varieties.Sugar provides energy for the life activities of human, and was an important fruit sensory quality . Six indOrganic acids were very important for fruit quality in terms of taste and contributed to the acidity of fruits . The conThe citric acid was the predominant organic acid in all varieties, with the contents from 2.95 ± 0.19 to 13.85 ± 2.38 g/100 g DW, followed by malic acid with the contents from 0.31 ± 0.02 to 2.37 ± 0.26 g/100 g DW. Oxalic acid and α-ketoglutaric acid were detected in all varieties with minor contents, while tartaric acid, lactic acid, fumaric acid and succinic acid existed in some varieties. Contents of fumaric acid were relatively low with values of 0.01 or 0.02 g/100 g DW. Citric acid was also identified as the major organic acids in wild red raspberry accessions from northern Turkey, in which small amounts of malic acids were also detected . MortazaThe sensory characteristics attributes, including appearance, color, flavor, taste and overall acceptability, and were presented in r = 0.95, p ≤ 0.001). The appearance had significantly negative correlation with the taste and overall acceptability, with the correlation coefficients of −0.52 and −0.55 (p ≤ 0.01), respectively. These findings indicated that consumers tend to have a higher overall acceptability for fruits that taste better, and their vision may be affected by the attractiveness of fruit, while ignoring the smell of fruit. Considering these factors, future sensory evaluation should strengthen the potential sensory bias caused by flavor preference.The correlation of five sensory characteristics  are shown in Ascorbic acid contents are one of the most important compositional parameters determining the nutritional and health related properties of plant foods . The resResearchers revealed that berry fruits are great dietary sources of phenols. Phenol may act as strong antioxidants and thus could help in the prevention of many diseases . The conSchulz et al.  detectedPCA is used to identify potential trait combinations . It extrAs reported in The comprehensive evaluation indexes were the accumulated sum of the factors’ score of each sample and the weight value of each principal component . TherefoComprehensive quality is a concept not only based on the single quality attributes but also on interactions among them. It includes the external characteristics , taste qualities , and also involves nutritional value (ascorbic acid and TPC) et al. . AccordiThe PCA results showed that TAC, SAR, pH, length, diameter, weight, sucrose and citric acid were the most important property indexes for processing attributes in the evaluating process. Although red raspberry comprehensive quality was closely related to these property indexes, the taste was affected by individual sugar, organic acids and SAR ,46,67. TThe ‘European red’, ‘DNS9’, ‘Bulgaskc’, ‘Canby’ and ‘Samodiva’ were placed into the first cluster (Ⅰ), and the rest of the red raspberry varieties was placed into the second cluster (Ⅱ). Ⅱ-A was comprised of 8 red raspberry varieties, Ⅱ-B included a total of 11 red raspberry varieties. In cluster (Ⅰ), ‘European red’, ‘DNS9’, ‘Bulgaskc’, ‘Canby’ and ‘Samodiva’ were grouped together because they had higher values of sucrose and SAR, and lower values of citric acid. In cluster (Ⅱ), Ⅱ-A was the red raspberry with higher fructose and glucose values, and Ⅱ-B was composed of other remaining varieties.Overall, significant diversity was revealed regarding the fruit quality and nutritional properties of the evaluated varieties by determining the physicochemical properties, bioactive compounds and sensory characteristics, and evaluating the comprehensive quality between different varieties based on the analysis of PCA and CA.According to PCA, the top five red raspberry varieties based on the final rank of comprehensive quality were ‘European red’, ‘DNS9’, ‘DNS4’, ‘Canby’ and ‘Beijing32’, and the top five red raspberry varieties for fresh utilization were ‘DNS9’, ‘European red’, ‘Bulgaskc’, ‘Canby’ and ‘Samodiva’. The second group of PCA was consistent with the results of CA for fresh-eating or processing to juice or other products directly, because they had suitable SAR, however, other varieties with relatively low SAR were unsuitable for fresh eating and need to adjust their excessive sour taste during processing.The ‘DNS4’ and ‘Beijing32’ had better appearance and bigger fruits, ‘European red’, ‘Schopska’ and ‘Fertod zamatos’ had higher contents of ascorbic acid. The ‘Boyene’, ‘DNS1’, ‘DNS5’ and ‘Beijing10’ contained more TPC, and these varieties might release more commercial potential abilities in the future.


Diastasis recti abdominis (DRA) is a prevalent condition in the postpartum period. To date, there is scant knowledge on how DRA influences physical, mental, and emotional health. This study investigates primiparous women`s knowledge about DRA, concerns about abdominal appearance, and perceived abdominal muscle strength, comparing women with and without reported DRA.This was a cross-sectional comparison study. Data were collected by a web-based questionnaire, mainly through social media in Norway. To be included in the study women had to be primiparous 6–8 months postpartum. The questionnaire contained questions regarding women`s knowledge about DRA, perceived protrusion, received treatment, concerns with abdominal appearance and muscle strength. Abdominal body image was measured through the shape concern questions from The Eating Disorder Examination questionnaire (EDE-Q 6.0). Demographic and other descriptive variables are presented as means with standard deviations (SD) or as frequencies with percentages. Chi-square test of independence and independent sample t-tests were used to compare differences between women with and without abdominal protrusion for categorical and continuous variables, respectively.Our sample consisted of 460 women. Knowledge about DRA was reported by 415/440 (94.3%) women. A total of 73.3% reported to have been worried during pregnancy about abdominal appearance postpartum. Mean degree of concern about present abdominal appearance was 5.5/10 (SD 2.4). Almost 80% experienced weaker abdominal muscles than pre-pregnancy. Ninety-six women (20.9%) reported a protrusion along the midline of their abdomen. Significantly more women with protrusion reported weaker abdominal muscles than women without protrusion. The most frequent treatment women with protrusion reported were exercises for the abdominal muscles (92.6%). Mean score on the EDE-Q, shape concern questions, was higher in women with reported protrusion (mean score: 2.37 (SD 1.6) than women without protrusion (mean score: 2.14 (SD 1.4), p = 0.175.Primiparous women are concerned about abdominal appearance both during pregnancy and after birth. Those reporting abdominal protrusion are less satisfied with their abdominal appearance and they report weaker abdominal muscles than women without protrusion. This study may contribute to improved knowledge about women`s health concerns, and assessment of DRA should be part of routine follow-up of postpartum women. Pregnancy and childbirth bring anatomical and morphological changes to the lower back, pelvic girdle, abdomen and pelvic floor . The mosDiastasis recti abdominis (DRA) is defined as an impairment with midline separation of the two rectus abdominis muscles along the linea alba, and is diagnosed by measuring the distance between the medial border of the two rectus abdominis muscles, the inter-recti distance (IRD), 5. CandRecent research has documented a connection between increased media attention and body image concerns among pregnant and postpartum women –17. A syThe aims of the present study were to investigate primiparous women`s knowledge about DRA, whether they have concerns about abdominal appearance and, perceive impaired abdominal muscle strength 6–8 months postpartum. Further to study whether there are differences between women with and without reported abdominal protrusion regarding knowledge about DRA, concerns about abdominal appearance, and perceived abdominal muscle strength 6–8 months postpartum.This was a descriptive cross-sectional comparison university initiated and conducted study. The study was conducted between March 2019 and August 2020. Healthcare clinics in Oslo and Akershus county, Norway and social media (Facebook and Instagram) were used to recruit women. The participants signed up by sending an email to the researcher or by clicking on a registration link. Before inclusion they had to confirm that they fulfilled the inclusion criteria. They subsequently received an email with the informed consent and a link to the electronic questionnaire.Inclusion criteria were primiparous women 6–8 months postpartum with a single or multiple pregnancy, any mode of delivery, and who were able to understand a Scandinavian language. Exclusion criteria were multiparous women, being < six months and > eight months postpartum, and being < 18 years old. In addition, we excluded responses with no answers, duplicates and no postpartum data.The participants were invited to respond to an