There is a lack of genetic knowledge among health care professionals especially in some developing countries such as Indonesia. Based on our experience, genetic disorders receive less attention in medical education and professionals. This study aims to determine the familiarity and literacy of genetics among medical students in Indonesia.A total of 1003 Indonesian medical  students completed the Rapid Estimate of Adult Literacy in Genetics (REAL-G) questionnaire with a total score of seven for familiarity and eight for genetic literacy. The Mann-Whitney U test was used to compare the familiarity and genetic literacy scores between pre-clinical and clinical students.± 0.96 and 6.37 ± 0.83, respectively. Genetic familiarity was higher (p = 0.043) among clinical students than pre-clinical students, while there was no significant difference in genetic literacy (p = 0.362) between pre-clinical and clinical students. Genetic familiarity does not impact the level of genetic literacy. However, medical students’ genetic literacy is influenced by demographic characteristics, such as age, sex, university type, genetic learning experience, university accreditation, and university location.The average scores of familiarity and genetic literacy were 5.63 In general, Indonesian medical students have relatively good familiarity and literacy in genetics although further study is necessary to accurately measure the genetic familiarity and literacy in medical students and general public. Genetic and genomic technologies have been rapidly developing in low- and middle-income countries in the Asia-Pacific region . This deGenetic services in Indonesia are relatively challenging, with limited facilities and expertise , 3. AnotGenetic literacy is defined as sufficient knowledge and understanding of genetic principles that can be measured from the familiarity of genetic terminology, clinical skills, and factual knowledge about genes and hereditary traits . GeneticThis study was a cross-sectional study with a consecutive sampling method. An online questionnaire was shared with medical students in Indonesia through the Indonesian Medical Student Association. A total of 1016 medical students from 47 universities in Indonesia filled an electronic survey between May and September 2019 however, a total of 13 questionnaire were incomplete. Total 1003 respondents were grouped into pre-clinical and clinical students based on the current medical education system in Indonesia. Detailed is shown in Fig. The the familiarity and genetic literacy in our study were assessed using the Rapid Estimate of Adult Literacy in Genetics (REAL-G) validated in a previous studies –17. The REAL-G consisted of 8 genetic terms, that are, genetic, chromosome, vulnerability, mutation, variation, abnormality, hereditary, and sporadic. The medical students’ level of familiarity was measured using a seven-point Likert-scale, ranging from ‘very unfamiliar’ (1), ‘moderately unfamiliar’ (2), ‘slightly unfamiliar’ (3), ‘familiar’ (4), ‘slightly familiar’ (5), ‘moderately familiar’ (6), and ‘very familiar’ (7). The score was based on respondents’ average perceived familiarity across the eight terms. A total score of correct answers of eight multiple-choice questions related to the terms was obtained to measure the genetic literacy, with a maximum score of eight and a minimum score of zero. The questionnaire is provided as a supplementary file.Analysis was performed using the SPSS v.25 for Windows. Pearson’s chi-squared test was used to compare the variables between pre-clinical and clinical students. We used the Kolmogorov-Smirnov test to analyze the normality of the distribution of the numeric data. The Mann-Whitney U test was conducted to compare the familiarity and genetic literacy scores between pre-clinical and clinical students. Kendall’s Tau test measured the correlation between familiarity and genetic literacy. Factors affecting each term of genetic literacy were determined using the regression analysis. The variables included in the regression analysis are the previously variables analyzed by bivariate analysis and resulted as p-value < 0.25.This study included 1003 students of 738 pre-clinical and 265 clinical students. The demographic information of pre-clinical and clinical students is displayed in Table On average, the respondents’ familiarity total score was 5.63 ± 0.96 out of 7. As shown in Table ± 0.95, p = 0.003), grade-“A”-accredited universities , and students with genetic learning experience  had higher score of genetic familiarity than other students. While in clinical students, the genetic familiarity score was significantly higher in the students from the universities outside of Java Island  than the universities in Java Island.Table Both pre-clinical students (6.06 ± 1.042) and clinical students (6.06 ± 0.979) were more familiar with the term “hereditary” compared to other terms. In general, both groups were also least familiar with the term “sporadic” while pre-clinical students (4.30 ± 1.750) were significantly less familiar compared to clinical students . Although both groups were least familiar with the term “sporadic,” more than half of the students answered the term “sporadic” correctly. Only a small number of students in both groups  could correctly answer the question term “variation” although most of the students admitted their familiarity with the term “variation”.± 0.83 . Genetic literacy score was higher in students from universities outside Java Island  than students from the universities in Java Island. Furthermore, students from public universities  also had a significantly higher level of genetic literacy than private universities.  and clinical students  from public universities scored significantly higher on genetic literacy assessment. Pre-clinical students younger than or 21 years old  also had a significantly higher genetic literacy than those older than 21 years  and “sporadic” (p = 0.006)  was found between familiarity and genetic literacy scores. However, the result was not significant (p = 0.192). In general, there was no association between familiarity and genetic literacy scores. Then, we separately analyzed the correlation between familiarity and genetic literacy scores for each term. Table Based on the results of the binomial regression test, the first term (genetic) was significantly affected by age , location of university , and familiarity . The university location was the most dominant variable. Students from universities outside of Java Island, younger than or age 21, and students with higher familiarity scores of the “genetic” term could answer correctly. The third  and fifth term  were significantly influenced by the university accreditation. Students from grade-“A”-accredited university could answer the term “vulnerability” correctly . However, students from grade-“C”-accredited university were more likely to answer the question about “variation” correctly . The seventh term (“hereditary”) was significantly affected by familiarity (p = 0.014). Students with higher familiarity score of term “hereditary” could answer correctly . However, there was no significant effect of variables on terms number 2 (chromosome), 4 (mutation), 6 , and 8 (sporadic)  to evaluate the genetic literacy of medical students in Indonesia with a more systematic collection of respondents.


Pyrus betulifolia Rehd.) variety and has a stronger red color that is retained in the mature fruit—as an experimental model. We compared the phenotype and gene expression patterns of the two varieties and found no significant differences at the early stage of fruit development. However, although the red color of ‘Xinli 7′ fruits began to fade 107 days after full bloom, that of ‘Xinqihong’ fruits persisted until the time of harvest. Transcriptome sequencing identified 639 genes that were differentially expressed between the two varieties, including genes related to light, calcium, and hormone signaling . Moreover, anthocyanin biosynthesis genes were downregulated as the red color of ‘Xinli 7′ fruits faded. These results provide insight into the molecular basis of color differences in red pears that can be useful for improving its fruit quality and commercial value.Red pear is a popular fruit that is appreciated for its attractive and distinctive appearance and mild flavor. In this study, we investigated the mechanism underlying the red coloration of pear skin using the ‘Xinqihong’ cultivar—which was selected as a spontaneous bud sport mutant of the ‘Xinli 7′( Fruit peel color is a key trait for judging fruit quality. Anthocyanin is a water-soluble flavonoid and natural colorant that accumulates in many plant tissues, including fruit peel  and flowAsian red-skinned pears develop their characteristic color when they are nearly ripe , in contPAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX), and UDP glucose:flavonoid 3-O-glucosyltransferase (UFGT) as well as the MYB, basic helix-loop-helix (bHLH), and WD40 transcription factors (TFs)  > 1)  .Pears with a red color are appealing to consumers . SpontanPbMYB10 showed significantly different expression between the two cultivars; the low expression level in ‘Xinli 7′ suggests that its downregulation contributed to color fading in this cultivar.We carried out a comparative analysis of ‘Xinqihong’ and ‘Xinli 7′ in order to identify the molecular basis for their differences in coloration. The anthocyanin content of ‘Xinqihong’ was higher than that of ‘Xinli 7′ at 107 and 127 DAFB, which was in accordance with their color difference parameters. We performed a transcriptomic analysis to identify DEGs that were potentially responsible for the observed difference in peel color between the two cultivars. Anthocyanin biosynthesis involves the regulation of structural genes , most ofPbPIF3 was significantly upregulated in ‘Xinli 7′ compared to ‘Xinqihong’. PIF3 regulates light signaling and anthocyanin biosynthesis in fruit. PIFs and HY5 act antagonistically in many regulatory processes, but PIF3 requires HY5 to bind to the promoter of anthocyanin biosynthesis genes and induce their expression [Genes encoding three WD40 genes as well as bHLHs were identified as DEGs. pression ,31. Thuspression . Thus, lPbERFs   . Under bin pears . Ethylenin pears ,35,36. Eethylene . Our difethylene ,39,40. Tethylene . In pearuit peel . ConsistThe RNA-seq analysis revealed 16 genes involved in calcium signaling . The fraThe red color of red pear is determined by the pattern of anthocyanin accumulation during fruit development. In the present study, we found that the ‘Xinqihong’ variety stayed red even at maturity and fruit quality was unchanged, in contrast to ‘Xinli 7′ in which the red color faded at the late stage of fruit development. Light, calcium, and hormone signaling were found to be involved in the regulation of the color difference between the two cultivars . These fP. betulifolia Bunge rootstock were collected at Jiaodong Peninsula Regional Experimental Park , which is in a region with a warm temperate continental monsoon climate, an average annual precipitation of 672.5 mm, and average annual temperature of 12.6 °C. For each sample, the skin of 10 fruits was removed and immediately frozen in liquid nitrogen and stored at −80 °C for total RNA isolation and measurement of pigment content.Samples of healthy and uniform 3-year-old ‘Xinqihong’ and ‘Xinli 7′ pear plants that had been grafted onto g for 20 min, the supernatant was transferred to a clean tube and the absorbance at 510 nm was measured with a spectrophotometer . The anthocyanin content was calculated using the equation Ca = 1000*A*V/(a*b*W), where Ca is the total anthocyanin content (mg/g), A is the absorbance value, V is the extraction solution volume, a is the absorptivity of anthocyanin (0.0775), b is the thickness of the colorimetric ware, and W is the fresh weight of fruit skin. Chlorophyll and carotenoid contents were calculated as previously described [Approximately 1 g of fruit peel was ground to a fine powder in liquid nitrogen and extracted with 5 mL of extraction solution (1% HCl in methanol) at 4 °C for 12 h. After centrifugation at 12,000× escribed ,45,46. DThe color of apple peel was determined with a portable color difference meter . The L*, a* b*, and C* values at the equator of the fruit were obtained, where L* is the brightness of the color; a* is the red–green coordinate value, which varies from −80 to 100 from green to red ; b* is the blue–yellow coordinate value, which varies from −80 (blue) to 70 (yellow) ; and C* is the color saturation (representing color purity).Total RNA was extracted and purified using the RNAprep Pure Plant Kit  according to the manufacturer’s instructions. RNA quality was verified by spectrophotometric measurement on a NanoDrop 2000C instrument , and the integrity and purity of a 5 μL RNA sample were verified by electrophoresis on 1–1.5% agarose gel. First-strand cDNA was synthesized using the PrimeScript RT-PCR