Precision medicine is “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person.” Among many medical specialists involved in precision medicine, the pathologists play an important and key role in the implementation and development of molecular tests that are in the center of decision of many therapeutic choices. Besides many laboratory procedures directly involved in the molecular tests, is fundamental to guarantee that tissues and cells collected for analysis be managed correctly before the DNA/RNA extraction. In this paper we explore the pivotal and interconnected points that can influence molecular studies, such as pre-analytical issues ; diagnosis and material selection, including the calculation of nuclei neoplastic fraction. The standardization of sample processing and morphological control ensures the accuracy of the diagnosis. Tissue or cytological samples constitutes the main foundation for the determination of biomarkers and development of druggable targets. Pathology and precision oncology still have a long way to go in terms of research and clinical practice: improving the accuracy and dissemination of molecular tests, learning in molecular tumor boards for advanced disease, and knowledge about early disease. Precision medicine needs pathology to be precise. Pathology is evolving to meet patient needs and be a central driver of personalized healthcare. The main objective of personalized medicine is to identify patients who are candidates for specific treatments. The individualized approach has transformed the oncological therapeutic scenario  and progTraditionally, pathologists have been at the forefront of cancer diagnostics with knowledge of cancer biology, morphology and prognosis . In precPrecision oncology reveals an accelerated pace, with great financial investment and a rapidly growing pharmacological arsenal. The dynamic and changing environment has allowed gaps in the standardization and validation of molecular diagnostic procedures. Pathology plays an important role in the implementation and development of molecular tests—precision medicine needs pathology to be precise.in situ” based techniques; integrate the molecular data in the original diagnostic report (translation to the clinicians); and educational activity ; diagnosis and material selection, including the calculation of nuclei neoplastic fraction. The standardization of sample processing and morphological control ensures the accuracy of the molecular diagnosis.Suboptimal processing can alter morphological, immunohistochemical and molecular characteristics of histological and cytological samples . The magFor all samples, the primary pre-analytical factors whose thresholds somehow affect molecular results can be grouped in: cold ischemia, fixation, decalcification and time of paraffin block storage .Cold ischemia is the time that occurs between the removal and fixation of tissue and is especially problematic for large surgical specimens. It affects gene expression at RNA and protein levels and mutation analysis at DNA level, thus being a major factor in molecular pathology . The recThe fixative worldwide accepted for tissue sample preservation is neutral buffered formalin (NBF). NBF penetrates tissue at around 1 mm/h, and fixation will only start when penetration occurs. NBF should only be used 24 h after dilution to 4% w/v, in order to reduce the effect of polymerization, and guarantee a stable 4% concentration. For cytological samples, fixation is commonly achieved with alcoholic-based fixatives . As the Decalcification is frequently not mentioned in the pathology report, though it can severely affect molecular analysis. To preserve the integrity of the nucleic acids and proteins, areas suspected of neoplasia in bone pieces should be processed without decalcification whenever they can be isolated. For smaller specimens, as bone biopsies, it is recommended to use Ethylenediaminetetraacetic (EDTA) as a decalcifying agent .Regarding the duration of paraffin block storage, the following thresholds were demonstrated: DNA ≤5 years; RNA ≤1 year; Protein ≤25 years for IHC, < 10 years for platforms requiring protein extraction .During the microscopic examination the pathologist should correlate the clinical information with morphology and recognize if there is the need for molecular analysis. The use of tissue in small specimens should be rational, avoiding unnecessary sections and complementary studies . In some countries, as in our experience, molecular studies are done in central laboratories that received material from different hospitals. In this situation is advisable to have a dedicated molecular pathology that evaluates the available material before proceeding to any molecular study.The following criteria are relevant in this phase: 1) Slide/sample for molecular analysis must accurately represent the diagnosis of the pathological report; 2) Most representative slide/sample: show all the characteristics of the neoplasm such as morphological type and staging; 3) Avoid samples with extensive areas of tumor necrosis, inflammatory infiltrate and fibrosis; 4) It is recommended that the pathologist marks the area of the section containing neoplasia on the hematoxylin and eosin (H&E) slide at the time of diagnosis.To avoid gross sampling errors, the pathologist must directly certify that the block sent for analysis corresponds to the patient and the diagnosis. In patients with multiple samples, the most recent tissue should be used . It is aIn our molecular laboratory, a specific and validated protocol of cutting sections has been optimized and is currently in use. First we perform sections that will be used for extraction of nucleic acids—at 10 μm thickness, and only after we do a 3 μm section and stain with H&E for morphological evaluation. The quantity of extraction sections obtained varies according to the molecular assay and/or biomarker: nine sections for Next Generation Sequencing (NGS) panels requiring DNA and RNA isolation ; 4–6 sections for Sanger sequencing studies requiring only DNA isolation  four sections for real-time PCR based studies ; 3–4 sections for studies requiring only RNA isolation .The percentage of neoplastic cells present in the selected sample must be estimated for morphological control . Tissue Recommendations for morphological control: 1) The evaluation of the percentage of malignant cells corresponds to the percentage of malignant nuclei ; 2) The In estimating the percentage of viable malignant nuclei, regions with necrosis or inflammatory cells or desmoplastic stroma or mucus should be avoided and excluded for molecular analysis. If this is not possible, it is necessary to consider that these non-neoplastic nuclei are viable and dilute the percentage amount of malignant neoplastic DNA . Tumor hThe macrodissection is an important tumor cell enrichment technique, highlighting the