electronic survey (SurveyXact) via their personal phone or computer. Up to three reminders were sent to non-responders.The questionnaire was a new web-based questionnaire developed for the present study, containing a combination of validated instruments and new questions developed from a focus group of a convenient assembly of parous women. The response options for the new questions were a mix of 11-point Likert scales, close-ended, and semi-close-ended questions. The new questions and response categories were piloted among members of our research group and women in the focus group, and the questions were revised accordingly for clarity.Response to all 162 questions in the questionnaire required a maximum of 30 min to complete. However, less time was required for those who reported no abdominal, low back-, or pelvic girdle pain, or pelvic floor dysfunction.Participants` demographics included age, height, pre-pregnancy weight, current weight, weight gain in pregnancy, single/twin pregnancy, time since birth, mode of delivery, child`s birth weight and length, physical activity level (frequency and min/week) and self-reported health, smoking habits, workload, breastfeeding, menstrual cycle, education level, and ethnic origin.www.fhi.no/morogbarn. We have not been able to find specific questionnaires on physical activity in the postpartum period.The questions about exercise frequency and exercise duration were from The Physical Activity and Pregnancy Questionnaire, which has been found to be an acceptable measure of habitual physical activity and exercise among pregnant women at group level . QuestioHave you heard about separation of the abdominals/ DRA related to pregnancy or the postpartum period? Response options: “yes” or “no”.Only women responding yes to the above question, were asked to respond to the following questions;Where did you hear about DRA? Response options: “scientific literature”, “the health service system ”, “friends/ acquaintances”, “social media”, “TV/ media”, “women’s magazines”, or “other”. Multiple response categories were allowed.Have you tried one or more treatments to decrease the separation between your abdominals postpartum? Response options: “yes” or “no”.Only women responding yes to the above question were asked to respond to the following questions;What type of treatment have you tried to resolve DRA? Response options: “surgery”, “application of specific creams to the abdomen”, “external support/corset”, tape (e.g. kinesio tape)”, “electrical stimulation”, “exercises for the pelvic floor muscles (PFM)”, “exercises for the abdominals”, or “other”. Multiple response categories were allowed.From where/whom have you found/received treatment/exercises for DRA? “internet sites/ social media”, “physiotherapist”, “naprapath/chiropractor/osteopath”, “fitness center/ personal trainer”, or “others (family/friends)”. Multiple response categories were allowed.How would you describe your DRA now compared to before treatment? The participants rated their recovery on a scale from − 5 to 5, where − 5 represented “very much worse”, 0 represented “unchanged”, and 5 represented complete recovery.All women responded to how they perceived their abdominal appearance using categorical responses and numeric rating on scales from 0 to 10.Do you experience a protrusion along the midline of your abdomen? “no, never”, “yes, sometimes”, “yes, all the time”, or “do not know”. This variable was dichotomized to yes and no, where “do not know” was classified as no.How would you describe the strength of your abdominal muscles? “stronger than pre-pregnancy”, “the same as pre-pregnancy”, “somewhat weaker than pre-pregnancy”, “much weaker than pre-pregnancy”, or “do not know”.Do you feel that the skin on your abdomen is flabby/lax? “no”, “yes, somewhat”, or “yes, to a great extent”.Have you developed striae on your abdomen during pregnancy/postpartum? “no”, “yes, a few”, “yes, several”, or “yes, a lot”.Only women responding yes to flabby/lax skin and/or striae rated bothersomeness on a scale from 0 to 10 where 0 represented “not at all” and 10 “to a great extent”. In addition, all women answered the following questions;Have others (e.g. friends or family) been concerned about your abdominal appearance postpartum? “no”, “yes, to some degree”, or “yes, to a great extent”. In the comparison analysis between women with and without reported protrusion this variable was dichotomized into “yes” (to some degree/ great extent) and “no”.Do you find there is too much focus from the media, TV, internet, magazines, about having a flat abdomen postpartum? “strongly disagree”, “disagree”, “neither”, “quite agree”, or, “agree to a great extent”. In the comparison analysis between women with and without reported protrusion this variable was dichotomized into “yes” (quite agree/agree in great extent) and “no” (neither/disagree/strongly disagree).While you were pregnant, were you worried about how your abdomen would look postpartum? “no”, “yes, to some degree”, “yes, to a great extent”. In the comparison analysis between women with and without reported protrusion this variable was dichotomized into “yes, worried” (some degree/great extent) and “no”.To what extent are you concerned about your abdominal appearance today on a scale from 0 to 10 where 0 represents “not at all” and 10 “to a great extent”?Overall, how satisfied are you with your abdominal appearance postpartum on a scale from 0 to 10 where 0 represents “very dissatisfied” and 10 “very satisfied”?The Eating Disorder Examination questionnaire (EDE-Q 6.0) is a self-reported measure of eating disorder psychopathology focusing on the previous 28 days and consisting of 28-item divided into four subscales. The Norwegian EDE-Q version has shown good test-retest reliability with a Spearman’s correlation coefficients of 0.82–0.91 for the subscales. Norms for the subscale; shape concern for healthy women is 1.8 (SD 1.6) and 4.7 (SD 1.4) in women with an eating disorder . Due to All statistical analyses were performed using SPSS statistical software package version 24 . Demographic and other descriptive variables are presented as means with standard deviations (SD) or as frequencies with percentages. Shapiro-Wilk test was used to test normality of distribution. Paired sample t-tests were used to compare the participants mean score for continuous variables pre-pregnancy, during pregnancy and postpartum. Chi-square test of independence (with Yates Continuity Correction) and independent sample t-tests were used to compare differences between women with and without protrusion for categorical and continuous variables, respectively. P-value was set at < 0.05.Our sample comprised 460 women, recruited mainly through social media in Norway. Flow diagram of included women with reason for exclusion are presented are Fig. Table Knowledge about DRA was reported by 415/440 (94.3%) women. Friends and acquaintances, social media and health personnel were the most common sources of this information. Approximately 20% of the women who knew about DRA had tried one or more treatment options to reduce it postpartum. The most frequently reported treatments were PFM exercises (84.1%) and abdominal muscle exercises (82.9%). These were mostly assessed through social media 44/82 (53.7%). No change in abdominal recovery after treatment was reported in 14/81 (17.3%) women and 23/81 (28.3%) reported complete recovery.Table Almost 80% reported weaker abdominal muscles after childbirth compared with pre-pregnancy levels.Four hundred and thirty-three women responded the EDE-Q, subscale shape concern questions. Mean subscale score for included women was 2.19/6.0 (SD 1.48). A mean subscale score above 4.7 was reported in 32/433 (7.4%) women.Ninety-six women (20.9%) reported a protrusion along the midline of their abdomen. There was no statistically significant difference in background variables between women with and without protrusion. In women who reported protrusion the prevalence of Caesarean section was 31.1% compared to 20.1% of women with vaginal delivery (p = 0.053). One or more treatment methods were tried in 27/92 (29%) of women with reported protrusion. The most frequent reported interventions were exercises for the abdominal muscles (92.6%) and the PFM (81.5%). The exercises were mostly sourced through social media 14/27 (51.9%). 