Rehmannia glutinosa Libosch and differentiation induction potential of some substances purified from this herbal, it finds difficult to seek research that investigated the effect of hot water-extracted R. glutinosa Libosch (RGE) on proliferation and cardiogenic differentiation of mesenchymal stem cells, even though it has commonly been used for a long time because of its function as a restorative and as a critical role in cardiovascular treatment in traditional.Vietnamese medicine tried and tested certain bioactive compounds from plants to increase the rate of tissue immunomodulation, regeneration, and differentiation. Although there are many research papers discovered about phytochemicals of Our research indicated that RGE has many predicted bio-pharmacological effects, and the RGE is demonstrated that it is non-toxic to UC-MSCs (IC50 = 1274 ppm). It also stimulates the proliferation and migration of UC-MSCs at various concentrations, especially at the RGE concentration of 50 ppm, during four days of treatment. On the other hand, the RGE can induce the cardiac pre-differentiation process from the fifth day to the fifteenth day after treatment, which was proven through both molecular and cellular (morphology evidence) levels like the up-regulation of GATA4, Nkx2.5, cTnT α-MHC, Desmin genes; the expression of Desmin protein, the appearance of two-nuclei cells, connecting process of adjoining cells, the cytoplasmic striations.The RGE could either stimulate proliferation–migration of MSCs or induce the cardiac pre-differentiation process. This extract can be classified as non-toxic to the UC-MSCs. Rehmannia glutinosa Libosch (RG) is a carbohydrate-rich plant in the family of Scrophulariaceae and is a famous folkloric medicinal plant in Vietnam. Research works in the past revealed that RG contains many components in its extract [R. glutinosa Libosch on mesenchymal stem cells even though residents of Eastern countries often use RG with a form like crude extract instead of using the purified individual components. Moreover, there is a tendency, which is more and more popular in research, to use the plant extract for proliferating and differentiating on the MSCs with the various promising outputs from many different plants such as Glycyrrhiza glabra, Rhizoma Drynariae, Foeniculum vulgare [ extract , which i extract , hypogly extract , anti-se extract , cardiov extract , heart f extract , and tum extract . We can  extract ,9; and o extract ; on neur extract . There i vulgare , so the in vitro or in vivo. In this day and age, they have been used for various clinical trials for dealing with many kinds of sickness, especially the MSCs use for treating COVID-19 recently [Besides, although the use of bone marrow-derived mesenchymal stem cells (BMSCs) has been popular in research and clinical studies, umbilical cord mesenchymal stem cells (UC-MSCs) can become an alternative source to BMSCs due to non-invasive collection protocol and easy accessibility. They have also emerged strongly to the attractive therapy for many diseases because of their abundant sources, self-renewal ability, immunoregulation ability, differentiation into different kinds of mature cells or functional cells, and even non-mesodermal cell types either recently –14. Sincthe vivo microenvironment when using MSCs or tumor-forming when embryonic stem cells are used [in vitro from stem cells in the last decades, despite the differentiation into function cardiomyocyte from MSCs is controversial due to the failure in the majority of researches in the past, especially when scientists use herbal plant for differentiation purpose [Cardiovascular disease-related mortality accounts for the most significant percentage of deaths world-wide. The critical reason for this situation is that cardiomyocytes' s insufficient capacity to proliferate and regenerate themselves. As a result, following a cardiac attack, these resident cells are replaced by fibroblasts and non-contractile scar tissue, resulting in heart muscle tissue contractile dysfunction and heart failure. A variety of techniques are used to address this issue, including allogeneic heart transplantation and stem cell transplantation. However, stem cell transplantation therapy has certain drawbacks, such as organ trapping and spontaneous differentiation in are used . With th purpose ,17.HowevThe roots of RG were collected between January and February 2020 from Thanh Hoa, Vietnam. The sample was identified by Dr. Nguyen Viet Thang (Hue University), and the voucher specimen is deposited in the Sub-department of Applied Biology, Department of Biology, College of Sciences, Hue University (voucher number – D06).The RGE was carried out using Jane C-J Chao's procedure  was modiThe total carbohydrate content in RGE was determined by a phenol-sulfuric acid assay using glucose as a standard.Briefly, 100 mg RG powder and 5 mL of 2.5 N HCl were boiled in a water bath for 3 h for hydrolyzing, followed by cooling. After solid sodium carbonate was added for neutral, the solution's volume was made up to 100 mL and centrifugation. Three experiment tubes were set by 0.1 mL sample and 0.9 mL water; 0.2 sample and 0.8 mL water; 1 mL water (blank). Next, 5 mL of 96% sulphuric acid was added to each tube and shaken after 10 min. After that, these tubes were incubated at 25–30 °C for 20 min, and the colors were read at 490 nm. Finally, the sample's total carbohydrate content was calculated using the standard graph ,19.The RGE after extraction and storage in the above condition was split into 3 mL in a tube for GC–MS analysis.The RGE was tested by the drug, cosmetic, and food quality control center of Thua Thien Hue province (HueQC). The composition was analyzed by the Gas Chromatography-Mass Spectrometry method (GC–MS).Umbilical cords were collected from Hue Central Hospital with the approval of the Research Ethics Committee of Hue Central Hospital and transported to the Stem cells Laboratory, Department of Biology, College of Sciences, Hue University in 0.9% normal saline containing 100 U/mL penicillin and 100 mg/mL streptomycin at 4 °C with written consents of mothers and families.2 sections. These fractions were rinsed with PBS and subsequently suspended in StemMACS™ MSC Expansion Media Kit XF . Incubation was performed at 37 °C in a humidified atmosphere containing 5% CO2, and the medium was replaced every three days. The morphology of UC-MSCs was followed with Olympus CKX31SF inverted microscope .Initially, blood vessels were removed in saline, and the umbilical cords were sliced into 1–2 cm2.When the cell population's confluence reached around 80%, the cells were trypsinized, counted, and re-seeded into culture dishes at a density of approximately 1000 cells/cmFor CFU-F culturing at passage 2–3, the cells were seeded at a density of 100 cells per well. Colony-forming unit-fibroblast (CFU–F) was stained with Giemsa solution and counted by ImageJ.The attached cells from passage culture were detached by trypsin digestion, washed in PBS, and reacted with FITC-conjugated or PE-conjugated monoclonal antibodies  against human CD34, CD45, CD73, CD90, and CD105 for 30 min in the dark at room temperature. The cells were washed twice in PBS, and at least 10000 events were collected with FACSCanto . The data were analyzed with FlowJo software.in vitro differentiation assay and assay to determine the induction of the proliferation - migration of UC-MSCs.The UC-MSCs at passage 3 were treated with RGE for primary assays, including This treatment divided the UC-MSCs population into three groups by various concentrations of RGE such as 50 ppm, 100 ppm, 150 ppm, respectively, R1, R2, R3 groups.2 incubator with 5% CO2 at 37 °C for 24 h for growth.Cell cytotoxic assay was performed for testing the cytotoxicity of the RGE against UC-MSCs. Initially, UC-MSCs were re-suspended with the fresh medium and counted. After that, UC-MSCs were seeded on a 96-well plate at the density of 10,000 cells/well and incubated in a COThe viability rate of UC-MSCs was evaluated through Trypan blue assay like afore-research . After tFinally, the online software named Quest GraphTM IC50 Calculator  , was useFor proliferation analysis, UC-MSCs were passage cultured in StemMACS™ MSC Expansion Media Kit XF . Cells were visualized using an Olympus CKX31SF inverted microscope (4× objective