Kit  according to the manufacturer’s instructions and stored at −20 °C for quantitative real-time PCR analysis.Actin gene was used as an internal control. Primers are listed in The cDNA templates were reverse transcribed using total RNA extracted from 5 developmental stages of the 2 red pear cultivars: Xinli 7 (87 DAFB), Xinqihong (87 DAFB), Xinli 7 (97 DAFB), Xinqihong (97 DAFB), Xinli 7 (107 DAFB), Xinqihong (107 DAFB), Xinli 7 (117DAFB), Xinqihong (117 DAFB), Xinli 7 (127 DAFB), and Xinqihong (127 DAFB). qRT-PCR amplification was carried out as follows: 95 °C for 5 min, 45 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s using the Roche 480 real-time PCR system  at the standard mode with the FastStart Essential DNA Green Master kit. All reactions were performed in triplicate at a volume of 20 µL, containing 2 µL of 10-fold diluted cDNA, and the pear http://daehwankimlab.github.io/hisat2/, accessed on 11 February 2016), which was also used to align paired-end clean reads to the reference genome. The mapped reads of each sample were assembled with StringTie (v1.3.3b)  using a reference-based approach. FeatureCounts v1.5.0-p3 was used to count the number of reads mapped to each gene. The FPKM of each gene was calculated based on the length of the gene and number of reads mapped to the gene. Six RNA-seq libraries were constructed for 3 developmental stages of the 2 red pear cultivars: Xinli 7_1 (87 DAFB), Xinqihong_1 (87 DAFB), Xinli 7_2 (107DAFB), and Xinqihong_2 (107 DAFB), Xinli 7_3 (127 DAFB), and Xinqihong_3 (127 DAFB). The transcriptome raw reads have been deposited at NCBI  under accession numbers PRJNA715346.For library construction, 1 μg of RNA per sample was used as the input material. Briefly, mRNA was purified from total RNA using poly-T oligo-coated magnetic beads. First-strand cDNA was synthesized using random hexamer primers and Moloney murine leukemia virus reverse transcriptase. Second-strand cDNA was synthesized using DNA polymerase I and RNase H. Overhangs were converted into blunt ends with exonuclease/polymerase. After adenylation of the 3′ ends of DNA fragments, an adapter with a hairpin loop structure was ligated for hybridization. cDNA fragments 370–420 bp in length were selected and PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and an index (X) primer. PCR products were purified (AMPure XP system) and library quality was assessed on the 2100 Bioanalyzer system . Clean reads were obtained from raw data by removing reads containing the adapter or poly-N along with low-quality reads. Q20, Q30, and the GC content of the clean data were calculated. Reference genome and gene model annotation files were downloaded from the genome website. An index of the reference genome was constructed using Hisat2 v2.0.5 . Values are represented as the mean ± SD of 3 independent biological replicates. Data were analyzed with Duncan’s test, and


We aim to assess attitudes toward a COVID-19 vaccine and vaccination status in cancer patients and to explore additional factors such as the level of information and comprehensibility and accessibility of this information, anxiety symptoms in general and toward COVID-19, and general health literacy.We included 425 outpatients  of the Psychosocial Counseling Center for Cancer patients of the Department of Medical Psychology and Medical Sociology, Leipzig. We recorded attitudes toward a COVID-19 vaccine and vaccination status via self-report. The impact of psychosocial factors, including anxiety (GAD-7), COVID-19-specific anxiety  and health literacy (HLS-EU-Q16) were analyzed with point-biserial correlations using Pearson’s r.We found that the vast majority (95.5%) reported being vaccinated against COVID-19 and that overall trust in safety and protective effects of a COVID-19 vaccine was high (90.9%). The vaccination readiness among nonvaccinated cancer survivors was low to very low with “fear of side effects” the most mentioned (72.2%) reason against a COVID-19 vaccine. There was no significant correlation between vaccination status and fear or anxiety symptomatology, and health literacy. Obsessive thoughts about COVID-19 was significantly higher in nonvaccinated cancer patients.Majority of respondents are positive about COVID-19 vaccine, accompanied by a very high rate of COVID-19 immunization in our sample. Further studies with a larger sample of nonvaccinated cancer patients should further investigate the relationship on fear and vaccination hesitancy and align communication strategies accordingly. We recorded attitudes toward a COVID-19 vaccine and vaccination status. In addition, we assessed potential associated factors such as the level of information, comprehensibility and accessibility of this information, anxiety symptoms in general and toward COVID-19, and general health literacy.The population of this cross-sectional study includes outpatients of the Psychosocial Counseling Center for Cancer patients of the Department of Medical Psychology and Medical Sociology, University Medical Center Leipzig, who had previously agreed to be contacted regarding scientific studies.Patients were eligible for study participation if they had a confirmed diagnosis of cancer, were at least 18 years of age at the time of diagnosis and who were fluent in written and spoken German. All participants gave written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Research Ethics Committee of the University of Leipzig (Ref. 266/21-ek).Eligible patients received a study information letter, together with a letter of consent and the paper and pencil-based questionnaire by mail. Using a prepaid envelope, patients could return the completed questionnaire including informed consent. A response form was included, in case patients did not wish to participate. Patients were able to indicate their reason(s) for non-participation and sent it back to the study team.COVID-19 immunization was covered by questions on vaccination status, number of vaccination(s) received. In addition, perceived reasons for or against COVID-19 vaccination, and questions about disease history regarding COVID-19 infection in oneself or a close relative was recorded via self-generated questions.We used a self-generated nine-item scale with statements about COVID-19 vaccination and vaccinations in general. Respondents were asked to indicate their agreement on a five-point Likert scale ranging from 1 “strongly disagree” to 5 “strongly agree”. Higher scores indicate greater confidence in COVID-19 vaccination and vaccinations in general, as well as in the relevant parties making recommendations in this regard.We inquired about the source(s) from which respondents obtained their information regarding COVID-19 vaccination. In addition, we used a self-generated scale where respondents indicate their agreement or disagreement on items such as the level of information, comprehensibility and accessibility of this information. Items were rated on a five-point Likert scale ranging from 1 “strongly disagree” to 5 “strongly agree”, with higher scores indicating higher agreement.We assessed General anxiety disorder symptomatology using the validated German version of the Generalized Anxiety Disorder Scale  and categorical variables .Comparisons between participants and non-responders were performed with t-test for the continuous variable age and Chi-square test for the categorical variable gender. Point-biserial correlations using Pearson’s r was performed to calculate the relationship between vaccination status and anxiety symptoms and health literacy.limited health literacy  vaccination” and “level, comprehensibility and accessibility of information on COVID-19 vaccination” were recoded. Response categories 1 “strongly disagree” and 2 “disagree” were combined into one category coded as 1 “disagreement”, 3 “neither nor” into one category coded as 2 “neither nor”, 4 “agree” and 5 “strongly agree” into one category coded as 3 “agreement”. Response categories from the Health Literacy scale 1 “very easy” and 2 “fairly easy” were merged into one category coded as “1”, 3 “fairly difficult” and 4 “very difficult” into another category coded as “0”. The sum score is ranging from 0 to 16. Patients with at least 11 answers were considered in the final analysis. We then calculated the mean, multiplied it with 16 and clustered patients into the following categories: inadequate (0–8 points), problematic (> 8–12 points) and sufficient health literacy (> 12–16 points), subsuming the first two categories into In all analyses, two-sided p < 0.05 were considered significant. Data analyses were performed with IBM SPSS Statistics 27 .Patient recruitment was carried out from 2021 September to 2021 November. Out of 744 eligible patients, 438 (response rate: 59.0%) participated in the study  and 60.5% were female. Breast cancer was the most frequent cancer diagnosis (38.6%), and respondents’ median time since diagnosis was 4 years (IQR 3 to 5 years). The majority of patients (67.5%) were not under treatment. Study participants were older than non-responders (p < 0.001) with no significant differences in gender (p = 0.307). Table Mean age was 61.4 years  received a vaccination against SARS-CoV-2. The majority (90.4%) is double-vaxed with the vaccine Comirnaty® . Table Most vaccinated respondents indicated four or more reasons for COVID-19 vaccination (65.6%). Figure The majority (97.2%) of nonvaccinated respondents indicated more than two reasons against a COVID-19 vaccination. Figure The vast majority of respondents indicate that they “believe a vaccination against COVID-19 reduces the risk of contracting COVID-19.” (90.9%) and that “vaccinations protect against contagious diseases.” 93.8%). The most trusted sources of information on COVID-19 vaccines are expert groups, e.g., Ständige Impfkommission (STIKO) and treating physicians with both 85.2%. Table .8%. The The main sources of information on COVID-19 vaccination indicated by respondents were “persons from the health care sector” (75.0%) and both “expert panels” and “everyday media” (63.0%). Figure In the total sample, the majority of patients 75.9%) feel well informed regarding COVID-19 vaccination, compared to those who disagree (11.2%) and those who are undecided (12.9%). In addition, the majority of respondents indicated that the information about COVID-19 vaccines is well understood 68.4%) and readily available (71.0%). Figures .9% feel 8.4% and From 397 participants, 211 (49.6%) met criteria for minimal, 121 (28.5%) for mild, 47(11.1%) for moderate and 18 (4.2%) for severe anxiety symptomatology. From 418 participants, 411 (98.3%) did not reach a total score of ≥ 7 and thus may have problematic symptoms and probable dysfunctional thoughts about COVID-19. From 412 participants, 51 (12.0%) met criteria for inadequate, 142 (33.4%) for problematic and 219 (51.5%) for sufficient health literacy.For means on fear and anxiety symptomatology and health literacy of the total sample, see Table N = 999 French cancer patients, in which the majority (76.9%) reported “fear for their health” as the main reason for vaccination  cancer patients across various cancer types. Our results demonstrate that the vast majority is vaccinated and “protection against severe course, disease consequences of infection” was the most mentioned reason among the vaccinated. This is in line with findings from a cross-sectional study among N = 1000 adults, in which physicians and expert panels such as the RKI and Bundeszentrale für gesundheitliche Aufklärung (BZgA) are trusted from a medium to high degree  and cancer site. Second, patients had a wide range on time since diagnosis (up to 61 years) and thus we were able to include patients who were currently under treatment and those who were not. Lastly, we could gather data on a very topical and important issue among a highly relevant patient sample within our health care system.However, this study has also limitation. One limitation lies in the small sample size of the subgroup of nonvaccinated respondents. Group comparisons should, therefore, be regarded as a trend. Second, we only surveyed cancer patients, who presented themselves at our counseling center. Those patients are already more integrated in the health care system, and therefore, the representativeness of the sample is limited. Conclusions only should be drawn for this special target group.In this study among cancer patients, the vast majority reported being vaccinated against COVID-19 with a positive attitude toward the vaccination. Considering obsessive thoughts about COVID-19 and vaccination status, we found first indications of a relationship. Further studies with a larger sample of nonvaccinated cancer patients should investigate whether this relationship can be verified, how and why those thoughts evolve and which role communication of responsible parties  and persons  takes. Targeted and tailored communication can help to further improve education, promote valid information, and build trust.


Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (ΔOCTgfbr2) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice  by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 ( Retinal degeneration is among the leading causes of blindness worldwide ,2. IntriTGFβ signaling controls a plethora of cellular responses such as proliferation, differentiation, tissue homeostasis, morphogenesis and regeneration . In addiTgfbr2 was notably upregulated following photoreceptor degeneration , w, wMapk7,ignaling .Ngf is dependent upon TGFβ2 treatment in vitro and is significantly enhanced in the juvenile retina of a mouse model with increased TGFβ signaling activity .The number of degenerating photoreceptor cells was determined using TUNEL . This method was performed on retinae of one-month-old animals, as described previously ,50. TUNEEyes of three-month-old mice were carefully enucleated and fixed for 24 h in Ito’s fixative . The eyet-test in Excel . One-way ANOVA analyses were performed in SPSS  if more than two individual groups were compared (post-hoc test: Bonferroni). p values ≤ 0.05 were considered to be statistically significant.Data are expressed as mean ± SEM. Statistical comparative analyses between the mean variables of two individual test populations were performed using a two-tailed Student’s


In conclusion, in NSTE-ACS, E/(e’×s’) is a powerful predictor of clinical outcome, particularly if it is accompanied by worsening after 6-weeks.It has been shown that the E/(e’×s’) index, which associates a marker of diastolic function  and a parameter that explores LV systolic performance , is a good predictor of outcome in acute anterior myocardial infarction. There are no studies that have investigated the prognostic value of E/(e’×s’) in a non-ST-segment elevated acute coronary syndrome (NSTE-ACS) population. Echocardiography was performed in 307 consecutive hospitalized patients with NSTE-ACS and succesful percutaneous coronary intervention, before discharge and six weeks after. The primary endpoint consisted of cardiac death or readmission due to re-infarction or heart failure. During the follow-up period (25.4 ± 3 months), cardiac events occurred in 106 patients (34.5%). Receiver operating characteristic (ROC) analysis identified E/(e’×s’) at discharge as the best independent predictor of composite outcome. The optimal cut-off value was 1.63 . By multivariate Cox regression analysis, E/(e’×s’) was the only independent predictor of cardiac events. Kaplan–Meier analysis identified that patients with an initial E/(e’×s’) > 1.63 that worsened after six weeks presented the worst prognosis regarding composite outcome, readmission, and cardiac death (all  Patients admitted to hospital because of an acute coronary syndrome (ACS) constitute a heterogeneous population with a varying risk of future cardiac events ,2. Non-SMany parameters for the indirect assessment of LV filling pressures have been used to assess cardiac outcomes in patients with coronary artery disease. Recent studies have shown tissue Doppler imaging (TDI) to be a strong predictor of adverse outcomes in several cardiac diseases. A significant decrease of systolic mitral annulus velocity (s’) and/or early diastolic mitral annulus velocity (e’) has been observed in patients with ischemic heart disease, even if cut-off values for peak systolic and diastolic velocities which detect significant coronary artery stenosis are still unclear ,6. SeverThe idea of a complex marker that incorporates several tissue Doppler parameters was used by several study teams. Our group proposed a new TDI index, E/(e’×s’), that associates a marker of diastolic function (E/e’) and a parameter that explores LV systolic performance (s’). We demonstrated the utility of this index to assess the LV filling pressure in a heterogeneous population of cardiac patients, regardless of LVEF  and to iThe Cardiology Department of Timisoara Institute of Cardiovascular Diseases is an invasive hub for 15 non-invasive cardiology departments. In the period November 2017 to January 2019, 2758 patients were admitted to the department for percutaneous coronary intervention (PCI). These patients were all included in a clinical registry. We analyzed prospectively 439 consecutive patients with NSTE-ACS and successful PCI, in sinus rhythm, hospitalized in our clinic. Successful PCI was defined as residual stenosis ≤20%; non-culprit intervention was performed during the same hospitalization. Patients with inadequate echocardiographic images, previous myocardial infarction, open-chest surgery, cardiac pacemaker/defibrillator, significant valvular heart disease, renal failure (serum creatinine >1.3 mg/dL), and non-cardiac disease with a life expectancy <1 year were excluded from the study group. The remaining 307 patients formed our study population. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of Institute of Cardiovascular Diseases Timisoara (protocol code 1658 and 28 March 2014). Informed consent was obtained from all participants.The following clinical variables were recorded and included in the prognostic model: age, sex, mean arterial pressures, heart rate, body mass index, peak high sensitivity cardiac troponin I level (hs-cTnI), and N-terminal pro-brain natriuretic peptide (NTproBNP) levels. Prescription of the main therapeutic classes was also recorded.For study purposes, five cardiovascular risk factors were considered as follows: hypertension , cardiovascular disease heredity, smoking , diabetes mellitus (fasting glycemia >126 mg/dL or in drug treatment), and hypercholesterolemia (>200 mg/dL or in drug treatment).Echocardiography was performed after PCI, at hospital discharge, using a Vivid 9 General Electric, Milwaukee, WI system, USA. Left atrial volume (LAV) and indexed LAV to the body surface area (LAVI) were determined according to current guidelines . LVEF waThe TDI program was set in pulsed-wave Doppler mode. In the apical four-chamber view, a 4–5 mm sample volume was positioned sequentially at the lateral and septal corner of the mitral annulus . Peak e’All measurements were performed by an experienced echocardiographer. The inter- and intra-observer variability for E/e’, s’ and E/(e’×s’) were examined. Measurements were performed in a group of 30 randomly selected subjects by one observer at two separate times and by two investigators who were unaware of the other’s measurements and of the study time point.Patients were followed for ≥24 months. The primary event consisted of cardiac death or hospital readmission for HF or re-infarction. Cardiac death was defined as either a death directly related to cardiac disease, mainly congestive HF, re-infarction, or sudden death. The follow-up information was obtained from electronic medical files or by telephone contact with the patients or their family members.t-test  or Mann–Whitney U test . Proportions were compared using χ2 test and Fischer’s exact test. Receiver operating characteristic (ROC) curves for predicting cardiac events were determined for different parameters, and area under the ROC curves (AUC) were compared. Patients who died of non-cardiac causes were censored at the time of death.Data were expressed as mean ± standard deviation (SD) for continuous variables and as proportions for categorical variables. Continuous variables were compared between groups using an unpaired p-value < 0.05 was considered significant. Intra-observer variability and inter-observer variability for E/e’, s’ and E/(e’×s’) were measured by Bland–Altman analysis, and intraclass correlation coefficients. We used STATA Statistics/Data analysis, MP 12.0  as statistical software.Uni- and multivariable Cox regression models were created to correlate clinical, biological, and echocardiographic findings to the primary endpoint. For multivariable Cox regressions, three models with an incremental number of confounders were constructed. Model 1 included either E/(e’×s’), s’, or E/e’ and the following confounders: age, gender, mean blood pressure, heart rate, body mass index, prior coronary artery disease, family history of cardiovascular disease, diabetes mellitus, current smoker, hypercholesterolemia, and hypertension. In Model 2, we also included variables obtained from the laboratory , coronary angiography , and ACS category. Model 3 additionally included all significant echocardiographic parameters . Harrell’s c-statistics was calculated from univariable Cox regression for all measures included in the multivariable Cox regression to compare the predictive potential of baseline predictors. The cardiac event-free survival rates were calculated using the Kaplan–Meier analysis, and the event rates were compared using the log-rank test. A In the period of November 