22% of the women with reported protrusion who had tried any form of treatment for DRA (n = 27) reported no improvement and 11% reported complete recovery.Table There was no significant difference in mean score on the EDE-Q shape concern questions in women with reported protrusion (mean score: 2.37 (SD 1.6)) compared to women without protrusion (mean score: 2.14 (SD 1.4)), p = 0.175.The results of the present study showed that primiparous women are concerned about abdominal appearance both during pregnancy and in the postpartum period and most seek advice of treatment through social media. Women reporting protrusion of the abdominal wall are less satisfied with their abdominal appearance and they report weaker abdominal muscles than women without protrusion. In addition, those reporting abdominal protrusion report weaker abdominal muscles than women without protrusion.Most of our study participants knew about DRA and this information mostly came from social media. Eriksson-Crommert, Petrov Fieril, and Gustavsson (2020)  found thWe found that more than 2/3 of our participants agreed or strongly agreed that there is too much media focus on regaining a “flat abdomen”. Studies have shown that media influence on body image is common in women and adolescent girls, 27. CoyAlthough several studies have investigated how postpartum women feel about their body, 29, as Almost 80% of our sample reported weaker abdominal muscles. This is based on women`s perceptions only, and not clinical assessment. There are few clinical studies of abdominal function after birth and there is a great diversity in how the studies assess abdominal muscle strength. Gilleard and Brown (1996)  assessedThe mean subscale score for shape concern the total sample in our study was higher than the mean subscale score for healthy Norwegian women (n = 1845); 1.8 (SD 1.6) . AlthougIn the present study there was no clinical assessment with observation of protrusion. Self-report of DRA and protrusion may be considered less valid than assessment by health personnel. However, the prevalence of 26.5% in women reporting abdominal protrusion in the present study is in line with previous research, although a bit lower than was found in a clinical study from the same country . SperstaWe found that women reporting protrusion reported significantly weaker abdominal muscle strength postpartum compared to pre-pregnancy. Several clinical studies comparing women with and without DRA confer with these results, 12, 35.We found no difference in bothersomeness about striae or lax skin between women with or without reported protrusion. However, women with reported protrusion showed a tendency of more lax skin than women without reported protrusion. Lax skin may be a marker of weak connective tissue and subsequent risk factor for DRA . FurtherThe findings of less satisfaction with abdominal appearance in women reporting protrusion confer with a qualitative study finding that women with increased IRD might experience body dissatisfaction . Also, KAs response rate and generalizability is not possible in web-based surveys, we may compare our participants with the official statistics of the Norwegian Institute of Public Health`s Medical Birth Register. In 2019 54 407 births were registered. 43% of these were primiparous, 98.5% were single deliveries and 15.9% gave birth by Cesarean section. Mean age in primiparous women was 29.7 years (SD 4.8), women’s mean BMI prior to pregnancy was 24.6 (SD 4.9) and the baby`s mean birth weight was 3500 g (SD 592). Our sample is comparable to the official characteristics of these background variables in Norwegian primiparous women, which may be considered a strength of the study. A limitation is that no power calculation was performed for the present study, however we consider the study`s sample size a major strength and that 92% responded to the entire questionnaire. Further, the use of DRA with a protrusion as an inclusion criteria for being classified with DRA in comparison with no DRA, may indicate a more robust diagnoses compared with a simple question on whether the women experience DRA. Inclusion of women with twin pregnancy, delivery between the 26th and 30th gestational week, and child`s birth weight (1000-1499 g) might be considered a limitation. However, the numbers of women in these groups are few and we therefore decided to include them and report the result of these subgroups separately. The results may serve as background for future studies.Although background variables of the participants are comparable with the total population, we cannot ensure generalizability as selection bias may have occurred due to recruitment mainly through social media as more women with more concerns about this topic might have been recruited. Our participants had a high educational level. Norway has a high uptake of mobile phones, and access to internet and social media is also commonly used by new mothers in other countries , but thiOur study found that primiparous women are concerned about abdominal appearance and that women reporting DRA with protrusion experience reduced abdominal strength in comparison with pre-pregnancy level. In the postpartum period women learn about exercises for the abdominals from social media where there is no quality assurance of the information. There is a need for future follow-up assessments of women in the postpartum period concerning DRA, abdominal strength, and abdominal body image. Further high quality RCTs on the effect of different exercise programs in prevention and treatment of DRA are warranted to be able to guide women returning to exercise after childbirth.


Gender (boy) and being employed influenced the source, but not the quantity of caffeine consumed. One-fifth (21.2%) of adolescents consumed more than the recommended European Food Safety Authority (EFSA) safe level (3 mg·kg−1·day−1). Taste, energy, and temperature were the main motivators for consumption, and increased energy, excitement, restlessness, and sleep disturbances were reported effects following caffeine consumption. This study provides information on caffeinated product consumption among New Zealand adolescents, some of whom consumed caffeine above the EFSA safe level. Public health initiatives directed at adolescents may be important to reduce potential caffeine-related harm.Adolescents may be particularly vulnerable to the effects of caffeine due to a lack of tolerance, their small size, changing brain physiology, and increasing independence. Concerns about adolescent caffeine consumption relate to potentially serious physiological and psychological effects following consumption. Motivations driving caffeine intake are not well understood among adolescents but are important to understand to reduce harmful behavioural patterns. This study explored caffeine consumption habits  of New Zealand adolescents; and factors motivating caffeine consumption and avoidance. The previously validated caffeine consumption habits questionnaire (CaffCo) was completed by 216 participants (15–18 years), with most (94.9%) consuming at least one caffeinated product daily. Chocolate, coffee, tea, and kola drinks were the most consumed sources. The median caffeine intake was 68 mg·day Globally, at least 70% of people consume caffeine, making it the most frequently and widely consumed mental stimulant. Similarly, 71–96% of New Zealanders over the age of 15 have been reported to consume caffeine. Caffeine occurs naturally in tea, coffee, and chocolate, or it may be artificially added to products such as soft drinks, energy drinks, sports gels, and medications . Sleep iIndividual responses to caffeine vary according to genetic and environmental factors, as well as age, sex, and habituation to caffeine ,5. Adole−1·day−1 by Health Canada and the European Food Safety Authority (EFSA) . e effect .A total of 216 participants with a mean age of 16.6 years ± 0.82 years (range 15–18 years) completed the questionnaire and their results were analysed. −1·day−1 [52.3–81.7 mg·kg−1·day−1].p = 0.005; Cohens r = 0.165), whereas boys were 2.18 times more likely to consume energy drinks  than girls. There was a trend for girls to consume more coffee (p = 0.066) and chocolate (p = 0.084) than boys. Participants who were in paid employment were 1.93 times more likely to drink coffee than those not in paid employment . Those in paid employment were also 3.23 times more likely to drink caffeinated RTDs than those who were not employed .Girls were 2.25 times more likely than boys to consume tea (p > 0.05). The median estimated daily intake of caffeine from tea was higher in girls than in boys (p = 0.019). Conversely, the median estimated daily intake of caffeine from kola drinks and energy drinks was higher in boys than girls (p = 0.003).p = 0.021 and p < 0.001, respectively). The analysis of the type of coffee consumed found a trend toward the increased intake of filter/plunger coffee in employed adolescents (p = 0.065) compared to those who were not employed.The median estimated daily intake of caffeine from coffee and caffeinated RTDs was higher for adolescents who were in paid employment than those who were not employed (n = 216), 58.8% and 31.3% reported never/infrequently drinking tea and coffee, respectively, due to the flavour. Moreover, 45.3% of participants reported not consuming coffee due to concerns about becoming dependent on it; whereas only 17.0% gave this as a reason for not consuming tea. Participants avoided chocolate, kola drinks, and energy drinks due to health concerns such as high sugar content . There were 85.4%, 72.0%, 58.3%, and 37.1% of respondents who had never considered taking caffeine tablets, caffeinated sports supplements and gels, caffeinated RTDs, and energy drinks, respectively.Of the total respondents (p = 0.425) who exceeded the EFSA safe-caffeine intake level of 3 mg·kg−1·day−1 [p < 0.001). Tea drinkers were 