lens) and images were captured . Cell number was assessed and compared using automated cell identification methods. This method already is demonstrated that it was sensitive enough to accurately detect both more minor changes in cell numbers and a more comprehensive range of cellular densities than spectrophotometric analysis of crystal violet-stained cells . Accordi//Convert Image to 8-bitrun("8-bit");//Remove Noiserun("Despeckle");//Adjust Brightness and ContrastsetMinAndMax;run("Apply LUT");//Apply Phansalkar Local Thresholdrun;setAutoThreshold("Default");//Watershedrun ("Watershed");//Count Objects (i.e. Cells)run;For migration analysis, UC-MSCs were passage cultured to gain 70% confluence before a scratch assay was performed as described by previous research . BrieflyUC-MSCs population after induction was evaluated with similar assays to our previous research paper , includiBriefly, the shape index can measure cellular morphology . This inEquation:ImageJ software was used to figure out the proportion of cells that were directed to alignment and orientation through θ angle ,29. Thiswww.ncbi.nlm.nih.gov/tools/primer-blast/). All reagents and primers were purchased from PHUSA Biochem Company, Vietnam.The total RNA extraction was performed using InviTrap® Spin Universal RNA Mini Kit , according to the manufacturer's instructions. Reverse transcription was reacted by Promega GoScript (TM) Reverse Transcriptase (Promega Corporation) and random hexamer primers to form first-strand with RNA templates. cDNA was diluted with Nuclease free water. Subsequently, PCR reactions used PHUSA Taq_500 and specific primers . These pInitially, UC-MSCs were fixed with methanol for 10 min at −20 °C, washed three times with PBS, followed by incubating at 4 °C. Primary antibodies, which were incubated with the cell for 1.5h at 37 °C, were diluted before (1:100). The next step was 30 min incubation with secondary antibody (diluted 1:200). Slides were taken photo by using diaminobenzidine substrate and counterstaining with hematoxylin.All data is shown as mean +standard deviation (SD), and differences between samples were determined by Student's t-test. One-way ANOVA, Turkey's HSD post-test among selected pairs of groups were also analyzed and performed with R software for Mac OS. Values with a p < 0.05, p < 0.01, p < 0.001 were considered statistically significant.Data from the phenol - sulfuric acid method was used for making a standard linear curve by glucose content. Forward, carbohydrate concentrations in the samples were calculated based on the standard curve .From the inferred carbohydrate content given in The GC–MS spectrum was presented in Several bioactive compounds from these plants have a specific role as bioactive mediators in regulating the rate of cell division and differentiation. In these compounds, most of them resemble afore-research, which is guanosine , methyl The cultured UC-MSCs population displayed a typically homogeneous fibroblast-like morphology . This ceAfter 3–5 days of culture, cells begin to divide and form small colonies around the original cell. From 7 to 10 days, CFU-F clusters form with a cell count greater than 50 cells per colony and its diameter more than 1 mm. Then, staining with Giemsa and counting with ImageJ software. The number of colonies constantly increased from the primary culture to the fourth passage .From the result was given by Quest GraphTM IC50 Calculator , the cytThe result about the rate of cell viability after treatment indicated the statistically significant difference between treated cell viability (cell group treated with RGE of concentration from 200 ppm to 6400 ppm) and cell viability in the control group . We deteThe cell number was counted by ImageJ software revealed the power of RGE on proliferation stimulation by all three various concentrations . At the The reduction of wound area when treating with RGE reflects this extract's ability to stimulate the migration and proliferation of UC-MSCs. Results this assay displayed through photomicrograph  and ImagInitially, the ability of RGE to induce the cardiogenic pre-differentiation from UC-MSCs was exhibited through the morphological changes traced by shape index and the orientation of the cell population after treatment. This work revealed that the shape index of the treated UC-MSCs constantly falls in the period after treating with RGE, for details from around 0.75 at the 0 days to approximate 0.25 at the 18 day under treating . This ouBesides the result, which was quantitated by ImageJ software, the morphology of UC-MSCs was also visualized and shown in On the contrary, the orientation of the UC-MSCs population did not change during this assay. This result showed through the random distribution of θ angles from 10° to 90° of all cell groups GATA4, Nkx-2.5, cTNT, α- MHC, Desmin. GAPDH was reference genes and used as internal reference controls for normalizing these bands for semi-quantitative by ImageJ software. The semi-quantitative analysis result revealed the expression of the five above genes and the silence of α- cardiac actin gene.After performing the RT – PCR assay, the photos of electrophoresis were captured for analyzing these concerned bands . AccordiFollowing the verified upregulation of some cardiac-specific mRNA, the result of differentiation was confirmed on the specific protein expression. Due to our laboratory's condition, we proved the expression of Desmin by immunocytochemistry, which is a particular protein of cardiac muscle tissue. This assay indicated that the RGE-treated groups after treatment expressed Demin protein, while cells in the control group did not display Desmin .Initially, these results regarding the tests on the phytochemicals of RGE revealed that RG is a carbohydrate-rich plant and has many different bioactivities that were predicted through the components in RGE obtained in GC-MSs result and other previous publications. Among these predicted effects pharmacologically, it is easy to guess that this extract has positive effects on the mesenchymal stem cells because MSCs play a prominent role in the recovery process of many diseases. Thus, unsurprisingly we discovered that RGE in this research could not have the cytotoxicity effect on the UC-MSCs population. This result about the cytotoxicity is contrary to a previous result that RGE can have cytotoxicity effect on a cancer cell line .All the tested RGE concentrations not only are safe for the growth of UC-MSCs but also have proliferation – migration stimulation effect on the UC-MSCs. Significantly, the RGE concentration of 50 ppm displayed the most potent stimulation ability on proliferation and migration. These results were confirmed through the scratch assay and the cell counting assay, and these results are also similar to previous publish that RG can stimulate the proliferation of cells ,48. HoweAfter that, for investigating the cardiogenic differentiation, the cell population after treatment was evaluated the morphology and the orientation of the whole population from the fifth day after treatment. It is clear that from the fifth day, the morphology of the cell has significant changes, which is become more elongated and turn to tubule-shaped from the fibroblast-shaped and spindle-shaped of MSCs, which were display through the photos and the reduction in the shape index and this results also is the same with many previous cardiogenic differentiation researches . BesidesAlthough the alignment is the main specific character of the mature heart muscle tissue, either this differentiation strategy or common differentiation strategy, which is 5-azacytidine use, had limited due to failure in making the alignment . BesidesGATA4 and Nkx-2.5 mRNA, which is similar to other published data [GATA4 and Nkx-2.5 mRNA at a low level [Nkx-2.5, cTNT, α- MHC, Desmin. Among these, GATA4 and Nkx-2.5 are early markers and were shown to modulate specific genes during the early-stage development of cardiogenic differentiation [Nkx2.5 and GATA4 clears the path to access these promoters for active transcription [From the results of PCR assay, it is evident that our UC-MSCs population do not express the hed data , while sow level . This contiation . Thus, tcription .cTnT, α- MHC were also up-regulated in all three treated groups. The semi-quantitative results revealed