2017 to January 2019, 307 consecutive patients hospitalized for NSTE-ACS who underwent successful PCI were included in this prospective study. All patients received standard management according to the institutional recommendation. The mean age of our study sample was 61 ± 12 years and 70.7% were males (217 patients). In this study, no patient was lost to follow-up. During the follow-up period (25.4 ± 3 months), cardiac events occurred in 106 patients (34.5%). The first cardiac event was represented by cardiac death in seven patients (2.28%) and readmission in 99 patients (32.2%). Of our patients, 72 (23.4%) were hospitalized for heart failure, and 17 (5.5%) had a non-fatal re-infarction. During the follow-up, cardiac death occurred in 22 patients (7.16%).p < 0.001).Patients’ baseline characteristics are presented in p < 0.001). The baseline s’ and E/e’ ratio were also significant for predicting composite outcomes . A statistical comparison of the ROC curves demonstrates significant differences between E/(e’×s’) and s’ (p = 0.009) and between E/(e’×s’) and E/e’ (p = 0.003). All the other analyzed echocardiographic parameters presented a lower AUC. The optimal cut-off value for E/(e’×s’) at discharge to predict the composite outcome was 1.63 (74% sensitivity and 67% specificity).p < 0.05) are shown in (p = 0.007).The echocardiographic variables that predicted cardiac events on univariate Cox regression analysis , the E/(e’×s’) ratio remained the only independently parameter associated with the composite outcomes and hospital readmission, respectively. Other echocardiographic measures were not associated with cardiac events and readmission for HF and/or re-infarction . HoweverModel 1 is adjusted for age, gender, mean blood pressure, heart rate, body mass index, prior coronary artery disease, family history of cardiovascular disease, diabetes mellitus, current smoker, hypercholesterolemia, and hypertension.Model 2 is adjusted for previous mentioned variables in Model 1 as well as laboratory data , coronary angiography , and acute coronary syndrome category.Model 3 is adjusted for all previous mentioned variables in Models 1 and 2, additionally left ventricular ejection fraction, systolic pulmonary artery pressure, left atrial volume, indexed left atrial volume, E velocity, E/A ratio, and e’ velocity.p = 0.027), and along with E/e’ provided the highest c-statistics among all baseline predictors (0.721 for E/(e’×s’) vs. 0.679 for E/e’, p = 0.009). Adding E/(e’×s’) to all the variables included in Model 1 resulted in a significant increase in the c-statistics than Model 1 alone (Model 1 with E/(e’×s’): 0.730 (0.69–0.77) vs. 0.689 (0.65–0.72), p = 0.034). Model 2 significantly improved when adding E/(e’×s’) ratio: 0.741 (0.70–0.78) with E/(e’×s’) vs. 0.722 (0.68–0.76) without E/(e’×s’), p = 0.041. Adding E/(e’×s’) to the variables included in Model 3 displayed a significant increase in the c-statistics when compared to Model 3 alone (0.761 (0.72–0.80) vs. 0.743 (0.70–0.77), p = 0.047). When E/(e’×s’) was already included in the models, we identified no significant changes in Harrell’s c-statistics by adding all the analyzed echocardiographic parameters.Harrell c-statistics revealed that the E/(e’×s’) index contributed with a higher c-statistics compared with s’ velocity . The subgroup of patients with an initial E/(e’×s’) ratio >1.63 and worsening after six weeks presented the worst prognosis regarding composite outcome  ratio in 140 patients (45.6%). Of these patients, 63 (20.5%) presented the initial value of E/(e’×s’) greater than 1.63. However, as shown in  outcome a, hospit outcome b, and ca outcome c, respecIn 30 patients with ischemic heart disease, Bland–Altman analysis demonstrated the intra- and inter-observer agreements were good for E/(e’×s’), E/e’, and s’ measurements. The intraclass correlation coefficient for inter-observer and intra-observer variability was 0.90 and 0.89 for E/(e’×s’); 0.92 and 0.90 for E/e’; and 0.93 and 0.92 for s’, respectively.To the best of our knowledge, this is the first study investigating the value of E/(e’×s’) ratio to predict cardiac events  in patients with NSTE-ACS who underwent successful PCI. E/(e’×s’) provided superior prognostic information when added to all other analyzed predictors in the current population. Patients with an initial E/(e’×s’) > 1.63 and worsening after six weeks presented the worst prognosis regarding composite outcome, hospital readmission, and cardiac death.The clinical importance of predicting prognosis in patients with NSTE-ACS has been increasing. In patients with NSTE-ACS, the rapid recognition of high-risk patients who may benefit from an intensified treatment strategy, risk stratification improves effectiveness of care to those in highest need ,4,12. InPulsed TDI is widely used and validated in the literature to analyze cardiac function, and it is characterized by easy accessibility and strong reproducibility. TDI has proven incremental prognostic value with respect to routine clinical, laboratory, and imaging information. This new technique does not require tracing of endocardial contours, unlike LV volumes and LVEF. Wang et al. demonstrated in a heterogeneous population of cardiac patients a better prognostic value for both e’ and E/e’ than for wall motion score index and LVEF . An incrA significant decrease of e’ and s’ velocities has been observed in patients with myocardial ischemia, even if cut-off values for maximum systolic and diastolic velocities that detect significant coronary lesion are still unclear ,6,10. InLV diastolic dysfunction is usually the result of altered LV relaxation with or without reduced restoring forces (and early diastolic suction), and increased LV chamber stiffness, which increases LV filling pressures. Conceptually, it is very difficult to separate relaxation from contraction, and it can be considered together as part of a continuous cycle. The energy generated during systole is stored in myocardial collagen fibers, and following relaxation, the ventricle uncoils, creating LV suction. There seems to be a relation of proportionality between decline in contractile function and reduction in recoil, with parallel changes in the extracellular matrix ,27.Our group proposed several years ago a new TDI index, E/(e’×s’) that associates a marker of diastolic function (E/e’) and a parameter that explores LV systolic performance (s’). Recent studies have shown E/(e’×s’) to be a strong predictor of adverse outcomes in several cardiac diseases. We demonstrated the utility of this index to assess the LV filling pressure in a heterogeneous population of cardiac patients, regardless of LVEF  and to iIn patients with acute myocardial infarction and severe LV systolic dysfunction, current guidelines recommend to repeat echocardiography 6–12 weeks after initial hospitalization and to measure for systolic function evaluation only LVEF, but not s’ wave ,18. We rClinical studies have clearly demonstrated that NT-proBNP and troponin levels are increased after episodes of ischemia ,28,30. EOur results should be considered in the context of several limitations. The most important limitation of this study is its small sample size. Therefore, larger studies are needed to validate our results. The use of standard echocardiographic assessment instead of more sophisticated methods  could be considered both a limitation and a strength of the study. The limitation is that strain imaging has proved to be more sensitive for detecting subclinical abnormalities of both systolic and diastolic function. The strength of our parameter is its ease of use, thanks to its availability in most of the modern echo machines, making it readily applicable for the bedside assessment of patients. We limited the analysis at two sites , and we did not examine anterior and posterior velocities that might have provided additional information. We are conscious that the E/(e’×s’) ratio is a global index of cardiac function that unmasks a precocious systolic and diastolic myocardial suffering, and it cannot provide regional information about perfusion. The study center functioned as a tertiary invasive center, and therefore, the study population may not reflect a general population of patients with NSTE-ACS.In summary, our findings indicate that in patients with NSTE-ACS who underwent successful PCI, E/(E’×S’) is an important independent prognostic index of cardiac events . E/(e’×s’) provided superior prognostic information when added to other clinical, laboratory, and echocardiographic predictors. Patients with an initial E/(e’×s’) > 1.63 that worsened after six weeks presented the worst prognosis regarding composite outcome, hospital readmission, and cardiac death.