5.89 times more likely to consume over the safe level than non-tea drinkers (p < 0.001). Similarly, energy drink consumers were 2.44 times more likely to consume over the safe caffeine level than non-energy drink consumers (p = 0.021). A trend also exists between the consumption of caffeinated RTDs and exceeding the safe level of caffeine intake (p = 0.054).There were 21.1% of participants  reported experiencing at least one type of perceived effect following caffeine consumption, the most frequent being “increased energy” (56.3%), “excited” (53.5%), “restlessness” (44.7%), “urge to urinate” (44.2%), and “sleep disturbances” 41.4%; .Our findings indicate that the proportion of adolescents who consume caffeine daily may have risen from 71% in 2008/2009 to 94.9%, as observed in the present study . Chocola−1 is similar to that of Americans aged 13 to 17 (52 to 66 mg·day−1) [−1) and South Korean adolescents aged 15 to 18 (30.04 mg·day−1) [−1 has been reported among 32,000 European adolescents aged 10 to 18 [The median daily caffeine intake in this study of 68.3 mg·dayg·day−1) ,21 but hg·day−1) . Convers10 to 18 , possibl−1·day−1) is well below the adverse effect level (3 mg·kg−1·day−1) suggested by EFSA [n = 3310) [n = 508) [The median caffeine intake in this study   and 12.2n = 508) . While sn = 508) .We found that nearly one-third (31.6%) of adolescents consumed energy drinks, a ten-fold increase over that reported in 2008/2009 (3.1%) . This isWe found boys had greater caffeine intake from energy drinks than girls, which is consistent with previous studies in the USA, Canada, and Poland ,30,31,32Caffeine intake from tea was 2.25 times higher in girls than boys, which may relate to the idea that tea is perceived as a more “feminine” drink ,31. Thesp = 0.065) towards increased filter/plunger coffee intake in those employed. Again, this trend may be due to improved accessibility of coffee at their place of employment [Although the current study found no difference in the total caffeine intake between those who are employed and those who are not, employed adolescents were 1.93 times more likely to drink coffee and 3.23 times more likely to drink caffeinated RTDs than those who were not employed, with a trend ; however, participants were less concerned about becoming dependent on tea (17.0% of non-consumers gave this as a reason to avoid tea) despite both products providing a significant amount of caffeine  .n = 1006) that adolescent boys ingested coffee “to stay awake” [n = 166) found that energy provision was the main motivating factor regardless of whether they liked the taste of caffeinated beverages [Coffee drinkers and energy drink users identified the energising effects  as the main reason for consuming these beverages. This is consistent with previous findings from a Saudi Arabian study  reported experiencing at least one type of perceived effect following caffeine consumption, with the most frequently reported being increased energy, excitement, restlessness, the urge to urinate, and sleep disturbances. Previous reports have suggested that adolescents consuming caffeinated products expect to experience increased energy ,29, altep < 0.001) than participants who reported no adverse effects following caffeine consumption. Since these participants reported consuming these products for their energizing benefits, they are aware of the caffeine content of these products and expect to experience these effects following consumption. Adolescents have a good understanding of the links between energy and coffee and a good knowledge of the effects of caffeine, including short-term increases in energy, rapid heart rate, jitteriness, and insomnia [The adolescents who reported perceived adverse effects had a higher intake of coffee and energy drinks  to support healthy caffeine habits among adolescents by increasing their “caffeine literacy” .Although the study has significant strengths, including using CaffCo, a New Zealand-validated questionnaire to determine caffeine intake patterns in New Zealand, there are also limitations. The use of a regional-based convenience sample and the relatively small sample size may have affected the accuracy of the caffeine consumption data and reduced the generalisability of results. Another limitation is that caffeine non-consumers may have felt the questionnaire was not applicable to them and therefore they did not participate, leading to an overestimation of the proportion of adolescent caffeine consumers. Future research should use a larger, more diverse sample, allowing for cluster analysis to better identify caffeine user types and determine how caffeine consumption patterns change over the life course.Most New Zealand adolescents regularly consume caffeine, with the majority having a moderate intake. Coffee is the main contributor to daily caffeine intake, followed by chocolate and tea. Both gender and employment status affect the source but not the overall level of caffeine intake. Taste, energy, and temperature were the main reasons adolescents chose to consume caffeinated products, with a small number of adolescents using sports supplements due to peer pressure or instead of illicit drugs. Over the past decade, there has been a ten-fold increase in caffeine intake from energy drinks. A fifth (21.1%) of New Zealand adolescents had a daily caffeine intake above the recognised safe level, which represents a three-fold increase over the past decade. Individuals consuming over the safe, recognised caffeine level had a higher intake of coffee, tea, and energy drinks than those who did not exceed the safe intake level and were also more likely to experience adverse effects following caffeine intake. Adolescents who consume greater than the safe level of caffeine would benefit from strategies designed to improve caffeine literacy to reduce the risk of caffeine-related harm.


There is a modest relationship between speech perception skills and perceived hearing handicap in individuals with hearing loss. In this study, an attempt is made at linking psychoacoustic results and speech perception skills to understand the subjective handicap and quality of life.To investigate how speech perception in noise , difference limen frequency, temporal modulation transfer function, hearing handicap inventory for adults, and quality of life in auditory neuropathy spectrum disorder differs from sensorineural hearing loss. Further we attempt to discern attributed factors of hearing handicap in auditory neuropathy spectrum disorder and sensorineural hearing loss.A cross-sectional study with comparative and correlational research designs were utilized. Eighty-four participants were grouped into sensorineural hearing loss (n = 49), and auditory neuropathy spectrum disorder (n = 35) was sub-grouped into mild, moderately severe, and severe. We evaluated signal to noise ratio-50, difference limen frequency, and temporal modulation transfer function. In addition, hearing handicap inventory for adults, and quality of life questionnaires were administered.Mild auditory neuropathy spectrum disorder showed impairment in speech perception and discriminating frequency, which were similar to the severe sensory neural hearing loss. Temporal resolution impairment in auditory neuropathy spectrum disorder mild was significantly higher than in each sub-groups of sensorineural hearing loss. The severity of the hearing handicap in was similar to severe sensorineural hearing loss, quality of life was equally affected in sensorineural hearing loss and auditory neuropathy spectrum disorder. In sensorineural hearing loss, signal to noise ratio-50 was positively related, and quality of life was negatively related to hearing handicap. In auditory neuropathy spectrum disorder, no contributory factors were related to hearing handicap.Mild auditory neural pathology demonstrates impairment in speech recognition and psychoacoustic skills similar to severe cochlear pathology. In sensorineural hearing loss, hearing handicap is predicted from quality of life and speech perception, but none of the contributory factors predicted hearing handicap in auditory neuropathy spectrum disorder. Hearing loss has a significant impact on hearing handicap and eventually affects the quality of life. Iwasaki et al.Auditory neuropathy spectrum disorder (ANSD) is a type of SNHL. The onset of hearing loss in ANSD at a later part of their life affects speech understanding ability,Psychoacoustics combines the study of acoustics and auditory physiology to determine the relationship between a sound’s characteristics and the auditory sensation that it provokes.Difference limen frequency (DLF) is another psychoacoustic test which assesses the ability to detect the smallest change in frequency between the two tones. GoldsteinNordvik et al.,The present study is focused on how the perceptual skill (SNR-50), psychoacoustic skills (TMTF and DLF), quality of life, and hearing handicap differed between ANSD and SNHL subjects. If the scores are different among groups, then to what extent auditory neural pathology subjects have impairment against their counterparts