the high expression of these markers compared to the expression of both GAPDH as control and other early markers. We also performed the PCR reaction to indicate the expression of Desmin mRNA during treatment. Unfortunately, in this work, although 5 out of 6 surveyed specific genes as either soon marker or later marker upregulated under RGE treatment, the last later marker is α- cardiac- actin was not expressed at any cell groups. This lack of expression can bring surprise due to the α- cardiac- actin one primary marker responsible for gathering muscle proteins and coordinating contractile reaction in the mature heart muscle tissue. This gene's silence can prove the failure in differentiation into functional cardiomyocytes from UC-MSCs by using bioactive compounds form RG [cTnT, α- MHC, GATA4, Nkx-2.5, Desmin could be confirmations of differentiation into cardiomyocyte-like cells [Besides, some mature myocardial markers like  form RG . Howeverke cells .R. glutinosa Libosch in this research has a large proportion of carbohydrate contents and has diverse pharmacological effects are predicted from the result of GC–MS. This extract is categorized as non-toxic to UC – MSCs with IC50 value is 1274 ppm. All the tested concentrations of RGE can stimulate the proliferation–migration process during the initial four days of treatment and induce the cardiogenic differentiation from the fifth day of treatment, which is demonstrated through the reduction in shape index, some specific morphology , the local orientation, the expression of specific genes , and the expression of Desmin protein.The hot water-extracted


We aimed to investigate the relation between the time intervals of the flow velocity waveform of ductus venosus (DV-FVW) and cardiac cycles. We defined Delta A as the difference in the time measurements between DV-FVW and cardiac cycles on the assumption that the second peak of ductus venosus (D-wave) starts simultaneously with the opening of the mitral valve (MV). As well, we defined Delta B as the difference of the time measurements between DV-FVW and cardiac cycles on the assumption that the D-wave starts simultaneously with the closure of the aortic valve (AV). We then compared Delta A and Delta B in the control and fetal growth restriction (FGR) groups. In the control group of healthy fetuses, Delta A was strikingly shorter than Delta B. On the other hand, in all FGR cases, no difference was observed. The acceleration of the D-wave is suggested to be generated by the opening of the MV under normal fetal hemodynamics, whereas it precedes the opening of the MV in FGR. Our results indicate that the time interval of DV analysis might be a more informative parameter than the analysis of cardiac cycles. Fetal growth restriction (FGR) caused by placental dysfunction is a challenging disease, and the appropriate timing of delivery in FGR cases is still controversial because it requires a balance between the risks of prolonged exposure to hypoxemia, which may lead to fetal demise, and the risks of prematurity to adapt to dramatic changes in respiratory and cardiocirculatory circumstances ,2,3,4. IIn the progression of fetal deterioration, a Doppler evaluation of the fetal venous system, including ductus venosus (DV), plays a pivotal role in close fetal monitoring ,6. DV-PITo elucidate these advantages of DV-FVW assessment, we aimed to conduct a meticulous investigation regarding the relation between the time intervals of DV-FVWs and fetal cardiac cycles, based on the clinical question as to whether the D-wave is generated at the timing of the opening of the atrioventricular valve.A cross-sectional study was performed on 60 normal fetuses, as a control group, from 25 to 33 gestational weeks at Osaka Metropolitan University Hospital, from December 2020 to February 2022. The fetuses showed normal morphology, and estimated fetal weights were within ±1.5 SD of the normal mean, according to local reference ranges, in all cases .n = 23) were also retrospectively analyzed at both Osaka Metropolitan University Hospital and Osaka City General Hospital from April 2011 to November 2021. FGR was defined as an estimated fetal weight (EFW) < −2.0 SD of the local reference range and with an elevated umbilical artery PI > 95th percentile of the reference range [FGR fetuses  and just above the mitral valve (MV)  and (iii) and that between (ii) and (iv); Delta B was defined the sum of each absolute value of the difference between (i) and (v) and that between (ii) and (vi).p < 0.05 was considered statistically significant.We compared Delta A and Delta B in the control and FGR groups. We also divided both groups into two sub-groups at examination: at ≤ 28 + 6 gestational age (GA) and >28 + 6 GA. We used the SPSS statistics version 20 for statistical analysis . Comparisons between the control and FGR groups were performed using the Mann–Whitney U-test, and Out of the 45 FGR cases, 3 cases were excluded during the study period: 1 case had a ventricular septal defect that was diagnosed postnatally, 1 showed fetal tachycardia, and 1 case resulted in fetal demise. All parameters were obtained from 23 of the total FGR cases. The decision for delivery was made based on a non-reassuring fetal status judged by cardiotocography, and all FGR cases were delivered by cesarean section. Sixteen cases in the FGR group were included from a previous study . Table 1The measurements of cardiac parameters in the control and FGR groups are shown in The present finding that Delta A was apparently shorter than Delta B in normal fetuses suggests that the acceleration of the D-wave is generated simultaneously by the opening of the MV under normal fetal hemodynamics. In contrast, in the FGR group, no difference was observed between Delta A and Delta B, and this result suggests that the acceleration of the D-wave starts before the opening of the MV. Furthermore, in contrast, Delta A was significantly increased in those severe FGR cases which necessitated the early termination of pregnancy.We previously demonstrated the systolic/diastolic time ratios of DV-FVWs and cardiac cycles, using similar methods as those in the FGR study, and the most significant decrease in systolic/diastolic ratios was observed in the DV-FVWs in severe FGR cases . FurtherTo mention, theoretically, ventricular relaxation and high atrial pressure lead to the opening of the MV, thus accelerating venous forward velocities in the D-wave ,14, and Based on the findings in the present study, representative alterations in Doppler measurements of DV-FVWs and cardiac cycles are shown in Efforts for the understanding of complexities in multifactor-derived DV-FVWs have been undertaken by some investigators and from other perspectives. The nadir between the S-wave and the D-wave, the starting point of the acceleration of the D-wave, has been investigated via Doppler examination using blood flow velocity. Turan et al. studied the blood flow patterns of the DV-FVWs in fetuses at risk for cardiac dysfunction and speculated that the velocity of the nadir between the S-wave and the D-wave (they named this trough the ‘v-wave’) reflects ventricular relaxation and holodiastolic filling . Later, The limitations of the present study are that the data were retrospectively obtained from FGR fetuses and that it was not possible methodologically to obtain precise, simultaneous measurements of both DV-FVWs and cardiac parameters. Furthermore, the low statistical power might have had some effects on the results of the comparison of cardiac parameters such as ICT, ET, IRT, and MPI. The present study also lacks any speculation as for the pathogenesis of preeclampsia, which has substantial effects on fetal growth and placental function ,23. HoweWe revealed that the acceleration of the D-wave may be generated simultaneously with the opening of the MV in physiological circulation, while, in contrast, the acceleration of the D-wave will start during the isovolumetric relaxation period in severely growth-restricted fetuses, thus suggesting that the alteration in time intervals of DV-FVWs will reflect not only cardiac cycles, but also pathophysiological systemic changes in FGR cases. We believe that the time interval analysis of DV-FVWs may provide a more comprehensive insight into the pathophysiological conditions of severe FGR fetuses.