Morphology is the most visible and distinct character of plant organs and is accepted as one of the most important tools for plant biologists, plant breeders and growers. A number of methods based on plant morphology are applied to discriminate in particular close cultivars. In this study, image processing analysis was used on 20 grape cultivars  “, “Morşabi“, “Müşgüle“, “Nuniya“, “Royal“, “Sultani çekirdeksiz (Sultanina)“, “Yalova incisi“, “Yerli beyazv“, “Yuvarlak çekirdeksiz“) to classify them. According to image processing analysis, the longest and the greatest projected area values were observed in “Antep karası“ cultivar. The “Sultani çekirdeksiz“ cultivar had the least geometric mean diameter. The greatest sphericity ratios were observed in “Yerli beyaz“, “Erenköy beyazı“ and “Amasya beyazı“ cultivars. According to principal component analysis, dimensional attributes were identified as the most significant source of variation discriminant grape cultivars from each other. Morphological differences between the cultivars were explained by sphericity and elongation variables. According to elliptic Fourier analysis (EFA) results, grape morphology largely looks like ellipse and sphere. However, there are some cultivars that look similar to a water drop. The cultivars with similar morphology were identified by a pair-wise comparison test conducted with the use of linear discriminant analysis, and they were presented in a scatter plot. According to cluster analysis, present grape cultivars were classified into seven sub-groups, which indicated great diversity. Grape is one of the oldest horticultural crops. It is also one of the most cultivated horticultural plants, along with apple, citrus and banana. Total world grape production in 2018 was 79,000,000 metric tons, up by 6.5% from 74,000,000 tons in 2017. China was the largest producer of grapes, with 13,397,000 tons of production, followed, respectively, by Italy with 8,514,000 tons, the USA with 6,891,000 tons, Spain with 6,673,00 tons, France with 6,198,000 tons and Turkey with 4,000,000 tons of production [Worldwide, about 57% of grapes are used to make wine, 36% are consumed as fresh table grapes and 7% as dried grapes .Vitis vinifera L. are widely distributed, mainly in the temperate and subtropical regions in the world, including Mediterranean countries, Central and Southern Europe, to southwestern Asia. It is estimated that 10000 known grapevine varieties are distributed in grape growing areas throughout the world, and around 13 varieties dominate world production and cover more than one-third of the world’s vineyard area [Common grapevines, Vitis vinifera L. subsp. sylvestris) and local cultivars are available [Vitis vinifera L. ssp. sylvestris Gmel. and included multiple introgression events from the wild to domestication [The neighboring regions of the Caucasian area, including Turkey, have a long history of viticulture and possess a great diversity of local grape cultivars into different products. In Turkey, a large number of wild grapevines  is gained more importance with the improvements of computer performance along with decreases in the cost of digital imaging hardware and software more recently. Thus the method widely contributed digital image processing applications for agriculturally relevant morphological analyses in different crop species ,33,34,35This study was conducted to investigate the size and shape features describing the physical attributes of 20 grapevine cultivars and to reveal shape distinctions with Elliptic Fourier descriptors modeling the closed contour of the cultivars.The study was conducted at the commercial farm Kemerhisar, Nigde province in middle Turkey during 2020. The geographical coordinates of the experiment location were 37.8318° N latitude and 34.6001° E longitude, and 1120 m elevation above sea level. All 20 grape cultivars were harvested from a 40-year-old wire-trained vineyard on the date of 12.09.2020. On the same day of harvest, samples were transported in cold-chain to Advanced Technology Research and Implementation Center of Mersin University. Daily irrigations from May to September and other management practices (herbicide and fertilizer applications and pruning) were conducted according to the farm manager’s criteria. Herbicides were periodically applied between rows to control weeds. Vines were pruned with shears in February each year. The berry skin color of grapevine cultivars is given in Twenty grape cultivars used in present experiments are presented in ®Pro 5.0 software was used to determine morphology and dimensional attributes of the grape cultivars. For image processing, 0–255 threshold range was applied to monochrome images and dimension analysis was automatically performed. Calibration was made over the ruler to convert pixel units into millimeters. With the present analyses, length , width , thickness , projection area , equivalent diameter , perimeter  and circularity (C) were automatically measured. Dimensional and area measures are presented in SigmaScanFor Elliptic Fourier analyses (EFA), 40 grape image files were used for each cultivar. EFA analysis was conducted in different phases with the use of MORPHOLOGY (version 1.03) software . In phasFor image processing, grape images were converted in 24-bit *.bmp format. Four modules were used to obtain morphological data. In module I (ChainCoder), image processing and morphology contour codes were generated. In module II (Chc2Nef), contour codes were normalized and Elliptic Fourier descriptors were obtained. In module III (PrinComp), descriptors were subjected to PC analysis and PC scores were obtained. In module IV (PrinPrint), morphology variations of grape image contours were visualized.Each variable of morphological characteristics of grape cultivars was subjected to analysis of variance (ANOVA) and significant means were compared with the use of Duncan’s test at 5% significance level. All variables of morphology and dimensional attributes were subjected to Principal Component Analysis (PCA) and differences between the cultivars were presented in scatter plots based on component scores. With PC analysis, significant variables revealing morphology and dimensional differences of the cultivars were identified and ordered. SPSS 20.0 software was used for statistical analyses.Normalized contour codes by Elliptic Fourier analysis (EFA) were subjected to multivariate variance analysis (MANOVA) with the use of PAST v.4.02 software. Morphological differences between grape cultivars were explained by Hotelling’s pair-wise comparison tests, including verified Bonferroni values and Mahalanobis distances. In linear discriminant analysis conducted with the use of principal component (PC) scores, functions revealing morphology differences of the grape cultivars were determined and similarity relationships were presented in scatter plots. Such similarities were also put forth by hierarchical cluster analysis with the use of Euclidean similarity index and the grape cultivars, with morphology similarities presented in a dendrogram.Morphology and dimensional attributes of the grape cultivars measured at horizontal and vertical orientations are provided in The greatest length average was observed in “Antep karası” and the lowest in “Bahçeli karası” cultivar . The greResults of principal component analysis are provided in Vitis vinifera L.) germplasm collection in Iran by using PCA.In the scatter plot presented in Thusly, in previous studies, differences in cultivars were put forth with principal component analysis in walnuts ,59,60,61The contour codes obtained by Elliptic Fourier Analysis were subjected to principal component analysis (PCA), and morphology differences between the grape cultivars were explained by two principal components . The totp > 0.05). If the Mahalanobis distance value provided in pair-wise comparison is low, then the similarity between the cultivars is high. Similar findings were also reported by Demir et al. [Cornus mas genotypes.According to MANOVA results in r et al.  for CornIn Results of cluster analysis conducted with the use of group centroids of discriminant functions are provided in The projected area, equivalent diameter and perimeter values measured at horizontal orientation were greater than the values measured at vertical orientation. Elongation ratio averages of 20 grape cultivars are not dependent on diameter, area and perimeter of the cultivars. Elongation averages of the cultivars exhibited large variations. The greatest length-to-width ratio was 1.56. There were some cultivars with a morphology quite close to the sphere. When the geometrical dimensions of the cultivars with a high sphericity average were assessed, it was observed that they were both large and small sizes. Principal component analysis revealed that present grape cultivars were distinguished from each other based on dimensional characteristics rather than morphology. Dimensional variables constituted the most significant source of variation between the grape cultivars. The most significant shape variables explaining the variation between the cultivars were identified as sphericity and elongation.Elliptic Fourier analysis revealed that present grape cultivars looked similar to an ellipse or sphere in morphology. There are also some cultivars that looked similar to a water drop because of widening at the peduncle or fruit base. Linear discriminant analysis and cluster analysis clearly demonstrated similarities between the grape cultivars. In terms of morphology, grape cultivars were classified into 7 sub-groups. The first sub-group included “Bahçeli karası“, “Cevşen“, “Dimrit“, “Royal“, “Çavuş“ and “Amasya beyazı“ cultivars; the second group included “Erenköy beyazı“ and “Yerli beyaz“ cultivars; the third group included “Crimson“, “Nuniya“, “İzabella“ and “Müşgüle“ cultivars; the fourth sub-group included “Hafızali“ and “Karaşabi“ cultivars; the fifth sub-group included “Kırmızı“, “Yuvarlak çekirdeksiz“, “Sultani çekirdeksiz“ and “Morşabi“ cultivars; the sixth sub-group included “Yalova incisi“ and the seventh sub-group included the “Antep karası“ cultivar.