who have an auditory sensory pathology in the above-mentioned skills? Further, the relationship between hearing handicap and each of the measurements was ascertained in each group to determine the attributing factors for hearing handicap. It is hypothesized that mild pathology in the inner ear has impairment similar to the severe degree of auditory neural pathology, and each of the measured skills attributes for hearing handicap. The study aimed to investigate the potential attributing factors of hearing handicap in individuals with SNHL and ANSD. The following objectives were formulated: a) To compare each of the measured skills between the SNHL and ANSD group and b) To find the relationship between hearing handicap and measured skills and identify the potential attributing factors for hearing handicap.A cross-sectional study with comparative and correlational research designs was utilized. The study was a part of a research project which was approved by the ethics committee with the reference number SH/CDN/CC/ARF-59/2019-20. A purposive sampling method was used. The procedures involved in the study were explained to the participants, and informed consent was obtained before the data collection. A total of 84 participants were recruited in the study. We grouped into two based on the site of pathology. Group I (n = 49) included participants with confirmed cochlear hearing loss.1Hearing thresholds ranging from 30 dBHL to 90 dBHL in both ears symmetrically.2Normal middle ear functioning as indicated by type “A” tympanogram with elevated reflexes (ipsi- and contra-) at 500 Hz to 4 kHz (an octave) or absent reflexes.3Absent TEOAEs or DPOAEs based on hearing loss.4Tone decay test: patients who heard the tone for one complete minute without a change in loudness.5Native speakers of Kannada.Those participants who fulfilled the following conditions were included in the study:We sub-grouped these participants into three based on degree of hearing loss: mild , moderately severe  and severe .Group II included 35 individuals with confirmed auditory neuropathy spectrum disorder.1They complained of reduced hearing sensitivity and difficulty understanding speech.2They met the diagnostic criteria of auditory neuropathy spectrum disorder as per Starr et al.3Hearing thresholds ranging from 30 dBHL to 90 dBHL in both ears symmetrically.4Normal middle ear status indicated by type “A” tympanogram and robust otoacoustic emission.5Impaired neural response reflected in auditory brainstem response and middle ear reflexes were recruited in the study.6Native speakers of Kannada.All the participants met the following inclusion criteria:Further, the ANSD group was sub-grouped into three based on degree of hearing loss mild , moderately severe  and severe .Exclusion criteria for group I and II: participants having any history of systemic illnesses and complaints of any psychological or neurological problem were not included in the study.1A)Behavioral tests:Quantitative measures: The procedure of data collection included quantitative and qualitative tests. The quantitative tests included behavioral tests and psychoacoustic tests. Behavioral tests comprised of pure tone audiometry, speech perception in noise (SNR-50). Psychoacoustic tests included difference limen frequency (DLF) and temporal modulation transfer function (TMTF). In addition, we assessed hearing handicap inventory and quality of life under the qualitative tests.SNR 50Stimulus preparation: We prepared speech shaped noise having a spectrum similar to that of a standardized sentence. The procedure given by Shetty and MendhakarA)Psychophysical tasks:Ten sentences embedded at different SNRs were randomized. Each sentence was presented at most comfortable level through a loudspeaker at 0° azimuths. We instructed the participants to repeat the sentence heard. We noted the SNR level at which the testing started (L) and the number of correctly recognized target words in each sentence. The total number of target words from all sentences was added (T). We also documented the total number of words per decrement (W) and SNR decrement step size in each sentence (d). The obtained values were substituted to the Spearman–Karber equation, given below, to determine SNR 50%.We used the Maximum Likelihood Procedure (MLP) to estimate the thresholds for TMTF and DLF. The laptop loaded with MLP was connected to an auxiliary input of audiometer. The output of the audiometer was delivered bilaterally through headphones at the participants’ most comfortable level.Temporal modulation transfer function: The TMTF stimulus developed by Lorenzi et al.We obtained the modulation detection threshold (MDT) in dB in terms of the following equation. We utilized the method adopted by Zeng et al.A)Qualitative measuresDifference limen frequency: The stimulus developed by Micheyl et al.We administered the Kannada version of the Hearing Handicap Inventory for Adults (HHIA)Hearing handicap inventory for adults: The HHIA questionnaire consists of 13 questions in emotional and 12 questions in the social domain, constituting a total of 25 questions. Each question was rated on a three-point rating scale “yes” as 4, “sometimes” as 2, and “no” as 0. The maximum overall score is 100. The maximum number of points for social and emotional subsections is 48 and 52, respectively. A score of 0 implies no handicap, while a score of 100 implies total handicap. A score ranging from 0% to 16% indicates no handicap; a score of 18%–42% indicates mild-moderate handicap. A score of above 44% indicates a significant handicap. We obtained scores for each domain.World Health Organization — Quality of life: The Kannada version of the World Health Organization – Quality of Life BREF versionExperienced audiologists did the evaluation in the department. Later the data for this study was conducted by the research associate in the lab. It took 30 min to perform all the tests on each participant. The basic audiological evaluation and the data collected were on the same day.2(2) = 8.242; p = 0.016). Further, we performed a post hoc Mann–Whitney U test to check in which sub-groups of SNHL had caused a significant difference in SNR-50. The mild sub-group of SNHL required significantly lesser SNR to achieve SNR-50 than moderately severe  and severe sub-groups . Though the SNR required to achieve 50% recognition in noise was less in moderately severe than severe subgroup, it failed to reach a significant difference . In the case of ANSD, a measurable SNR-50 was obtained from 13/19 participants of the mild sub-group. The median SNR required to achieve 50% recognition in them was 9.5 dB . However, no measurable SNR-50 was obtained from either moderately severe or severe sub-groups of ANSD.To achieve 50% recognition of speech, the SNR required was less in mild hearing loss (n = 10) than moderately severe (n = 32), followed by a severe degree (n = 7) of SNHL group. The median SNR required to achieve 50% recognition in mild group was −1 dB , moderately severe was 3 dB , severe group was 4 dB . A Kruskal Wallis test showed a significant difference between sub-groups for SNR-50 in SNHL (χU test. It was observed that mild sub-group of ANSD (median = 9.5) required a significantly higher SNR to achieve 50% speech recognition than mild  and moderately severe  subgroups of ANSD. In addition, though mild sub-group of ANSD (median = 9.5) required higher SNR to achieve 50% recognition than severe , it failed to reach significance. It purports that the mild degree of neural pathology in speech recognition has a similar problem with that of the severe degree of cochlear pathology.Further, to know the severity of impairment in the SNR-50 between the cochlear and neural site of the lesion, we compared the mild subgroup of ANSD (n = 19) with each of the subgroups of SNHL on SNR-50 using the Mann–Whitney 2(2) = 2.62, p = 0.270), 1000 Hz (χ2(2) = 3.141, p = 0.208), 2000 Hz (χ2(2) = 1.26, p = 0.53), 4000 Hz (χ2(2) = 2.22, p = 0.328). Similarly, for the ANSD group, there were no significant differences between subgroups on DLF at 500 Hz (χ2(2) = 5.29, p = 0.071), 1000 Hz (χ2(2) = 8.022, p = 0.051), 2000 Hz (χ2(2) = 1.938, p = 0.379), 4000 Hz (χ2(2) = 2.672, p = 0.263).The median and SD of DLF at each frequency from sub-groups of SNHL and ANSD are tabulated in U test revealed a significantly better frequency resolution in SNHL than ANSD on DLF at 500 Hz , 1000 Hz , 2000 Hz , and 4000 Hz .We combined the DLF data at each frequency as there was no significant difference between the sub-groups. This is true in case of SNHL and ANSD. Further a Mann–Whitney U test result revealed that the mild subgroup of ANSD required a significantly larger DLF for 500 Hz than each of the mild , moderately severe  and severe  subgroups of SNHL. In addition, a significantly larger DLF was observed in mild ANSD for 1 kHz than mild , moderately severe  and severe  sub-groups of SNHL. Further, a significantly larger DLF was required by the sub-group of ANSD for 2 kHz than mild , moderately severe  and severe  subgroups of SNHL. We observed a similar result for 4 kHz as the mild group of