Corynebacterium glutamicum is a bacterium widely employed in the industrial production of amino acids as well as a broad range of other biotechnological products. The present study describes the characterization of C. glutamicum proteoforms, and their post-translational modifications (PTMs) employing top-down proteomics. Despite previous evidence of PTMs having roles in the regulation of C. glutamicum metabolism, this is the first top-down proteome analysis of this organism. We identified 1125 proteoforms from 273 proteins, with 60% of proteins presenting at least one mass shift, suggesting the presence of PTMs, including several acetylated, oxidized and formylated proteoforms. Furthermore, proteins relevant to amino acid production, protein secretion, and oxidative stress were identified with mass shifts suggesting the presence of uncharacterized PTMs and proteoforms that may affect biotechnologically relevant processes in this industrial workhorse. For instance, the membrane proteins mepB and SecG were identified as a cleaved and a formylated proteoform, respectively. While in the central metabolism, OdhI was identified as two proteoforms with potential biological relevance: a cleaved proteoform and a proteoform with PTMs corresponding to a 70 Da mass shift. Corynebacterium glutamicum is an industrial workhorse capable of producing a broad range of biomolecules from a large pool of substrates1 and is currently the best option for production of L-alpha amino acids2. Amino acid production represents a multi-billion dollar market3, with an annual production of approximately 10 million tons worldwide2, with l-glutamate and l-lysine corresponding to 3.3 million tons/year and 2.2 million tons/year, respectively2. Besides amino acid production, C. glutamicum is also utilized in other biotechnological processes, such as the production of heterologous proteins4, organic acids5, carotenoids, isobutanol6, polymers7, and more recently, its potential in bioremediation processes has been investigated8.The bacterium 9. In C. glutamicum, the importance of PTMs was investigated previously revealing the crucial effect of oxoglutarate dehydrogenase inhibitor (OdhI) phosphorylation in the production of l-glutamate11. More recently, mass spectrometry proteomics of C. glutamicum showed the influence of l-glutamate-producing conditions on the succinylation and acetylation of C. glutamicum metabolic proteins, including OdhI12.Recent developments in proteomics have demonstrated the importance of post-translational modifications (PTMs) in several species of bacteria13. As a consequence of protease digestion, the information from several protein regions can be lost in bottom-up approaches, including PTMs. Furthermore, in this approach protein identity is inferred based on peptide identification, and this may cause ambiguity in cases in which peptides may belong to more than one protein13. In contrast, the top-down approach allows the identification of the full sequence of proteins. Moreover, analysis of intact forms of proteins permits the identification of proteoforms, defined as different forms of proteins derived from the same gene14. Such proteoforms can be generated by amino acid residue substitution, proteolytic cleavage, alternative splicing, and several types of PTMs15. Furthermore, different proteoforms of the same protein can have different function and affect biological processes of an organism16. The power of top-down proteomics has been applied to other bacteria17. For instance, top-down proteomics of Escherichia coli and the identification of proteoforms has been performed18. Despite the evidence of the relevance of PTMs in C. glutamicum metabolism, no top-down proteomic studies have been published to date. Here we carried out the first top-down proteomics analysis of C. glutamicum, using a precursor tolerant approach to identify and reveal PTMs and proteoforms.There are currently two main proteomics approaches that are referred to as bottom-up and top-down. Briefly, in the bottom-up approach proteins are cleaved by a protease  and the subsequent peptides are analyzed by mass spectrometry, whilst in the top-down procedure, proteins are examined in their intact forms19.As expected, a wide range of proteoform molecular masses could be seen in the pre-fractionated intracellular proteins, but GELFrEE was efficient in separating large proteoforms from smaller forms Fig. A. FractiC. glutamicum generated 5127 proteoform spectrum matches (PrSMs), providing the identification of 1125 different proteoforms related to 273 different proteins , −18 Da , and 32 Da . Interestingly, the proportion of E. coli identified peptide spectrum matches (PSMs) with -18 Da (3.8%) and 32 Da (0.8%) Δm were very similar to those found in this study. In contrast, the proportion of PSMs identified with 16 Da (24%) Δm in E. coli was greater than in C. glutamicum. However, it is worth mentioning that the high number of PrSMs identified with 15 Da Δm (6.11% of modified PrSMs) may be caused by miss identification of the 16 Da Δm, as will be further discussed in the next topics.Some of the most frequent Δm identified here were also reported as highly present in Proteins identified with Δm were analyzed using the String Cytoscape app, after clustering proteins according to their interaction nodes, each cluster was submitted to overrepresentation analysis and the most representative terms were related to different gene ontology terms such as protein-containing complexes, electron transfer activity and CYTH domain , pyrimidine metabolism, transmembrane helix, biosynthesis of secondary metabolites and thioredoxin domain. Moreover, eight ribosomal PrSMs presented two Δm, indicating the presence of at least two concomitantly occurring PTMs , and persulfide . The 28 Da mass shift may also correspond to di-methylation . Despite being difficult to differentiate between formylation and di-methylation, the higher number of Δm close to 27.99 Da and 27.98 Da . The presence of SDS artifacts was also reported in an intact proteomic analysis of E. coli, and suggested to be caused by incomplete removal of the detergent25. Regarding the −18 Da Δm, it may result from dehydration events and was predominantly identified in serine residues  represented by the blue lines in the protein sequence is shown, and it can be seen that the mass shift is localized to a very narrowed region of three amino acid residues, due to the several b-ions contained in this region.Secretion system protein (SecG) Fig. A and lar27. Both the SecG and MscL formylated PrSMs mentioned above were identified with the presence of N-terminal methionine. This suggests a possible mechanism of degradation of membrane proteins, including some with promising biotechnological applications. For example, the SecG protein is part of the Sec protein export pathway. There is a growing interest in the capacity of C. glutamicum to express and secrete heterologous proteins of biotechnological interest28. Furthermore, MscL and MscS are mechanosensitive channels, known for reacting to osmotic stress. Another mechanosensitive channel of C. glutamicum  plays a major role in l-glutamate efflux29.The 28 Da mass shift near the protein N-terminus of these proteins suggests the presence of N-terminal formylation  in the identified proteoforms. Recent evidence has suggested the N-terminal formylation of methionine as a signal for protein degradation. This mechanism has been hypothesized as a quality control of protein translation in bacteria30. Furthermore, according to pfam (https://pfam.xfam.org/), mepB has a domain belonging to the M23 metallopeptidase family (MEROPS) [130–226]. A well characterized member of MEROPS is the LytM protein from Staphylococcus aureus, a metallopeptidase involved in autolysis31. It was demonstrated that cleavage in the N-terminal chain causes its peptidase activity to be activated32. Moreover, MEROPS proteins have specificity to peptidoglycan polyglycine regions, some with suggested cell wall metabolism activity33. In agreement, the C. glutamicum mepB gene was described as part of the MtrAB regulon, a two component system implicated in osmoregulation and cell wall metabolism control34. Although it is not clear whether mepB is secreted or membrane bound, it has been suggested that its activity would occur extracitoplasmically30. Considering this, C. glutamicum mepB may have an important role in cell wall metabolism and/or heterologous protein secretion integrity, and its activity is likely regulated by cleavage of its N-terminal.Top-down proteomics is efficient in identifying cleaved proteoforms. For example, mepB, a membrane protein related to the metalloendopeptidase Q8NS24) was identified by a portion of its sequence with precursor mass of 14.464 kDa. This mass corresponds to the loss of 97 amino acid residues from its N-terminal region  cycle is an important step in l-glutamate production by C. glutamicum. It is well established that the decrease of 2-oxoglutarate dehydrogenase complex (ODHC) activity occurs in conditions that induce l-glutamate production37. Furthermore, ODHC activity was found to be regulated by the phosphorylation status of oxoglutarate dehydrogenase inhibitor . Thus, the phosphorylated proteoform of OdhI is unable to interact with ODHC, whilst, unphosphorylated OdhI interacts with ODHC inhibiting 2-oxoglutarate conversion to succinyl-CoA11  and dehydration (Δm = −18 Da). Conversely, two proteoforms were identified with potential biological relevance: one of them with N-terminal cleavage of three residues and another one with Δm of 70 Da . In the present study, the exact site of the modification resulting in the 70 Da mass shift in OdhI could not