The figure of merit up to ∼17 kΩ−1 is superior to those of the unpatterned film counterpart, our fabricated 180 nm thick ITO, as well as most reported thick metal grid TCEs. Our ultrathin TCE, firmly attached to the substrate, is mechanically more flexible and more stable than the film counterpart and ITO. As a flexible transparent film heater, it achieves comparable or even superior heating performances with previously-reported heaters and performs well in a thermochromic test.Transparent conductive electrodes (TCEs) are experimentally demonstrated using patterned few nanometer-thick silver films on zinc oxide-coated rigid and flexible substrates. The grid lines are completely continuous, but only 8.4 nm thick. This is the thinnest metallic grid we are aware of. Owing to the high transparency of both the grid lines and spacing, our TCE with an opening ratio (OR) as small as 36% achieves an average optical transmittance up to ∼90% in the visible regime, breaking the optical limits of both the unpatterned film counterpart and the thick grid counterpart . The small OR enables a low sheet resistance of ∼21.5 Ω sq Patterned few nanometers thick silver films with high optical transparency, electrical conductivity, mechanical flexibility and stability. Furthermore, the inherent fragility has hindered its potential applications to emerging flexible optoelectronics, which require not only high transparency and conductivity, but also strong mechanical flexibility.3 Indium-free transparent conductive oxides (TCOs) have relatively lower cost, but still suffer from the same problem with brittleness if relatively thick films are employed for good conductivities.4Transparent conductive electrodes (TCEs), which by definition transmit light  and conduct electrical current simultaneously, are essential elements for various optoelectronic devices, such as film heaters, touch panels, solar cells, and light emitting diodes (LEDs).5,6 Carbon-based TCEs, e.g., conductive polymers,7,8 carbon nanotubes,9,10 graphene,11,12 always have rather low carrier concentrations and thus inferior conductivities to the metallic TCEs.1,5,6 Bulk metals are good conductors but are opaque due to the high absorptivity and reflectivity; while micro-/nano-structured metals, such as nanowire networks,13–15 patterned grids,5,6 and ultrathin films (∼10 nm or less),5,6,15–17 are promising for replacing ITO because of the high light transmission and electrical conductivity, as well as the excellent mechanical flexibility. Metallic nanowire networks can be processed in solution with low cost. The nanowires are usually randomly and sparsely stacked and distributed. In this case, light can transmit through the network with low loss. However, such networks are actually discontinuous, and their conductivities are limited by wire-to-wire contact resistances. The three-dimensional (3D) geometry also enables air or chemicals to easily infiltrate into the network. Therefore, the nanowires, difficult to protect, are susceptible to chemical corrosion. We also found that the nanowire networks suffered from poor adhesion to the substrates.18–20 In contrast, metallic patterned grids and ultrathin films do not have those problems.5,6,15–17 Previously-reported metal grids had either regular patterns fabricated through electron-beam lithography or photolithography, e.g., rectangles,21–28 modified rectangles,29 hexagons,26,30etc., or irregular patterns with self-forming cracks/masks, e.g., titanium oxide,31 egg white,32,33 CA200,32 leaf venation,34,35 spider's silk web,35 electro-spun fibers,36 all paint containing colloidal silicon dioxide,37 cosmetic containing acrylic emulsion,37etc. In general, the grid lines are made narrow enough so that light can transmit through the sufficiently large spacing without loss and meanwhile thick enough to maintain a sufficiently high conductivity.21–27,30–37 Because of the opaque grid lines, the optical transparency is usually limited by the opening ratio (OR), which is defined as the ratio of the space area to the unit cell area. Sheet resistances, Rsh, less than 1 Ω sq−1 have been reported with metallic grids that have micrometer thick grid lines.22,32,33 In this case, the overall roughness is increased, which may cause shorting in some thin-film optoelectronics, e.g., organic solar cells or LEDs. The micrometer thick grid may have serious shadow effects.22,32,33 In comparison, few nanometers thick metallic films in a two-dimensional (2D) morphology are much smoother. However, it is challenging to deposit smooth and continuous metal films with both high optical transmission and high electrical conductivity.5,6,15–17 Due to the large surface energy difference between the substrate and the metal to be deposited, the substrate usually has poor wettability to the metal, leading to 3D Volmer–Weber growth mode in the initial stage of physical vapor deposition processes.38–41 Fortunately, different methods have been developed to successfully suppress the 3D growth mode and evolve into a 2D morphology at an ultralow thickness, such as treating the substrate surface with seed layers or surfactants , doping the metal,50,51 lowering the substrate temperature during deposition,52etc. It is clear that an increase in thickness results in an increase in film conductivity but a decrease in optical transmittance. However, a too thin film is also not favorable for optical transmittance because of the plasmonic absorption excited in island-like discrete metallic clusters. Only at a certain thickness, where a continuous film just develops, can the best transparency be achieved, though the transparency is still not very high absolutely because of the intrinsic metallic absorption and reflection. Hybrid TCE consisting of a 100 nm thick copper grid on top of a 2 nm thick nickel film can achieve greatly improved conductivity but degraded transmittance.17In order to replace ITO and other TCOs, carbon- and metal-based alternatives have been developed.Tave, up to ∼90% in the visible regime, much higher than the unpatterned film counterpart (Tave ∼ 84%) and the thick grid counterpart (whose Tave depends on the grid OR). The small OR allows a relatively low Rsh ∼ 21.5 Ω sq−1. The achieved figure of merit (FoM) is up to ∼17 kΩ−1, which is superior to that of a 2D film counterpart without any patterns (FoM ∼ 15 kΩ−1) and to that of a 180 nm thick ITO (FoM ∼ 0.6 kΩ−1). It is comparable to and even higher than those of most thick metal grid based TCEs reported.21,23–31 Throughout the whole paper, the FoMs are calculated using the following widely-accepted expression:53,54T550 is the optical transmittance at the wavelength of 550 nm. Our patterned sub-10 nm TCE, strongly attached to the substrates, demonstrates better mechanical flexibility than the non-patterned film counterpart, let alone the brittle ITO. Finally, our TCE is explored as a flexible transparent film heater, which achieves comparable or even superior heating performances as compared with previously-reported heaters.19,24,55–59 Therefore, our patterned sub-10 nm Ag films are highly promising for many applications, including emerging flexible and thin-film optoelectronics (where shorting can be avoided).Here in this work, in order to break the optical limits of both metallic patterned grids and ultrathin films mentioned above, we propose to combine their advantages and experimentally demonstrate distinct TCEs consisting of patterned few nanometers thick silver (Ag) films on both rigid and flexible substrates coated with zinc oxide (ZnO) seed layers. As far as we know, metallic grids thinner than ours have not been reported. With an OR as small as 36%, our ultrathin TCE still achieves an average optical transmittance, −1 by a magnetron sputter . Subsequently, Ag films with different thicknesses were deposited by another sputter at 3.7 Å s−1 . With the bottom ZnO seed film, continuous ultrathin Ag films could be achieved with thicknesses of only several nanometers. For comparison, Ag films of different thicknesses were sputtered directly onto the quartz substrates without the ZnO seed layer.Pieces of quartz (2 cm × 2 cm), serving as rigid substrates, were ultrasonically cleaned with acetone, isopropanol, and deionized water in sequence, each for 5 min. On top of each substrate, a ∼40 nm thick ZnO film was deposited at a rate of ∼0.4 Å sIn order to conduct patterning, UV photolithography was employed to define regular grid patterns into the photoresist , which was spin-coated at 6000 rpm for 40 s on top of a 4′′-diameter ZnO-covered quartz substrate. After Ag film deposition according to the above sputtering recipe, the PR was ultrasonically removed by acetone, leaving a uniform grid-patterned Ag film on top of the substrate. It was very easy to conduct this lift-off process, because the PR (∼1.3 μm in thickness) was much thicker than the sub-10 nm Ag film. For further removal of the remaining solvent, the sample was ultimately cleaned with isopropanol and deionized water in sequence.Pieces of PE (50 μm thick) were chosen as the substrates for flexible TCEs and transparent heaters. They were carefully attached to silicon wafers before the deposition of the ∼40 nm thick ZnO seed layer. After the patterned and unpatterned sub-10 nm Ag films were processed with the above-mentioned procedures, the PE substrates were peeled off from the wafers, resulting in flexible TCEs. Flexible transparent heaters were achieved by applying Ag conductive paste to the flexible TCEs in a two-terminal side-contact configuration, as shown in via two conductive aluminium supports . For flexibility tests, flexible TCEs were bent to different curvatures or to a fixed radius of curvature of ∼5 mm for 500 cycles Before demonstrating patterned few nanometers thick Ag films, we first investigated the development of sub-10 nm continuous Ag films without any patterns. As mentioned above, such a film with complete continuity is usually difficult to deposit due to the 3D Volmer–Weber growth mode in the initial deposition stage. In order to minimize this unfavorable stage, we introduced a ZnO seed layer between the Ag film and the quartz substrate so as to improve the wettability of the substrate. The seed layer was ∼40 nm in thickness, which was sufficiently thin and would not degrade the mechanical flexibility of the film or grid (to be shown in T(λ), of our fabricated ultrathin Ag films with different thicknesses on top of quartz substrates with and without the ZnO seed layers. In order to quantitatively demonstrate their optical transparencies, average transmittances, Tave, were calculated with the following equation and plotted in T(λ) was weighted with CIE “physiologically-relevant” luminous efficiency function, V(λ)  induced absorption, to be discussed in detail along with the morphologies of the Ag films on both bare and ZnO-seeded quartz substrates shown in Tave was improved first as the Ag thickness increased. Further increasing the film thickness degraded the visible light transmission due to the intrinsic absorption of Ag, leading to a lower Tave value. The samples without the seed layers behaved similarly but with smaller Tave values, as shown in Tave ∼ 84%, which was ∼20% better than the film deposited on the bare substrate . As the film thickness was further increased to 10.5 nm, Rsh decreased slowly to ∼10 Ω sq−1, to which Rsh of the sample without the seed layer approached. In this paper, the ITO used for comparison was deposited in our own laboratory through room-temperature sputtering without post-annealing (unless otherwise specified).In Rsh, which was too large to measure at the minimal thickness of 4.2 nm  and shown in f 4.2 nm . At the f 4.2 nm , while w reduced . In greaasurable . The optasurable . As the  nm film .p = 50 μm and line width w = 20 μm , could be well predicted with their ORs and the transmittance spectra of both the grid spacing, Tspace(λ) and the grid line , Tline(λ), through the following equation:In order to further improve the transparency of the 8.4 nm thick Ag film on top of the ZnO seed layer, periodically arranged