ANSD required larger DLF to discriminate the primary tone from its variable tones than mild , moderately severe  and severe  subgroups of SNHL. It infers that frequency resolution in the mild sub-group of ANSD was affected more than the severe subgroup of SNHL. To sum up, the individuals with ANSD required a significantly higher difference in frequency to differentiate two tones compared to those with SNHL  = 3.529, p = 0.129), 64 Hz (χ2(2) = 2.81, p = 0.245), and 128 Hz (χ2(2) = 3.65, p = 0.161). Unlike the SNHL group, there was a measurable score on temporal resolution only at 8 Hz and 64 Hz in mild sub-groups of ANSD.The median scores and SD on TMTF in individuals with SNHL and ANSD are tabulated in U test result revealed that mild subgroup of ANSD required a significantly higher modulation detection threshold for 8 Hz modulation frequency than each of the mild , moderately severe  and severe  subgroups of SNHL. Similarly, a significant higher modulation detection threshold was observed in mild ANSD for 64 Hz modulation frequency than mild , moderately severe  and severe  sub-groups of SNHL. It infers that the temporal resolution in mild ANSD was affected more than the severe subgroup of SNHL.In addition, we compared the mild subgroup of ANSD with each of the sub-groups of SNHL on modulation detection threshold at 8 Hz and 64 Hz. A Mann–Whitney 2(2) = 3.49, p = 0.175), the emotional domain of HHIA (χ2(2) = 1.497, p = 0.473) and total score of HHIA (χ2(2) = 2.757, p = 0.252).2(2) = 2.40, p = 0.301) and total score of HHIA (χ2(2) = 0.682, p = 0.711).U test result revealed a significantly higher severity of hearing handicap in ANSD than SNHL (χ2(1) = 6.347, p = 0.012) (2(1) = 7.842, p = 0.005) and emotional domain (χ2(1) = 4.249, p = 0.039).The scores in each domain of HHIA obtained between sub-groups were combined in each of the groups as there were no significant differences in them. This was done to compare the scores of each domain of HHIA between SNHL and ANSD. A Mann–Whitney = 0.012) . This waU test. We performed these comparisons to determine the severity of handicap in the mild sub-group of ANSD compared to which subgroups of SNHL. The mild sub-group (median = 68) of ANSD showed a significant higher hearing handicap than mild  and moderately severe  subgroups of SNHL. Also, though the total hearing handicap in the mild subgroup of ANSD (median = 68) had a higher score on total hearing handicap than the severe sub-group of SNHL (median = 54), it failed to reach significance .Further, we compared the mild subgroup of ANSD with each of the subgroups of SNHL on the total hearing handicap index using the Mann–Whitney 2(2) = 0.838, p = 0.658), health (χ2(2) = 0.014, p = 0.993), physical (χ2(2) = 0.091, p = 0.955), psychological (χ2(2) = 0.243, p = 0.885), environmental (χ2(2) = 1.72, p = 0.422), social (χ2(2) = 1.623, p = 0.444), and total QOL (χ2(2) = 0.743, p = 0.690) (2(2) = 0.468, p = 0.792), health (χ2(2) = 4.212, p = 0.122), physical (χ2(2) = 2.16, p = 0.340), psychological (χ2(2) = 2.383, p = 0.304), environmental (χ2(2) = 0.082, p = 0.960), social (χ2(2) = 0.006, p = 0.997), total QOL (χ2(2) = 0.130, p = 0.937)  . Further= 0.937) .Table 4M2(1) = 4.454, p = 0.035). In addition, though each domain of quality of life was affected more in ANSD than SNHL, it failed to reach significance in health (χ2(1) = 0.075, p = 0.785), physical (χ2(1) = .238, p = 0.626), psychological (χ2(1) = 0.238, p = 0.626), environmental (χ2(1) = 0.644, p = 0.422), social (χ2(1) = 2.899, p = 0.089) and total score of quality of life (χ2(1) = 0.486, p = 0.486).We combined the scores in each domain of quality of life obtained between sub-groups of SNHL, as there were no significant differences in them. Similarly, it was done for ANSD subgroups. Further, we compared the quality of life between SNHL and ANSD groups. The total score of quality of life was affected more in ANSD than SNHL , which wU test. We performed these comparisons to know the severity of quality of life in the mild sub-group of ANSD compared to which subgroups of SNHL. Though the quality of life was worse in the mild sub-group of ANSD than ieach of the sub-groups of SNHL, it failed to reach significance.We compared the mild subgroup of ANSD with each subgroup of SNHL on the total score of quality of life using the Mann–Whitney p = 0.022). In addition, we found a significant mild degree of negative correlation between total hearing handicap and a total score of quality of life . However, no correlation was found between total hearing handicap scores and an average of pure tone averages ., DLF at 500 Hz , 1000 Hz , 2000 Hz , 4000 Hz ; and TMTF scores at 8 Hz , 64 Hz  and 128 Hz .The attributing factors such as hearing thresholds, speech perception in quiet, SNR-50, modulation detection thresholds, difference limen of frequency, and quality of life in individuals with SNHL and ANSD were utilized to correlate with hearing handicap. This relationship was performed separately for SNHL and ANSD. We performed a Spearman's sign rank correlation coefficient between hearing handicap scores and attributing factors. In individuals with SNHL, we found a significant mild degree of the positive correlation between the total hearing handicap scores and SNR 50 , SNR 50 , DLF at 500 Hz , 1000 Hz , 2000 Hz , 4000 Hz  and TMTF scores at 8 Hz , 64 Hz , and 128 Hz . However, there was a significant mild degree of negative correlation between total handicap scores and total quality of life .In individuals with ANSD, no correlation was found between hearing handicap and any of the attributing factors: average of pure tone averages . It purports that the hearing handicap is equally affected irrespective of scores in the measured test from the samples of ANSD participants. This is because they tend to compare the present listening problem with their listening abilities prior to the hearing loss.Deterioration of the temporal processing begins by forth decade of life in individuals with normal hearing.The mild subgroup of ANSD participants required significantly higher SNR to achieve 50% recognition of speech than the mild subgroup of SNHL. However, there was no measurable SNR for 50% recognition in moderately severe and severe sub-groups of ANSD. In addition, the mild ANSD sub-group has a speech perception impairment similar to that of the severe sub-group of SNHL. Larger DLF was required as a function of frequency. This was true for each of the sub-groups of SNHL and ANSD. A significantly larger DLF was required in ANSD than SNHL for each frequency. Also, the severity of impairment in discriminating frequency in the mild subgroup of ANSD was similar to the severe sub-group of SNHL. In TMTF, there was no measurable modulation detection threshold for moderately severe and severe groups of ANSD in each of the modulation rates. Whereas mild sub-group of ANSD required a higher modulation detection threshold than the mild sub-group of SNHL at 8 Hz and 64 Hz, respectively. Besides, temporal resolution impairment in the mild sub-group of ANSD was significantly higher than in each of the sub-groups of SNHL. The hearing handicap was more significantly affected in ANSD than SNHL. The severity of the hearing handicap in the mild sub-group of ANSD was similar to the severe sub-group of SNHL. Quality of life was equally affected in both SNHL and ANSD groups. In the SNHL group, the SNR-50 was positively related, and QOL was negatively related to hearing handicap. Speech perception and quality of life were the contributory factors for hearing handicap in the SNHL group. Whereas, in ANSD, none of the factors are related to hearing handicap.Speech perception in noise, psychoacoustic skills, hearing handicap, and QOL was significantly affected more in ANSD than SNHL groups. Also, the severity of the mild sub-group of ANSD was similar to those participants of the severe sub-group of SNHL in each of the behavioral skills, psychoacoustic skills, hearing handicap, and QOL assessed. Further, the hearing handicap in SNHL was attributed to SNR-50 and quality of life. In ANSD, the hearing handicap is not related to any of the attributor factors undertaken in the study. The study provides insight into how much the mild ANSD subject suffer against their counterparts who have a sensory impairment. The finding purports that “mild” ANSD subjects have listening impairment similar to that of “severe” degree of SNHL subjects. The limitation of the study is the fact that in the sample of each group, we were unable to categorize their hearing handicap or QOL based on the scores of tests measured in the present study. This is because the sampled population rated the same degree of hearing handicap or QOL irrespective of the degree of hearing loss.This study provides insight into how much the mild ANSD subject suffers against their counterpart who has a sensory impairment. The finding purports that “mild” ANSD subjects have listening impairment similar to that of “severe” degree of SNHL subjects.All India Institute of Speech and Hearing, Mysuru.This study was funded by the The authors declare no conflicts of interest.