be identified due to the complexity of the spectra and limited fragmentation by MS/MS. Therefore, other modifications such as 5 methyl group additions (14 Da), and acetylation (42 Da) followed by formylation (28 Da) could not be excluded. As aforementioned, ODHC inactivation arises from the interaction of unphosphorylated OdhI and the OdhA subunit of ODHC. The T14 OdhI phosphorylation inhibits this interaction, resulting in ODHC activation10. OdhI has a fork-head associated (FHA) domain responsible for the recognition of a phosphothreonine residue. Furthermore, this domain is the region that interacts with the OdhA subunit of ODHC, resulting in its inactivation. Moreover, it was demonstrated that the phosphorylation of the OdhI threonine residue causes drastic changes in its conformation, leading to an auto inhibition of its function, consequently activating ODHC39. More recently, it was reported that K142 succinylation also affects the OdhI–ODHC interaction, hampering the inhibition of ODHC with impacts on C. glutamicuml-glutamate production40.Recently, the 70 Da mass shift was identified in the GM-CSF heterologous expressed protein in 40, we investigated such a modification in addition to N-terminal formylation (27.9949 Da + 42.0106 Da) as a possible cause of production of the 70 Da OdhI proteoform. However, these modifications resulted in a considerable loss of matched fragment peaks (data not shown). Therefore, it is improbable that this was the source of such a mass shift. Moreover, the suggested region for the 70 Da Δm is near to the known T14 phosphorylation site of OdhI, consequently raising the question if it could affect its interaction with ODHC. OdhI phosphorylation at T14 is mainly catalyzed by C. glutamicum PknG10. Considering the recognition capacity of the OdhI FHA domain of its phosphothreonine and the close location of this phosphorylation site with the 70 Da Δm proteoform, this modification may affect OdhI inhibition of ODHC, resulting in its activation or inactivation .Another protein with relevance to the glutamate production process is the heavy-metal-associated domain (HMA) containing protein . Its corresponding transcript was identified as up-regulated in a series of glutamate overproduction conditions, however its function in this process remains unclear42. To clarify C. glutamicum HMADP function, the Q8NL68 protein sequence was blasted against the UniprotKB reference proteomes plus Swiss-Prot database with default parameters in Uniprot, resulting in similarity to copper chaperone and heavy metal transport/detoxification proteins  and lysine (K47) near C51. Moreover, the C51 is the resolving residue and was suggested to be very important in the catalytic process of this peroxiredoxin46.Two oxidations of cysteines result in the formation of sulfinic acid, which is an irreversible modification and signal for protein degradation47. Therefore, there is a good chance that the peroxiredoxin protein of C. glutamicum undergoes oxidative modifications which could regulate its activity and integrity. Furthermore, the suggested modification by a ONE peroxidation suggests a possible role of this protein in cell membrane repair.Less common Δm values were observed in a peroxiredoxin (Q8NMS6). One proteoform with a mass shift of 154 Da was identified and N-terminal acetylation was also detected , described as thiol-disulfide isomerase and thioredoxins, was identified by two proteoforms, both with N-terminal cleavage of 26 amino acid residues. Interestingly, one proteoform was identified with approximately −2 Da mass shift in a region near two cysteines  and pre-culture was performed overnight in tryptic soy broth under 170 rpm agitation at 30 °C. Afterwards, the pre-culture was used to inoculate three flasks containing CGXII media 49 [start optical density (OD) = 1.0]. The samples were grown for 36 h under 170 rpm agitation at 30 °C. Growth measurements were assessed by OD600 using a spectrophotometer CLARIOstar .C. glutamicum cultures were harvested and centrifuged at 12,000×g for 10 min at 21 °C. Pellets were washed with CGXII medium minus glucose, by resuspending the pellet and centrifuging at 12,000×g for 10 min at 21 °C. Cells were then resuspended in lysis buffer , followed by physical disruption through maceration in liquid nitrogen. After maceration, the solution was centrifuged at 20,000×g for 15 min at 21 °C to separate insoluble particles, and the supernatant with soluble intracellular proteins was stored at −80 °C until further use. Proteins were then quantified by Qubit™ (Invitrogen). A pooled sample was prepared using equal protein quantities of each replicate and 500 µg of the pooled sample was fractionated by Gel-eluted Liquid Fraction Entrapment Electrophoresis—GELFrEE50, using a homemade cartridge with 12% resolving gel and 4% stacking gel. First, the sample was diluted in sample buffer 2× , then it was submitted to electrophoresis using a running buffer  and a constant current of 10 mA. Fractions were collected according to time (in minutes) after bromophenol blue elution (0 min). Subsequent collections were as follows: 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 45, 60, 75, 90, and 120 min. The molecular mass range of proteins from each fraction was assessed by SDS-PAGE51. GELFrEE fractions were then submitted to methanol/chloroform/water precipitation for SDS removal52. Briefly, four volumes of methanol were added to the fractions, followed by addition of 1 volume of chloroform and 3 volumes of water, with 30 s of vortexing subsequent to the inclusion of each solution. After water addition and vortex, the solutions were submitted to centrifugation at 20,000×g for 10 min at 21 °C, resulting in the formation of two layers, with proteins floating between them. Then, the superior layer was removed, with care to prevent disturbing the protein pellet, and 3 volumes of methanol were added, followed by gentle mixing and centrifugation at 20,000×g for 10 min at 21 °C. Subsequently, the supernatant was discarded, followed by a second wash with 3 volumes of methanol as described before, and the resulting pellet was dried in a laminar flow bio-hood. After drying, pellets were resuspended in 5% acetonitrile and 0.1% formic acid and stored in −80 °C before LC–MS/MS.m/z, using a dynamic exclusion duration of 30 s. Source-induced dissociation (SID) was used with 15 eV in MS1 and the first MS2, whilst for the second most intense ion, it was set to 35 eV. Resolutions were 240,000 and 120,000 for MS1 and MS2, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE53 partner repository with the dataset identifier PXD038038.Three technical replicates of GELFrEE fractions 0, 1, 2, 3, 4, 5, 7, 9 and 11 were submitted to top-down analysis on a nano-UHPLC Dionex Ultimate 3000 system coupled to Orbitrap Elite™ mass spectrometer  (LC–MS/MS). Analytical  and trap  columns packed with PLRPS 1000 A, 5 µM  were used for reverse phase separation. In the analytical column a tip of approximately 1 cm was pulled using a P-2000 instrument , to be used as emitter in the mass spectrometer. Both columns were kept at room temperature, controlled at approximately 22 °C. Samples were loaded using a flowrate of 3 µL/min for 10 min, under the isocratic condition of 5% Acetonitrile and 0.1% formic acid, then a gradient under flow of 0.230 µL/min was used to elute proteoforms from the column for MS analysis. The gradient was composed of solutions A (formic acid 0.1%) and B  and was created by: 5% B (0–10 min), 20% B (10–55 min), 55% B (55–60 min), 85% B (60–80 min) and 5% B (80–90 min). Acquisitions were made during the 90 min gradient in positive mode, and the top-2 most abundant ions were fragmented by stepped high-energy collisional dissociation (HCD) applying normalized collision energy of 25, using two steps, with collision energy width of 10, resulting in two separated fragmentations of ions with normalized collision energy of 20 and 30, and analysis of all resulting fragments together in the Orbitrap. Isolation width of precursors was 25 54. Identification and characterization of proteoforms were done using the TopPic suite software tool, version 1.455. Briefly, spectra were deconvoluted using TopFD with default parameters followed by identifications against a C. glutamicum ATCC 13032 reference proteome (11-2020), obtained from UniProt (https://www.uniprot.org/). Two mass shifts between −500 Da and 500 Da were allowed in the identification and the N-terminal forms were set as “NONE” (No modifications), “NME” , “NME-ACETYLATION” , and “M-ACETYLATION” . A false discovery rate strategy was adopted with a decoy database, resulting in the identification of only proteoforms with FDR below 1% at the proteoform spectrum matches (PrSMs) level. Proteoform characterization and annotation were followed by visual interpretation of TopMSV21 files. Detected proteins’ MW evaluation and retention times were performed using VisioProt-MS56.Thermo raw files were converted to MzML format using the MSConvert tool57 and ggplot258. Briefly, the number of identified mass shifts (Δm) was counted and the frequency of the most common Δm were defined based on rounded values. Then, the number of amino acid residues modified by each Δm was defined. Functional annotation and overrepresentation analysis were performed using the String Cytoscape app60, and DAVID61. Visual interpretation of DAVID overrepresented terms was also carried out using R computer language through the creation of a bubble plot with the tools present in ggplot2.Proteoforms identification data were analyzed in R 4.0.2 (R Core Team 2020) using the packages tidyverseSupplementary Figures.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.