blank squares with 100% transmission were introduced into the top Ag film, which were predefined in a photomask for photolithography. Since all the fabrication processes  were scalable, such ultrathin Ag grid TCEs could be made as large as possible. Limited by our laboratory facilities, we only demonstrated in inset of . With painset of . This wa Fig. S3a, the who21,30 while our ultrathin Ag grids achieved much higher transmittance than their ORs (though the exposed ZnO coating might introduce some reflection and absorption). This totally benefited from the contribution of the highly transparent 8.4 nm thick Ag film on the ZnO seed layer. According to T(λ) could be further increased if the ZnO seed layer was simultaneously patterned into grid with the top Ag film (where Tspace(λ) = 1). However, it could be predicted that T(λ) would not improve too much, especially in the visible and near-infrared wavelength range, because Tspace(λ) was already close to 1 in this wavelength range for the ∼40 nm thick ZnO layer  achieved almost the same T(λ), Tave and Rsh, which could indicate the dependence of the OR value rather than the specific grid geometry. For the sample with OR = 36%, Rsh ∼ 21.5 Ω sq−1 was not degraded too much from that of the Ag film and was still much smaller than that of the 180 nm thick ITO.As shown in versus T550 (instead of Rsh) were plotted in 21–37 Among our fabricated 8.4 nm thick Ag grid and film TCEs, the one with OR = 36% achieved the highest FoM of ∼17 kΩ−1, because of the best balance between Rsh and T550. Such a FoM could not compete with those achieved by >1 μm ultrathick grid TCEs with ultralow Rsh's,22,32,33 but was comparable to and even higher than those obtained by most grid TCEs with intermediate thicknesses between 30 nm and 500 nm.21,23–31T550 ∼ 90%, which only a few thick grid TCEs could surpass.25,28,30,33,35,36 The thinnest TCE we could find in the literature was 30 nm,23 which had an FoM of ∼10 kΩ−1 and a T550 of less than 80%. In comparison, our fabricated 8.4 nm thick Ag grid TCE with OR = 36% had much better performances in both FoM and T550. Considering the poor performance of our deposited 180 nm thick ITO, some better representative results were also included in i.e., T550 = 85.7%, FoM = 15.9 kΩ−1,29,49 and T550 = 92%, FoM = 28.9 kΩ−1.60,61 The FoM of the 8.4 nm thick Ag grid TCE with OR = 36% was much higher than that of our deposited ITO (FoM ∼ 0.6 kΩ−1) but was between the previously-reported ITO data in terms of both T550 and Rsh. This indicated that our ultrathin Ag grid TCEs was very competitive to replace ITO.In order to highlight the optical performances, FoMs 18–20 The ultrathin geometry could be protected from corrosion simply with an additional dielectric coating, e.g., a 10 nm thick ZnO film, while such a simple protecting method was not applicable to the randomly distributed nanowire networks. The few nanometers thick film or grid was also more advantageous in many applications, e.g., thin-film solar cells and LEDs, where shorting might be avoided.Furthermore, our 8.4 nm thick Ag film and grid were firmly attached to the ZnO-seeded substrates, which could not be peeled off with scotch tape /R0 (where R0 and R were measured resistances of the sample before and after bending) and plotted in Our few nanometers thick Ag films with or without grid patterns could be easily prepared on any substrates. In this work, we fabricated them on flexible polyethylene (PE) substrates with the same procedure for the samples on rigid quartz substrates. Vdc) was applied to the Ag paste contacts on the opposite edges of the grid TCE, where heat was generated through the Joule effect. Vdc = 3 and 5 V. The temperature at the sample center was recorded. A temperature up to ∼80 °C was achieved when the heater was powered by a 5 V bias. It was comparable and even superior to those obtained by many other previously-reported transparent thin-film heaters  with similar sizes and under the same biases.19,24,55–59 Our device showed rapid responses upon being powered and unpowered. Regardless of the applied voltages, it took ∼6 s for the temperature of our heater to rise from 10% to 90% of its maximum when the bias was turned on; and took another ∼7 s to drop from 90% to 10% when the bias was turned off. The homogeneous steady-state temperature profile shown in Vdc = 5 V, heat was generated and the letters faded away immediately. Within 6 s, the letters completely disappeared.To explore a practical application, we employed our few nanometers thick Ag grid TCE as a flexible transparent thin-film heater. As schematically shown in Tave up to ∼90% in the visible regime, much higher than the unpatterned film counterpart with Tave ∼ 84% and the thick grid counterpart with Tave mainly limited by the OR. The small OR or wide grid lines enable a relatively low Rsh ∼ 21.5 Ω sq−1. The achieved FoM is up to ∼17 kΩ−1, which is superior to that of a 2D film counterpart without any patterns (FoM ∼ 15 kΩ−1) and to that of our fabricated 180 nm thick ITO (FoM ∼ 0.6 kΩ−1). It is comparable to and even higher than those of most reported thick metal grid based TCEs.21,23–31 Our patterned sub-10 nm TCE keeps almost unvaried in terms of conductivity when bent with a curvature radius as small as ∼2 mm or after being bent 500 times at a bending radius of ∼5 mm. Such mechanical flexibility and stability are better than the non-patterned film counterpart, let alone the brittle ITO. Our TCE can be employed as a high-performance flexible transparent film heater, which achieves comparable or even superior heating performances with previously-reported heaters,19,24,55–59 and performs well in a thermochromic test. Furthermore, it has much stronger attachment to the substrate than the nanowire networks according to our previous experience.18–20 Because of the ultrathin geometry, our TCE can be easily protected from corrosion and potentially be incorporated into emerging flexible and thin-film optoelectronics.In conclusion, we have demonstrated experimentally distinct TCEs based on patterned few nanometers thick Ag films on ZnO-seeded rigid and flexible substrates. The grid lines are completely continuous, but only 8.4 nm thick. As far as we know, even thinner metallic grids have not been reported. Owing to the high transparency of both the ultrathin grid lines and the grid spacing, our TCE with an OR as small as 36% achieves a There are no conflicts to declare.RA-011-D1RA00549A-s001RA-011-D1RA00549A-s002


Thermal stability of lactase  enzyme has been studied by a variety of physico-chemical methods. β-galactosidase is the main active ingredient of medications for lactose intolerance. It is typically produced industrially by the Aspergillus oryzae filamentous fungus. Lactase was used as a model to help understand thermal stability of enzyme-type biopharmaceuticals. Enzyme activity (hydrolyzation of lactose) of β-galactosidase was determined after storing the solid enzyme substance at various temperatures. For a better understanding of the relationship between structure and activity changes we determined the mass and size of the molecules with gel electrophoresis and dynamic light scattering and detected aggregation processes. A bottom-up proteomic procedure was used to determine the primary amino acid sequence and to investigate changes in the N-glycosylation pattern of the protein. NMR and CD spectroscopic methods were used to observe changes in higher order structures and to reveal relationships between structural and functional changes. Quality assurance and stability testing of these complex protein type molecules demand extensive knowledge of their structure in order to provide effective and safe medications. Stability testing of immunoglobulin-based biologicals is relatively well established, but for other biologicals, like large, enzyme type drugs it has not yet been defined. Stability and quality assessments are usually based on simple activity tests, without any information on possible changes in the structure or in the posttranslational modifications of the enzyme. Stress measurements of the enzyme are usually performed in the form of a solution, although the formulations and technological processes are mainly present in the solid form.β-galactosidase, also called lactase is a hydrolytic enzyme that decomposes lactose, a disaccharide found in dairy products. It also catalyses the reverse of the reaction, called trans glycosylation . β-GalacEnzyme molecules like lactase have many different structural variants with different properties and resistance to external effects , depending on its source. . StructuDespite the pharmaceutical and commercial relevance of the lactase containing products , our knoThe main objective of the research is to reveal structural changes of the lactase enzyme under heat-stress conditions. This is important both for understanding fundamental aspects of enzyme activity changes under storage, and for practical considerations such as improving dosing, manufacturing, shipping and storage of biopharmaceuticals, like lactase containing products .22.1d-galactopyranoside (ONPG), MU-Gal  and the broad range SDS–PAGE molecular weight standards were obtained from VWR International . For the tryptic digestion Trypsin Gold, Mass Spectrometry Grade  and Rapigest  were used. Ultrapure water obtained from the MilliRO-hMilliQ-system  was used throughout all measurements. All other ingredients like the reagents, solvents, eluents were obtained from Sigma Chemical Company .The powder of β-galactosidase enzyme (Opti-lactase A-50 powder) derived from Aspergillus oryzae was purchased from Optiferm Gmbh . O-nitrophenyl-2.2The essence of the stress stability test is to model the behaviour of the sample under extreme conditions, such as high temperatures to determine and model extreme storage and production parameters , and pre2.3d-galactopyranoside (ONPG) as substrate [2CO3 solution to stop the enzymatic hydrolysis and to produce the maximum possible absorption due to the liberated o-nitrophenolate ion. The solution was left to cool to room temperature and the absorbance of o-nitrophenol was determined by UV–vis spectroscopy using an ATi Unicam UV2 UV/VIS Spectrometer  at 420 nm in three parallel measurements with five different stressed samples.The hydrolysing activity of the examined enzyme stored at various temperatures for various time periods listed above was estimated using o-nitrophenyl-β-ubstrate . ONPG isubstrate . 5 ng/mLubstrate ,25. The 2.4The average diameter of the proteins in the different samples was determined by dynamic light scattering (DLS) using Zetasizer NanoZS™ . Absorbances of a sample have been investigated before each particle sizing by single beam UV–vis spectrophotometer,  at λ max =633 nm, to determine the correct amount of sample scatterings vs. absorbances. Samples were illuminated by a HeNe laser , an avalanche photodiode (APD) detection angle was 173° (backscatter mode). Protein samples were dissolved in MilliQ water in the concentration of 1 mg/mL to reduce nonlinearity effects on measurements by increased viscosity of solvent with higher concentrations. Measurements were made performing 15 runs of 10 s each, an equilibration time of at least 5 min was set before the measurement started. Size measurements were made in triplicate and were used immediately after preparation. The experiments were carried out at a constant room temperature of 25 °C. All solutions were analysed without prior filtration to assess if aggregates were present up to 10,000 nm (detection limit). The diameter of the solved molecules is related to the properties of the solvent, so refractive index and viscosity were tested. The refractive index was measured by a clinical refractometer, the viscosity by a Brookfiled Ametek DVE viscometer  and it proved to be 1.031 and 0.8991 cP.2.5SDS-PAGE was carried out using a 10 % Tris-Glycin gel and a vertical Novex Minigel electrophoretic system. 