Green snow microorganisms play an important role in biogeochemical cycle and carbon sink processes and they can be a source of biotechnologically interesting cell factories. A wide temperature tolerance is a unique property of bacteria isolated from cold environments, which has received great attention in the last years. The present paper examines the growth and chemical profile flexibility for green snow bacteria exposed to different temperature and nutrient fluctuations. By applying high-throughput chemical phenotyping with FTIR spectroscopy we discovered chemical changes possessed by green snow bacteria when grown at high/low temperature and rich/minimal media.Temperature fluctuations and nutrient composition are the main parameters influencing green snow microbiome. In this study we investigated the influence of temperature and nutrient conditions on the growth and cellular chemical profile of bacteria isolated from green snow. Chemical profiling of the green snow bacteria was done by high-throughput FTIR spectroscopy combined with multivariate data analysis. We showed that temperature and nutrients fluctuations strongly affect growth ability and chemical profile of the green snow bacteria. The size of colonies for green snow bacteria grown at higher (25 °C) and lower (4 °C and 10 °C) than optimal temperature (18 °C) was smaller. All isolates grew on rich medium, and only 19 isolates were able to grow on synthetic minimal media. Lipid and mixed spectral regions showed to be phylogeny related. FTIR fingerprinting indicates that lipids are often affected by the temperature fluctuations. Growth on different media resulted in the change of the whole chemical profile, where lipids showed to be more affected than proteins and polysaccharides. Correlation analysis showed that nutrient composition is clearly strongly influencing chemical changes in the cells, followed by temperature. Climate change and global warming lead to the accelerated melting of glaciers, coastal snow fields, and overall decrease in the snow cover in Antarctica. This creates favorable conditions for blooming of snow algae, resulting in the appearance of green snow areas. The blooming of snow algae in Antarctica was recorded for the first time during expeditions in the 1950s ,3 and anActinobacteria and Proteobacteria, and genera Arthrobacter, Cryobacterium, Leifsonia, Salinibacterium, Paeniglutamicibacter, Rhodococcus, Polaromonas, Pseudomonas, and Psychrobacter. Interestingly, despite originating from the cold environment, all green snow bacteria were characterized by a wide range of temperature tolerance from 4 °C to 25 °C, and the optimum growth temperature of 18 °C.Green snow is characterized by the presence of a complex microbiome, where algae are the main colonizers and bacteria, archaea, and fungi are co-colonizing microorganisms ,7,8. GreProteobacteria and Bacteroides can degrade hydrocarbons and are applied in the biodegradation of oils and petroleum and bioremediation as a treatment of contaminated wastewater [Pseudomonas arctica isolated from the soil in King George Island, Antarctica [A wide temperature tolerance is a unique property of bacteria isolated from cold environments that has received great attention in the last years ,11. For stewater ,15,16. Astewater ,19,20,21tarctica . Due to The main aim of this study was to examine, in detail, the influence of different temperatures and nutrients on the growth ability and cellular chemical profile of the Antarctic fast-growing green snow bacteria. Bacteria were grown on rich complex medium—brain heart infusion (BHI) and minimal media with the different carbon sources and mineral composition. Growth on the selected media was done at 4, 10, 18, and 25 °C. For the profiling of the cellular chemical profile, Fourier transform infrared (FTIR) spectroscopy was utilized. FTIR spectroscopy is a rapid, non-destructive next-generation phenotyping technique widely used in medicine ,27,28, fForty-five fast-growing bacteria were isolated from the two green snow samples collected during the 7th Belarussian Antarctic Expedition (2014–2015). Identification of the isolated bacteria was performed by 16S rRNA gene sequencing, and characterization of enzymatic activity, optimum growth temperature and biochemical phenotyping by FTIR spectroscopy was reported previously . Bacteri2HPO4—24, KH2PO4—15; NaCl—2.5; NH4Cl—5. MGU minimal medium was prepared by mixing of 300 mL of 20% agar, 100 mL of M9 stock solution, 4 mL of 20% glucose, 4 mL of 0.1 M (CaCl2) and 4 mL of 0.1 M (MgSO4). X9 stock solution was prepared as follows (g/L): NH4Cl—20; NH4NO3—4; Na2SO4 × 10H2O—8; K2HPO4—12; KH2PO4—4; MgSO4 × 7H2O—0.4. XGU minimal medium was prepared by mixing of 300 mL of 20% agar, 100 mL of X9 salt stock solution and 4 mL of 20% glucose. All media components were autoclaved at 115 °C for 15 min. Two analogous minimal media, MGY and XGY, with glycerol as carbon source were prepared by added 4 mL of 20% glycerol instead of glucose (Cultivation of bacteria was performed on five agar media: complex medium brain heart infusion (BHI) , two types of minimal media with different nitrogen and phosphorus content and two different carbon sources, glucose and glycerol. For preparing minimal media, two types of salt solutions, M9  and a mo glucose .Cultivations were performed at four temperatures: 4 °C, 10 °C, 18 °C, and 25 °C by streaking cultures on all five agar-based media. The ability to grow on different media and at different temperatures was determined by daily visual evaluation of the growth at four areas of the streak plates: (1) area of inoculation and first streaks, (2) area of second streaks, (3) area of third streaks, and (4) area of fourth streaks. The evaluation of the growth was performed according to the score-based scale were score “0” indicates the absence of growth on all areas of the streak plates, score “1–2” indicates a growth only at the area of inoculation and first streaks but without single colonies, score “3–4” indicates a growth at the area of inoculation and first streaks and area of second streaks with single colonies below 0.5 mm in diameter, score “5–6” was for the growth at all areas of the streak plates with single colonies 0.5–1.0 mm in diameter, score “7–8” when the diameter of the colonies was 1.5–2.0 mm, and score “9–10” for colonies with a diameter higher than 2.5 mm. The cultivation time was for 3–12 days depending on the cultivation conditions and strains . Agar cug, 4 °C for 5 min; (2) at the last washing step, 50–100 µL of distilled water was added for resuspending the cell pellet; (3) 8–10 µL of the obtained cell suspension was transferred onto IR-light-transparent silicon 384-well microplate  in three technical replicates; (4) samples were dried at room temperature for 1 h.Sample preparation for FTIR spectroscopy measurements was done as previously described : (1) bac−1 with a spectral resolution of 6 cm−1 and an aperture of 5.0 mm.FTIR spectroscopy analysis was performed using the High-throughput Screening eXTension (HTS-XT) unit coupled to the Vertex 70 FTIR spectrometer  allowing to perform High-throughput Screening (HTS) transmission mode measurements. The spectra were recorded in the region between 4000 and 500 cm−1 and 1800–1700 cm−1, protein region at 1700–1500 cm−1, mixed region at 1500–1200 cm−1, and polysaccharide region at 1200–700 cm−1. Each of the spectral regions were preprocessed separately as follows: (1) second derivative spectra were obtained by the Savitzky–Golay algorithm [Prior to data analysis, the spectral data were quality checked. A quality test has been applied to pre-select good quality spectra for further analysis. This was done using the method published by Tafintseva et al. ,56,57. Tlgorithm ,63 with lgorithm ,65; (3) −1 were selected, and spectra were normalized by EMSC [−1, 2927 cm−1, 2925 cm−1, and 2854 cm−1 peaks representing –C–H (CH3)/–C–H (CH2)/CH2/CH3 stretching, and 1745 cm−1, 1743 cm−1, and 1741 cm−1 representing ester bond stretching, for mixed—1456 cm−1, 1454 cm−1, 1394 cm−1, 1380 cm−1, 1338 cm−1, 1336 cm−1, 1313 cm−1, 1311 cm−1, and 1305 cm−1 peaks representing CH2/CH3 bending modes or deformation of this functional groups modes, 1404 cm−1, 1402 cm−1, and 1400 cm−1 representing C=O symmetric stretching vibrations of COO− functional groups and 1243 cm−1, 1242 cm−1, and 1240 cm−1 representing –P=O stretching vibrations of phosphodiesters; and, for proteins, 1654 cm−1, 1548 cm−1, and 1546 cm−1 peaks characterizing the Amide I and Amide II bands of proteins [Correlation analysis was done to detect the effect of nutrients and temperature on different strains. To do that, all the data representing different growth conditions were analyzed together (put together as rows), as opposed to multi-block setting where data is put next to each other as separate blocks (put together as columns). The spectra were preprocessed as follows: the spectral regions 3050–2800 and 1800–900 cm by EMSC ,65. PCA  by EMSC  with 13,proteins . Peaks fPolaromonas sp. BIM B-1676, which had relatively small size colonies of about 0.5–1.0 mm in a diameter. Cultivation of isolates on BHI agar at 10 °C and 4 °C provided slightly smaller colonies and, in some cases, a considerable growth reduction was observed, as for example for Salinibacterium isolates , and isolate Leifsonia antarctica BIM B-1671 had a good growth, while all isolates of Cryobacterium, Paeniglutamicibacter, Polaromonas, Psychrobacter, Salinibacterium, Leifsonia (except Leifsonia antarctica BIM B-1671), and isolate Rhodococcus yunnanensis BIM B-1621 had little to no growth , Pseudomonas (6 isolates), Rhodococcus (3 isolates), and Leifsonia antarctica BIM B-1671 since other 26 isolates from genera Crybacterium (6), Salinibacterium (4), Psychrobacter (3), Paeniglutamicibacter antarcticus BIM B-1657, Polaromonas sp. BIM B-1676 and Rhodococcus yunnanensis BIM B-1621 (1) were not able to grow on these media.FTIR spectroscopy chemical profiling was performed for all isolates cultivated on BHI agar and cultivation on the minimal media was done only for 19 isolates from the genera In order to get a general overview over the chemical differences based on phylogeny, we performed multi-block principal component analysis  using dRhodococcus, Psychrobacter, Pseudomonas, and Polaromonas are clustered out from the main cloud formed by Cryobacterium, Leifsonia, Arthrobacter, Paeniglutamicibacter, and Salinibacterium isolates. In the main cloud we see that Arthrobacter and Cryobacterium isolates organized into two distinct clusters close to each other and Cryobacterium isolates clustered close to the Leifsonia, Paeniglutamicibacter, and Salinibacterium isolates, which did not show any clear clustering and were completely overlapping  C=O stretching at 1743 cm−1 and 1739 cm−1 (−1 and 1377 cm−1) identified to be significant on the loading plots for the mixed region. In addition, PO−2 asymmetric stretching of phosphodiesters in phospholipids at ~1230 cm−1 (PC1) and ~1250 cm−1 (PC2) is shown to be significant in this region  –C–739 cm−1 A. Also, s region B.Rhodococcus, Paeniglutamicibacter, Arthrobacter, and Cryobacterium grown on certain temperatures were clustered out (−1) and Amide II (~1550 cm−1) region as significant ones  and 1024 cm−1 (PC1), and 980 cm−1)  D.Rhodococcus and Cryobacterium isolates have very different lipid profile  and PO−2 asymmetric stretching of phosphodiesters in phospholipids at ~1230 cm−1 (PC1) and ~1250 cm−1 (PC2) are responsible for the distinctions of the cells grown on the minimal media (−1)  B.