Depression has major consequences for the entire family, among them emotional distress, disrupted daily routine and social damage caused by negative stigmas. The aim of this study was to explore the retrospective experiences of young adults who lived with a sibling with depression while they were adolescents. The present study adopted a qualitative-phenomenological approach. The research participants were recruited via purposive sampling on social networks across Israel from May to September 2022. Semi-structured interviews were conducted with 21 participants aged 18–29 who lived with a sibling with depression during their adolescence. Data collection continued until saturation of concepts was reached. The results underwent thematic analysis. Three themes emerged from the qualitative analyses: 1) “I felt like I was living in a minefield”: Adolescence while living with a sibling with depression; 2) “One step forward and two steps back”: Siblings’ coping strategies; 3) “My parents were not there for me when I needed them”: Participants’ experiences with their parents during their adolescence. The research findings indicate that adolescents who grew up with a sibling affected by depression had to cope with an acute family crisis, whose serious ramifications required emotional and social support. Mental health professionals and counselors working within educational institutions and the broader community should provide support and intervention for adolescents who have siblings struggling with depression. This intervention may take the form of individual or group therapy that aims to foster a sense of belonging and help affected individuals. Creating a supportive environment that meets the needs of the affected siblings is also crucial in addressing this issue effectively. Depression is a common and serious psychiatric disorder that finds expression in depressed mood, loss of pleasure and interest, feelings of emptiness, sleep disruptions, and loss of appetite . DepressNot only does depression affect the individual sufferer. It also affects the entire family. Young adults who lived with a sibling with depression are themselves at increased risk for distress and mental illness –10. ReseLiving in the same house as a sibling with depression is liable to have a negative impact on an individual’s emotional well-being, produce feelings of distress and anxiety, and cause the individual to perceive of the sibling as a burden –10. The Adolescents living with a sibling with depression are themselves at high risk of developing depression. There are several explanations for this, including genetics, similar life experiences, parental resources devoted to caring for the sibling with depression, and family climate . BarnettDespite its resemblance to adolescence, the period of emerging adulthood is marked by unique features that define it as a separate developmental state. Unlike adolescents, young adults have already reached physical and sexual maturity. They are legally responsible for their actions and can therefore decide what to do, where to work, and where to live. Yet unlike mature adults, they are not yet stable financially, personally, or professionally . SeveralMany studies have examined the experiences of adolescents with depression and the perspectives of their parents , 19. YetThe study was conducted according to the qualitative phenomenological approach, which attempts to obtain an in-depth understanding of the studied phenomenon by entering the world and experiences of the participants . The appTwenty-one young adults who lived with a sibling with depression while they were adolescents participated in the study. The participants, who ranged in age from 18–29 years old, all grew up in the same house as a sibling with depression . Most of the participants were single (90.47%) and none had children of their own. Inclusion criteria were: 1) lived in the same house with a brother or sister with depression when they were adolescents; 2) between the ages of 18 and 29 years old; 3) lived at home during adolescence (not at boarding schools); 4) biological siblings from the same two parents . These inclusion criteria were used to create a homogeneous group and reduce intervening variables. Exclusion criteria were: 1) brother or sister with a history of psychosis; 2) participants currently exhibiting suicidal intent or severe depression because of concerns about the adverse effects of the interview process; 3) participants who identified themselves as having depression .The research participants were recruited via purposive sampling on social networks  across Israel. Young adults who grew up with siblings with depression were examined. Participants responded to a letter posted on social media by sending an email with their details. After receiving a comprehensive explanation about the general research aims, interviewees signed an informed consent form. For the interview, they also were required to submit a medical record documenting their sibling’s history of depression during their own period of adolescence. Three participants who did not submit a medical document testifying to their sibling’s depression were eliminated from the study.The interviewers (IL and ML) were both females. IL is a psychotherapist (PhD) and ML holds a master’s degree in educational counselling. Both are experienced in conducting qualitative research. Prior to conducting the interviews, the interviewers reflected on the identities, social locations, assumptions, and life experiences they brought to the research endeavor and contemplated their interactions with the interviewees. The interviews took place between May and September 2022 and were conducted via the Zoom platform to minimize personal contact during the COVID-19 pandemic. The interviews lasted about one hour, on average.All interviews were recorded and transcribed verbatim. The interviews were conducted in Hebrew, and the transcripts were translated into English. Each translation was verified by two native speakers, one of whom is a professional translator. Once theoretical saturation was reached , no additional interviews were conducted.The qualitative data in this study were collected by means of in-depth, semi-structured interviews. The interviews were conducted based on an interview guide that included significant key areas but were flexible enough to allow a dialogue to develop between the interviewer and the interviewee, as well as to facilitate meaningful self-expression  Table 2Table 2.audio recordings. Phase six entailed describing the themes and the underlying story describing how the phenomenon was experienced and adding illustrative quotations [The data collected in the interviews were analyzed thematically in six phenomenological phases . In Phasotations .This study was approved by the Ethical Review Board of the Oranim College Research Ethics Committee, Israel) No. 117/2022) and conformed to the Declaration of Helsinki . InformeAnalysis of the interviews yielded three themes that shed light on the experiences of the research participants as adolescents growing up with a sibling with depression .The research participants described the period before their sibling was diagnosed with depression as “foggy”. During this period, they felt a lack of clarity and confusion regarding their sibling’s condition. Some of the participants mentioned their difficulty in understanding their sibling’s emotional distress. Often, they accused their sibling of disrespecting their parents, acting like a crybaby, exaggerating things or trying to attract attention. Others felt that their sibling was suffering but they had no way to help. The research participants described their complicated feelings during that period, particularly their anger, frustration, helplessness, and loneliness. After the depression diagnosis, the participants stated they were able to identify depressive symptoms in their sibling, such as social problems, diminished sharing and fewer emotional discussions, a tendency toward seclusion at home, suicidal thoughts and self-harm, profound sadness or loss of meaning in life. In most cases, the depressive symptoms identified after the diagnosis worsened with time, and the research participants’ sense of distress increased accordingly.When my sister came home from the army, all she did was cry, repeatedly state how bad things were for her, tell us that the other girls in the course did this or that to her and she couldn’t find any friends there…. For me this was just that she is a drama queen, she exaggerates everything, that’s how it started… at some point I really began to be angry with her because it was impossible to talk to her .Some of the participants felt that their sibling’s illness was an existential state of emergency that generated a storm at home. Participants described a situation in which they, as adolescents, experienced ups and downs in their lives but were forced to repress these due to the centrality of their sibling’s illness. Batia, 20 years old, describes her frustration because her brother’s pain did not leave any room for the rest of the family to cope with their problems. Indeed, a hierarchy of challenges emerged, and she felt she was at the bottom of the ladder:I felt like I was living in the middle of a huge minefield. I remember times when I felt I was not allowed to get angry, not allowed to feel insulted, not allowed to feel anything, because other people have things so much worse. I remember when my brother broke up with his last serious partner, and he just didn’t come home, and it was as if…. no one else was allowed to have any problems in life because his situation was so difficult .Within the storm and huge family crisis, many of the participants felt they had to remain constantly on alert. They felt they had to enlist themselves to help with the family crisis, even at the price of self-sacrifice and self-nullification. These participants put aside their personal commitments and desires, including social connections, hobbies, or attention from their parents. Doing this exacted a steep emotional price from them, and they experienced negative emotions such as anxiety, stress, and sadness. Yet nevertheless, the research participants felt they had no choice but to do their part.When my sister was receiving outpatient care, she was at home all the time and needed supervision all the time. Someone always had to be with her and keep her occupied. On many