100 μL of 1 mg/mL lactase solution was mixed to 100 μL SDS sample buffer  denatured at 80 °C for 5 min. 15 μL of each sample was used and the electrophoresis was carried out at 35 mA and 150 V for 90 min until the bromophenol blue has reached the lower edges of the gel. Protein bands were visualised by staining with Coomassie Brilliant Blue R-250 in 50 % methanol, and 10 % acetic acid staining solution, distained by a solution of 5% methanol and 10 % acetic acid for overnight. Broad-range SDS–PAGE molecular weight standards purchased from VWR were used as molecular mass standards .d-galactopyranoside) to test for β-galactosidase activity of the separated bands. This gel is soaked for 15 min in 100 mM sodium acetate buffer, pH = 5.0 with lateral shaking, 100 rpm. Then, it is incubated with the MU-Gal solution at 37 °C for 15 min. The zymogram gel is analyzed under UV light, λ = 365 nm to detect enzyme activity due to the release of 4-methylumbelliferone. This step should be performed quickly, as the fluorescent product diffuses within the gel, obscuring the bands.For the Zymography (correlation between activity and protein position) the gel is subjected to fluorometric analysis using MU-Gal  NMR. The samples were: lactase standard 100 mg solved in MilliQ water, 10 % D2O, 1% DSS (standard) added, pH = 7.45. Heated lactase at 80 °C for 14 days 100 mg solved in MilliQ water, 10 % D2O, 1% DSS (standard) added, pH = 6.96. Before the NMR measurements the samples were filtered five times with Eppendorf centrifuge tube equipped with a membrane of 10 kDa cut. After the filtration, the samples were diluted to 500 μL with MilliQ Water then measured immediately. NMR measurements were performed on a Bruker Avance III 700 MHz spectrometer equipped with a 5 mm Prodigy TCI H&F-C/N-D, z-gradient probe head operating at 700.05 MHz for 1H. All spectra were processed with Bruker TOPSPIN software. 1H NMR measurements DOSY experiments were performed at 14 °C in accordance with a previous NMR study which compares diffusion constants with the size of proteins [δ =5 ms, Δ = 300 ms. The strength of the diffusion gradient was linearly incremented in 32 equal steps, varying between 5 % and 95 % of its maximum value; the applied maximum gradient strength was 45.4 G/cm. The number of scans was adjusted for each sample to obtain reliable S/N ratios (16 for 1H and 40 or 160 for DOSY experiments). Each measurement was repeated at least three times. To investigate the diffusion constants signals were chosen from several regions in the aliphatic proton range, and the decay was processed with 2D Fourier transform.Data were acquired from two samples with two different NMR methods: 1D proteins , using t2.7Circular dichroism (CD) measurements were performed on a Jasco J-815 CD spectrometer equipped with a thermostable cell holder. Temperature was controlled and monitored with a Peltier type Jasco CDF-426 L thermostat unit. The substrate and different stressed enzyme mixtures were measured together for kinetic studies, to determine the reduction of activity in time. The possible manifestation in secondary structure changes due to the supposed temperature induced degradation was also intended for analyzation. Conformational changes in the secondary structure of protein were monitored in the region of 190–250 nm with a protein concentration of 50 μg/mL (0.60 μmol) in a quartz cuvette (Hellma) with a path length of 1 mm. The scanning speed, bandwidth and data pitch were set to 50 nm/min, 1 nm and 0.5 nm, respectively, while the number of accumulations for each recorded spectrum was set to 3 (within 600 H T voltage range) and averaged to get the complete spectrum.2HPO4 buffer, pH = 4.6, temperature 37 °C ± 0.05. Detection wavelength was set at 356 nm (ONPG CD maximum).The activity measurements were followed by CD spectroscopy alternatively, where the ligand ONPG hydrolysis could be measured selectively online, and the kinetic curve could be acquired directly. Kinetic measurements were performed as follows: ONPG (0.050 g / 10 mL) +1 μL enzyme stock solution (10 mg / mL) dissolved in 3 mL of McIlvaine citrate-Na2.8For the sequence and glycan identification bottom-up analysis was used. Five different parallel stressed samples were identified to inspect the permanence level of the changes. All reagents were used in relevant volume, comparable to the protein level. The samples corresponding to 100 μg of protein were solved in MilliQ water. For the S-S bridge reduction ammonium bicarbonate buffer at pH 8.0 containing 200 mM of DTT and Rapigest was added. The samples were successively incubated at 60 °C for 30 min, and then cooled at room temperature. Alkylation of cysteine residues by adding 200 mM IAA in 100 mM ammonium bicarbonate was carried out in the dark at room temperature for 30 min. The samples were left to be digested for 90 min at 37 °C by adding trypsin 1:50 w/w ratio considering the total protein content. Lastly, samples were acidified with cc. formic acid to stop the digestion. In the end the samples were centrifuged at 13447 G-force for 10 min and the phases were carefully separated. The trypsin digestion was carried out in solution without TFA in order to avoid the MS source contamination. The mass spectrometric measurements started right after the preparation as follows: 6 μL sample was subjected to nanoLC-MS/MS analysis using a Dionex Ultimate 3000 RSLC nanoLC  coupled to a Bruker Maxis II Q-TOF  via CaptiveSpray nanoBooster ion source. Peptides were separated on an Acquity M-Class BEH130 C18 analytical column  using gradient elution (4–50 % eluent B in 120 min) following trapping on an Acclaim PepMap100 C18, 5 μm, 100 μm × 20 mm  trap column. Solvent A consisted of water containing 0.1 % formic acid, while Solvent B was acetonitrile containing 0.1 % formic acid. Spectra were collected using a fix cycle time of 2.5 s and the following scan speeds: MS spectra at 3 Hz, while CID was performed on multiply charged precursors at 16 Hz for abundant ones and at 4 Hz for the ones with low abundance. Internal calibration was performed by Compass Data Analysis software 4.3  based on infusing sodium-formate. Data were processed by the Byonic software . Peptides were identified by searching against the Aspergillus Oryzae (W5ZSH9_ASPOZ) in the UNIprot database. Glycans were quantified with the GlycoPattern software .3The enzyme activity and various structural aspects of the lactase sample stored under various conditions  has beenThese results will be presented below in detail.3.1The changes in lactase activity due to thermal stress are shown in 3.2Having observed changes in lactase activity due to heat-stress, we have tried to understand the corresponding changes in protein structure. First, we have performed dynamic light scattering (DLS) measurements to characterize molecular size and possible aggregation. We have determined the average molecular size (hydrolytic diameter using a hard sphere model) and the polydispersity index. Note, polydispersity indicates the variability of the molecular size based on the DLS distribution ,30. The Increase of particle size is small at low temperatures and is likely due to protein unfolding. At higher temperatures (80 °C) large, 2−3-fold increase in average particle size can be observed. 3.3According to the information in the UNIprot database , the weiIn the first column a standard protein ladder was used to determine where the different masses of materials are located on the gel. There is no visible change in molecular weight in the case of 40 °C, 60 °C heat stressed samples, but at 80 °C a band appeared at higher (approximately 500 kDa) molecular weight, from which it can be concluded that the protein starts to aggregate and likely forms tri- or tetrameric structures, as suggested . It can 3.4The NMR measurements provided information on the structure and size of the protein. The large molecular size and heterogeneous composition of the lactase samples caused some practical difficulties regarding NMR measurements. For this reason, the classical Stejksal-Tanner equation fitting did not give useful results, as regression always resulted in a multicomponent fitting. Information on various size molecular components in the lactase sample . This revealed that multiple diffusion constants can be detected in the samples with differences both in size and distribution. The specified diffusion constants and the calculated masses are extrapolated (based on the work of Dudás and Bodor ) are sumThe 27 and 58 kDa size ranges in the measurements are not caused by the thermal stress; they are present in both samples from the beginning like presented earlier in 1H-NMR patterns change over time in the samples. Based on the DOSY spectra, the samples were found to be mixtures characterized by multiple diffusion constants. The molecule sizes determined based on DOSY are significantly larger in the stressed samples , which is the ratio of the CD and the UV signals and a dimensionless parameter, then the concentration differences disappear. Furthermore, if there is a slight but significant difference in either the CD or the UV spectrum, it appears on the g-spectrum . The fol3.6The lactase enzymes (the control and heat-stressed samples) were studied, after tryptic digestion, using LC–MS/MS analysis. Peptides were identified using the Byonic software. Very high, 95.02 % sequence coverage was observed for the control sample, while 91.4 % for the 80 °C stressed sample. Recent studies on other proteins  suggest t-test was used to identify those glycans, that showed significant (p = 0.05) difference between the control and the heat-stressed sample (Heat-stress caused a large (and irreversible) change in glycosylation. This was revealed by quantitative mass spectrometric analysis. For quantitative comparison 5 replicates of the control sample, and 5 replicates of the 80 °C heat stressed sample were analysed. The glycopeptides found in both samples were selected for further investigation. A d sample . The glyd sample . Note th4Our investigations indicate various changes occur when submitting the solid enzyme to heat stress. Heat stress causes significant decrease in enzyme activity. However, after dissolution, in several hours, enzyme activity somewhat recovers, suggesting the activity decrease is partly reversible. This can be explained by reversible aggregation of lactase molecules, suggested by the results of DLS and NMR measurements. In the case of oral medication, partial recovery of enzyme activity is not relevant, as lactase molecules do not spend sufficient time in dissolved form in the body. The amino acid sequence and the secondary structure of the enzyme did not show any change due to heat stress. Lactase is heavily glycosylated, and heat stress did change glycosylation significantly. LC–MS experiments revealed that various peptide-bound glycan chains were shortened due to heat stress. These are irreversible changes, changeing the physico-chemical properties of the enzyme and may increase tendency of aggregation.These studies suggest that both structural changes in the higher order structure of the protein and changes due to altered glycosylation are responsible for the decrease of activity. Understanding behaviour under heat stress may help to design better manufacturing, shipping, and storage conditions and improve our understanding of different types of lactases, like Kluyveromyces lactis used in dairy industry or even human lactase. The methodology developed here may serve as a model for investigating other enzymes of pharmaceutical interest.Márton Király: Writing - original draft, Visualization, Formal analysis, Investigation, Methodology. Borbála Dalmadi Kiss: Writing - review & editing, Methodology, Conceptualization. Péter Horváth: Investigation, Writing - original draft, Visualization. László Drahos: Software, Formal analysis, Writing - review & editing. Arash Mirzahosseini: Funding acquisition, Writing - original draft. Gyula Pálfy: Investigation, Visualization, Writing - review & editing. István Antal: Resources. Krisztina Ludányi: Conceptualization, Supervision, Funding acquisition, Writing - review & editing, Methodology.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.