−1) and Amide II (~1550 cm−1) regions, indicating a change in the protein structure and profile  as the most significant  and in the protein profile (1546 cm−1 and 1548 cm−1) was observed for Arthrobacter  and polysaccharide profile  was observed.To understand cellular phenotypic responses represented by the change in the chemical profile introduced by different temperature and nutrient conditions, we performed correlation analysis using standard PCA. Correlation analysis was done separately for each genus, pulling all the spectra of different media together. Correlation loading plots allow representation of the correlations among all variables available for the analysis, including spectral variables (peaks), design variables  and any other variables available for the samples. We are especially interested to see the influence of temperature fluctuations for each genus and each isolate. When analyzing correlation loading plots presented in posed to A,C,F,H. posed to A,F,H. Sorobacter A. For Le−1 and 1548 cm−1 and negative correlation with polysaccharide related bands 1240–1243 cm−1 was observed for Cryobacterium, Paeniglutamicibacter, and Psychrobacter  as it was observed for Cryobacterium, Polaromonas, Psychrobacter, and Salinibacterium , inorganic nitrogen, phosphorus, and some salts. These media and range of temperatures were chosen to mimic the availability of two nutrients and temperature scenarios in Antarctica, where minimal media and lower temperatures of 4 °C and 10 °C represent the current low nutrients’ availability and temperature scenario, and where rich complex medium and higher temperatures of 18 °C and 25 °C represent the scenario of elevated nutrients availability and temperature in the case of active snow melting due to the global temperature increase. In addition, these conditions are relevant for exploring the biotechnological potential of these bacteria.The obtained results show that most of the Antarctic green snow bacteria grow on the rich complex medium, while only a limited number of the isolates were able to grow on the synthetic minimal media. In addition, bacteria grown on the synthetic minimal media were characterized by smaller colonies and longer growth time. This could be an indication of a high level of nutrients’ requirements for the studied green snow bacteria, which in the case of minimal media were obviously lacking, for example some vitamins, trace elements, amino acids, etc. ,69,72,73The studied green snow bacteria could be considered as psychrotolerant as the best growth ability with the isolated colonies of a good size was observed at 18 °C. They could be characterized by a wide range of temperature tolerances; however, growth at lower and higher temperatures were suboptimal. It is important to note that growth reduction at lower temperatures (4 °C and 10 °C) was less significant than at a higher temperature (25 °C). It is well known that the size of colonies are determined by the individual cell metabolic kinetics, enzymatic activity, and cell population responses to environmental conditions . This coPseudomonas isolates showed equally good growth ability on all tested media and temperatures. The isolate Pseudomonas sp. BIM B-1635 was characterized by a lower growth ability at 4 °C and 10 °C when cultivated on BHI agar and absence of growth when cultivated on minimal media. Therefore, we assume that this isolate is distinctly different from other Pseudomonas isolates. Such growth behavior is common for Pseudomonas bacteria as they are usually characterized by a wide growth temperature tolerance and are able to grow on complex and synthetic media [Pseudomonas species is 28 °C, while they can tolerate temperatures as low as 4 °C and as high as 45 °C [Arthrobacter bacteria, which showed good growth at 4–18 °C and reduced growth at 25 °C, while temperatures of 20–30 °C were often reported as optimum for these bacteria isolated from non-polar regions [Psychrobacter and Salinibacterium bacteria, for which the often reported optimum temperature is above 20 °C [Leifsonia isolates, where Leifsonia antarctica BIM B-1671 did not grow on minimal media while other isolates did. According to the previously reported studies, most of the Leifsonia species usually grow well on media containing rich organic nitrogen sources—yeast extract or peptone—but some species require the presence of growth factors such as amino acids or vitamins [Leifsonia antarctica BIM B-1671 belongs to another species.The temperature-induced growth response of the studied green snow bacteria was genus and species-specific. Thus, among the studied green snow bacteria, almost all ic media . Thus, ias 45 °C . Unusual regions ,81. The ve 20 °C ,82,83. Ivitamins . Thus, wHigh-throughput FTIR spectroscopy combined with the multivariate data analysis was utilized to investigate temperature and nutrients-induced changes of the cellular biochemical profile of the green snow bacteria. FTIR spectroscopy is a next-generation phenotypic technique widely used for rapid, non-invasive biochemical phenotyping of microorganisms ,85,86. IPsychrobacter, Pseudomonas, and Polaromonas were clearly separated from the Gram-positive genera; (2) Rhodococcus isolates clustered out of the main cloud because they phylogenetically belong to the order Mycrobacteriales, while other genera belong to the order Micrococcales; (3) overlapping of genera Leifsonia and Salinibacterium can be explained by the previously reported phylogenetic similarity between these two genera, especially between Salinibacterium species and species Leifsonia rubra and Leifsonia aurea [Paeniglutamicibacter and Arthrobacter isolates could be due to the re-classification of Arthrobacter antarcticus to Paeniglutamicibacter antarcticus [Pseudomonas fluorescence BIM B-1668 clustered out from all other Pseudomonas isolates and overlapped with isolate Leifsonia antarctica BIM B-1671 could be explained by potentially sequencing-related misidentification. Taxonomic differences in polysaccharides are more known for fungi, and in the case of bacteria, differences in the polysaccharides were reported mainly associated with the cell-wall structure [The analysis showed that the chemical profile differences related to phylogeny were observed among bacteria grown on BHI medium. For example: (1) Gram-negative bacteria represented by genera ia aurea , and (4)arcticus . The clearcticus . Interesarcticus . Multi-b−1, 1743 cm−1, and 1745 cm−1, indicating a possible change in the total lipid content and, possibly, a type of produced lipids. In addition, it was correlated/negatively correlated with CH2 and CH3 stretching bands, indicating a change in the lipids’ length and branching. Thus, it could be concluded that lipids might be one of the main cellular building blocks involved in the nutrients/temperature adaptations. Further, the response to temperature fluctuations was more pronounced when bacteria were grown on the synthetic minimal media than on complex BHI agar. This is in accordance with the previously published reports, which showed that cultivation in rich complex media alters mainly the total lipid content and the length of the fatty acids chain, but there are no changes in a proportion of branched-chain fatty acids and fatty acid profile in general. In minimal synthetic media, though, there are changes in the pattern of fatty acids branching regarding the amount of branched-chain fatty acids compared to straited-chain saturated and unsaturated fatty acids [Under different nutrients and temperature conditions, the most considerable changes in the cellular chemical profile were associated with different media and, when only one medium was used, then low and high temperatures had a strong effect, which was often associated with lipid-related FTIR bands and lesser associated with protein and polysaccharide bands. The correlation analysis indicated that the whole chemical composition is changing under different nutrients and temperature growth conditions. Correlation analysis showed that nutrients are clearly a stronger parameter influencing chemical changes in the cells, followed by temperature. It is known that temperature may considerably affect the amount and profile of the intracellular metabolites in microorganisms ,91,92. Pty acids . HoweverOur study shows that under the temperature rising conditions, green snow bacteria will contribute to the microbial flora of Antarctic regions as they are able to grow at higher temperatures and can utilize different carbon sources. Due to the fact that the overall cellular chemical profile was changing to a different extent under various temperatures, further study would be needed to understand in detail how these green snow bacteria alter their chemical composition. Changes in lipid profile and content are especially interesting.In conclusion, we identified that Antarctic green snow bacteria are psychrotolerant and able to grow at lower and higher temperatures. All bacteria were able to grow on complex rich medium, and only few of them grew on minimal synthetic media. Alteration in nutrients induced bigger changes in the cellular chemical profile than temperature. The most significant taxonomic differences were observed in lipids, while smaller differences were observed in proteins and polysaccharides. It could be concluded that lipids might be one of the main cellular building blocks involved in the nutrients/temperature adaptations.