occasions she would lock herself in the bathroom or in her room and my parents would cry … that was always so chaotic, with everyone crying and screaming because they were convinced she would never come out. As the person who was closest in age to her, I was always the one who had to deal with her, take her places, stay with her .A significant number of research participants witnessed behaviors such as self-harm, verbal and physical violence directed towards them or their parents, and hospitalization in psychiatric wards. These situations led to intense fear, worry, stress, anxiety, instability, and insecurity within their own homes. The research participants did not know how to handle the reactions of their siblings, who were required to perform various actions even when they were struggling with depression. The unexpected responses surprised the research participants, causing them panic and shock.During a youth movement trip, I began telling my sister how I felt about her condition, but she showed me her cuts, silencing me. I felt hurt and scared by her actions because this was the first time I had witnessed her self-harming behaviour. I was upset but tried to reassure her by asking her whether she would hurt herself again .Participants in the study viewed their sibling’s illness as an existential crisis, creating turmoil at home and resulting in feelings of emotional distress, frustration, and loneliness. They described their adolescence as a time of suppressed emotions due to the importance of their sibling’s illness. Amidst this family turbulence, many felt a constant need to be on alert and were willing to put the family’s needs above their own, even at the cost of personal sacrifice.In the midst of the storm and the great rift, many of the siblings felt they needed to remain constantly vigilant and ready to act. They felt they had to mobilize themselves to help the family, even at the cost of sacrificing their personal needs. These participants reported that their sibling’s illness "came at their expense”. They described pushing aside personal obligations and wishes, including social relationships, hobbies, or attention from their parents. Their sibling’s condition exacted many personal costs from them, including damaging their social relationships and eliciting negative feelings such as sadness, stress, pressure, and anger. Still, the research participants felt they had no choice but to carry out their obligations to the family.During the periods my sister was treated as an outpatient, she was always at home and always had to be supervised. I always had to be with her and keep her occupied. On many occasions she would lock herself up, lock herself in the shower, or lock herself in the room and my parents would cry…. Because I’m the closest to her in age, I tried to be with her because she always really trusted me and was very attached to me. So I always kept her occupied and stayed with her. Many times I would take her to the beach or I would just stay with her at home, trying to find things we had in common .The participants stated that their relationship with their siblings during that period was not reciprocal but rather focused on the sibling with depression. They took care of their sibling, devoted emotional resources and time, tried to engage the sibling in conversation, and showed concern for or physically protected their sibling. In contrast, the affected sibling was engrossed in his/her own world and was not emotionally available to pay attention to what was happening in the world of the research participants:I was totally there. I was fully engrossed, in action mode. I didn’t ask too many questions. Apparently I understood there really wasn’t anyone to talk to. It was an emergency situation… I often sat beside her all night long, I engaged in guided imagery, lots of discussions, I was totally drawn in and devoted myself totally, and afterwards I was totally drained .Other participants, in contrast, reported that their sibling’s difficulties frightened them to the point of avoidance. As young adults they recognized their sibling’s condition made them feel unwanted, rejected and sometimes even in the way at home. They expressed fears that society would assign a negative label to them because of their sibling’s illness. Hence they spent many hours at the homes of friends and relatives and devoted lots of time to demanding sports activities or hobbies. They defined their relationship with their sibling as “nonexistent”, possibly due to repression resulting from having trouble seeing their sibling in this situation. Despite the lack of communication between the siblings, the research participants reported feelings of guilt, anger, fear, helplessness, and distress when thinking about their sibling.At home they saw me as a guest, someone who comes home on weekends and holidays. I also wasn’t so involved in what was going on at home, certainly not in the lives of my siblings. As a child it was very difficult to communicate with my sibling on a certain level, I didn’t have the tools for that .Many participants felt the need to prioritize their family’s well-being over their own personal needs, sacrificing social relationships and hobbies. The illness of their sibling often led to strained social connections and feelings of sorrow and stress. Nevertheless, they saw their responsibilities towards their family as inescapable. On the other hand, some participants reacted to their sibling’s challenges with fear and avoidance. Internal feelings of rejection and concerns about societal stigmatization drove them to find solace with friends or immerse themselves in intense hobbies.The young adult research participants reported that when they were adolescents, their sibling’s depression was a factor in how close they felt to their parents, whether they were able to share experiences with their parents, and whether they could rely on their parents for support. Some of the participants described how they lost the positive and significant relationship they had with their parents before their sibling’s illness. Nili, age 28, reported that she had a good relationship with her parents before the onset of her sister’s illness. Yet after her sister began experiencing symptoms of depression, Nili said that her parents focused all their energy on her sister, leaving her feeling neglected. She recalled that “they no longer seemed really interested in me”.According to Ido, before the onset of his sister’s depression he had many problems in elementary school and relied on his parents to help him solve them. When his sister’s depression began to emerge and as it escalated, Ido stopped “causing problems” or asking for his parents’ help. In retrospect he described himself during high school as “a remote and easy kid that my parents did not need to deal with very much”. The feeling that the parents are not available or attentive to their needs came up in other interviews as well. For most of the participants, their relationship with their parents was marked by feelings of rejection, disappointment, and abandonment. Most noted that they yearned for a deeper, more inclusive, and more meaningful relationship with their parents. The research participants did not want to disappoint or make things any harder for their parents, who already were so sad and in so much pain, so they tried to please them and win their love. They tried to be “perfect children” and to avoid behavior or situations that might make their parents uncomfortable. Sarah commented on the need to meet her parents’ expectations and maintain an external image of being problem-free, even though inside she was struggling with many difficulties. She behaved this way to satisfy her parents:I excel and am wonderful and do everything I should, so that on the outside there is no reason to be ashamed of me, as opposed to… A brother with depression is something to hide, but about me they can brag all the time .Some of the participants reported that they tried to assume parental roles at home, such as watching their sibling, taking him/her to treatments, or doing all sorts of chores to make things easier for their parents. This desire to please their parents was typical of many of the research participants and caused them fatigue and burnout:On the weekends I would come home from university and spend lots of time there doing things my mother never had time for, like cleaning the house, taking the dog for a walk, doing laundry, anything that my mother could never find time for because she was busy with my sister 24/4 .Participants also had to cope with the challenge posed by their parents’ request that they conceal their sibling’s condition from people outside the family. Sometimes the parents directly asked them “to hide”, while in other cases the participants extrapolated this from their parents’ behavior. Hence, the participants were left alone to cope with difficult incidents and complex experiences, without being allowed to share their feelings and receive support from those around them. For many this led to isolation and sadness.They always told me not to tell anyone. I remember that my mother insisted that I keep this a secret, and also my father… It was also clear to me from their behavior and their language that this is what was expected of me. Today I understand how much this lack of communication, these scare tactics, influenced me .Many participants noted a deterioration in their positive relationships with their parents after their sibling’s illness. They felt rejected, disappointed, and abandoned. Many expressed a longing for a deeper connection with their parents and aimed to be the ’perfect child’ to please them, even amidst feelings of neglect. Additionally, they faced the challenge of keeping their sibling’s condition a secret, as requested by their parents.The effects of depression are not limited to the afflicted individual, for they also extend to the individual’s siblings. Young adults living in the same household with a sibling with depression are at increased risk of emotional distress, reduced social status, diminished self-esteem, and negative stigmatization of their family –10, 12. The participants in the current study reported feeling guilty as a result of their sibling’s behavior and inability to function, as well as anger, confusion, and instability. They described their adolescence as a period of constant tension, anger, and worry. The findings of this study are in line with the findings of previous studies describing the difficulties experienced by siblings living with a brother or sister with a chronic illness or mental health condition , 27, 28.Some of the participants in the current study reported that despite the difficult situation at home, they felt they must help the family, even if it meant relinquishing their own needs. Other participants, in contrast, chose to stay away and avoid the stormy atmosphere at home. Indeed, the participants adopted two main approaches to cope with this complex situation: merging or avoidance. Merging entails enlisting oneself to the benefit of the sibling with depression to the point of self-nullification, while avoidance involves cutting oneself off and distancing oneself from the sibling. The merging pattern helped the participants feel useful and valuable in helping cope with the distressful situation at home, but on the other hand it also had a negative impact on their personal lives.The findings of the present study demonstrate a consistent pattern reflecting the significance of the term "family burden" in characterizing the adverse impact on individuals who care for family members living with mental illness . Family In contrast, participants in the current study who adopted the avoidance pattern avoided communicating with their sibling with depression and sometimes even distanced themselves from the entire family in order not to be flooded emotionally. Participants who chose to cut themselves off from their sibling with depression expressed a fear that society would assign negative labels to them because of their sibling’s illness. The research literature contains abundant evidence showing that the individual with mental illness is not the only one who must cope with negative stigmas. Rather, a negative label is often affixed to the entire family in what is known as “stigma by association” , 43, sucIn our study, participants indicated that their relationship with their parents changed after their sibling became ill. The sibling’s difficulties made their parents less available to them and resulted in feelings of neglect, disappointment, loss, and pain. These findings are in line with other research evidence showing that parents’ lack of availability due to the illness of one of their children has a negative impact on the other children in the home , 28, 31.Moreover, the research participants reported having trouble witnessing their parents’ difficulties, which caused them deep pain and sorrow. They invested major efforts to bring their parents some degree of pleasure, to make things easier for them, and to spare them further pain. The research participants noted that they sought to please their parents in an attempt to compensate them. The findings of the current study reinforce previous findings indicating that quality of parental emotional support acts as a mechanism safeguarding the mental health of the siblings of those with mental health issues , 28, 44.Some of the features of the research participants’ relationships with their parents are reminiscent of the experience of losing someone close. This type of loss includes profound feelings of extinction, destruction, and loss. The parents and family members of someone with mental illness experience significant losses that engender grief . Such grThe strength of this study lies in the fact that it is a qualitative study focusing on participants who lived with a sibling with depression during their adolescence. The interview format allowed for a more detailed and in-depth investigation of the participants’ perceptions and perspectives, thus adding more information to previous findings regarding this phenomenon. This format led to the creation of a homogeneous group of individuals who lived with siblings with depression, in contrast to studies that examined different mental disorders or combined siblings and parents of the affected individual. Another strength lies in the age of the participants (18–29). The period of emerging adulthood has been the focus of very few studies in this context. Another major advantage of the qualitative approach is that the inquiry is broad, allowing the participants to raise issues that matter most to them. The current study sheds light on several important themes, such as the emotional effects experienced by the participants, their coping patterns that range from closeness to avoidance, and their longing for the relationship they had with their parents before their sibling’s illness emerged.This study also has several limitations. First, due to its qualitative nature, causal relationships could not be determined. Second, the sample comprised a small cohort of siblings in Israel, so that the results cannot be generalized to other population groups. Third, the data emerging from interviews conducted via Zoom may not be as rich as data emerging from face-to-face interviews. Nevertheless, face-to-face interviews were assessed as less beneficial because they required restrictions in the sampling process given the geographical spread of the participants. Fourth, this study focused on the siblings of people with depression. We created a homogeneous group using inclusion and exclusion criteria. At the same time, because the level of the siblings’ depression was not examined in the study, the intensity of the illness may have differed.Further research is needed to address the potential cultural, ethnic, gender, and age differences in the siblings’ experiences. The long-term experience of the research participants would be a valuable avenue to explore in the future.The findings of this study shed light on the experiences of adolescents who grew up with a sibling affected by depression. The participants felt confused, helpless, and anxious due to changes at home. Some described witnessing their sibling’s withdrawal, self-inflicted injuries, and turmoil at home. The participants described having to repress their own problems as teenagers and feeling lonely due to the unstable home situation. Young adults who lived with a sibling with depression during their adolescence reported that their sibling’s condition brought about changes in their routine and forced them to adjust their behavior to cope with the new situation. Some adopted merging behaviors and attempted to protect their sibling, while relinquishing their own personal desires. Others adopted avoidance coping patterns marked by evasion and escape and tried to conceal their sibling’s condition from others. After the sibling’s illness emerged, the participants distanced themselves from their parents. They felt their parents were no longer available for them and therefore avoided asking for help or sharing their negative experiences.The results of this study suggest that improving our understanding of adolescents who grew up with a sibling with depression may result in more positive and supportive relationships, highlighting the need to consider not only the patient with depression but also the siblings who are emotionally and behaviorally affected. Our findings indicate that intervention and policy approaches in educational settings and in the community are crucial in addressing these issues. Specifically, solutions are needed that can reduce the burdens on siblings by providing them more flexible and tolerant support. To build on the insights gained from the current study, future research should adopt a triangulation approach by conducting follow-up studies that examine the perspective of the sibling with depression as well as provide a more comprehensive understanding of their experiences. Additionally, considering various characteristics of the affected sibling and the entire family, such as age, gender, culture, and single parent status, could provide important context for understanding the impact of depression on siblings. Another recommendation for further research is to interview siblings at different ages and use various qualitative methods, such as diaries and videos, to gain a deeper understanding of their experiences. Lastly, gathering both quantitative and qualitative data at the same time may offer a more complete understanding of the impact of depression on siblings’ emotional well-being and coping patterns.


We have simulated BiCoO Multiferroic materials, which combine broken space inversion symmetry with time inversion symmetry, have become one of the fastest growing research topics . Two or BiCoOansition ,8,9. Sucaterials . By optiplitting . BiCoO3 factants . The Ne´3−xBiCoO3. Due to  texture ,15.On the other hand, the properties of thin films could be prominently affected by epitaxial strain derived from lattice mismatch between a thin film and the underlying substrate . In thisy, and We conducted first-principles calculations by the Vienna Ab initio Simulation Package (VASP) with projector augmented wave (PAW) pseudopotentials based on the density functional theory (DFT) ,18. The ous work ,22,23,24d NaNbO3 ,25,26.i and the corresponding displacement of atom i along the direction i runs over all the atoms in the unit cell. The structural symmetry of strained epitaxial phases are determined by the use of the FINDSYM program [The ferroelectric polarization is computed by means of Berry phase ,28. One  program .t < 1) is inconsistent with BaTiOThe perovskite BiCoOe factor ,(5)t=(rc radii) ,33, respAnalogous to the typical ABOeriments . Bi atomWe first calculate the evolution in the total energy with the in-plane lattice parameter for the low-energy states. The ground state is a bstrates  can provLet us now turn our attention to the phase transition from the ed BiFeO3, i.e., tansition ,38, can ubstrate . Moreovea are equal to 3.72, 4.03, 4.18, and 4.47 Å, respectively, which are redefined by the FINDSYM program.As the strain continues to increase, the y-axis.Note that the orientation of ferroelectric polarization tends to tilt away from the out-of-plane in the transition of At the same time, the pyramidal CoO5 in the otations ). With that Ref.  also repThe Nulations ,41 by coa) and (b) and presents a direct comparison between our theoretical predictions and the measured transition temperatures. Hence, we employ the conventional value of 4.8 eV for UThe calculated Neratures ,42. The We also solve the Hamiltonians to obtain TIn summary, we have used density functional theory calculations to investigate misfit strain-induced structural phase transition in epitaxial (001) BiCoO