critical role of the pathologist. This technique improving the accuracy of the molecular analysis, for direct sequencing or next generation sequencing . It is cSmall samples with limited tumor cell content (<30%) may permit morphological classification; however, the quantity of tumor tissue is not always sufficient for biomarker testing . The minIf samples have lower than acceptable levels of malignant nuclei or in the absence of viable tumour tissue, histological slides used for morphological diagnosis (H&E and immunostained slides) can also be an additional alternative, to extract material for molecular analysis. From our experience, we can extract DNA and RNA from previous H&E slides, while the use of previous immunostained slides is indicated only for DNA extraction. RNA obtained from previous immunostained slides, shows degradation and does not give viable results. The utilization of the archive slides must be reserved and discussed case by case. Furthermore, the scanning and digital archiving of the slides is mandatory to ensure medical-legal issues.When DNA is purified from a pure tumor cell population (100% tumor cellularity), the mutant allele frequency would be expected to be 50%. If a sample harbored 50% tumor cells and 50% non-tumor cells, the latter population harboring two copies of the wild type allele, the mutant allele frequency would be 25% . If in tIt is important to note that an intact diploid cell produces 6–7 pg of DNA and 10–30 pg of RNA. Input nucleic acid mass requirements for molecular testing are variable, with minimum recommendations ranging from of 1–10 ng , 50 ng, 100 ng, and 200 ng .Cytopathology samples ethanol-fixed, as smears and touch preparations, usually contain higher-quality nucleic acids than formalin-fixed, paraffin-embedded (FFPE) samples and are useful for molecular testing . For exaThe interobserver variability in estimating the percentage of neoplastic cells is 20%  and pathThe pathology molecular report should include the histopathological diagnosis, the estimated percentage of malignant cells present in the tissue used for DNA/RNA extraction, and the allele frequency. Diagnosis, classification, prognosis  and evenThe standardization of tissue sample handling at each step—sample processing and morphological control—ensures the accuracy of the diagnosis. Estimation of percent neoplastic cellularity can also affect care in other diagnostic settings, including assessment of copy number variation, chromosomal translocation, determination of residual cancer burden after neoadjuvant therapy, and other genomic aberrations that can be affected by contaminating normal DNA .Best practice guidelines aim to management tissue and support a complete molecular diagnosis, so that eligible patients may benefit from targeted therapy. A biomarker negative sample with a tumor cell content below the thresholds for analysis should be determined as inconclusive, requiring further assessment. Consequently, the proportion of cells may also inform the choice of molecular testing.Molecular therapy demonstrates remarkable response rates, making advances in reducing cancer mortality , althougIn this scenario, it is necessary to continue researching the complex interaction of tumor biology, microenvironment, and immune response; to disseminate molecular tests in clinical practice; to encourage molecular tumor board for advanced diseases; and to learn about early diseases. Pathology and oncology in precision medicine still have a long way to go in research and clinical practice.


The purpose of this study was to finetune a deep learning model, real-enhanced super-resolution generative adversarial network , and investigate its diagnostic value in calcified coronary plaques with the aim of suppressing blooming artifacts for the further improvement of coronary lumen assessment. We finetuned the Real-ESRGAN model and applied it to 50 patients with 184 calcified plaques detected at three main coronary arteries . Measurements of coronary stenosis were collected from original coronary computed tomography angiography (CCTA) and Real-ESRGAN-processed images, including Real-ESRGAN-high-resolution, Real-ESRGAN-average and Real-ESRGAN-median  with invasive coronary angiography as the reference. Our results showed specificity and positive predictive value (PPV) of the Real-ESRGAN-processed images were improved at all of the three coronary arteries, leading to significant reduction in the false positive rates when compared to those of the original CCTA images. The specificity and PPV of the Real-ESRGAN-M images were the highest at the RCA level, with values being 80%  and 61.9% , although the sensitivity was reduced to 81.3%  due to false negative results. The corresponding specificity and PPV of the Real-ESRGAN-M images were 51.9  and 31.5%  at LAD, 62.5%  and 43.8%  at LCx, respectively. The area under the receiver operating characteristic curve was also the highest at the RCA with value of 0.76 , 0.84 , 0.85  and 0.73 , corresponding to original CCTA, Real-ESRGAN-HR, Real-ESRGAN-A and Real-ESRGAN-M images, respectively. This study proves that the finetuned Real-ESRGAN model significantly improves the diagnostic performance of CCTA in assessing calcified plaques. Coronary artery calcium scoring is widely used in patient screening to enable a more personalized risk assessment ,2,3. HowOne of the main approaches for blooming artifact suppression is to improve the CCTA image spatial resolution. Various strategies for increasing the CCTA image spatial resolution to reduce the blooming artifact have been reported ,4,5,6. TOur recent study has shown that enhanced super-resolution GAN (ESRGAN) was able to effectively suppress the CCTA blooming artifact and improve the specificity and PPV by 10–40% for patients with heavy calcification in the coronary arteries . Its perhttps://github.com/xinntao/Real-ESRGAN (accessed on 26 April 2022) [https://data.vision.ee.ethz.ch/cvl/DIV2K/ (accessed on 1 February 2022)), Flickr2K (http://cv.snu.ac.kr/research/EDSR/Flickr2K.tar (accessed on 1 February 2022)) and OutdoorSceneTraining (OST) (http://mmlab.ie.cuhk.edu.hk/projects/SFTGAN/ (accessed on 1 February 2022)) with a total of 13,774 non-medical images were used for training the Real-ESRGAN model with 400,000 iterations, these images were sharpened before using them as ground-truth images for the training [The open-source, pre-trained ESRGAN  model by Wang et al. was used in this study. Its source code in PyTorch v1.7.0  was available at il 2022) . The Reail 2022) ,13,14. DFor finetuning Wang et al.’s Real-ESRGAN model to achieve better performance on calcium deblooming , 32 deidGeneration of two other CCTA datasets with an image size of 512 × 512 pixels based on the high-resolution images  (2048 × 2048 pixels) through average  and median (pixel) binning  approaches for further image noise reduction.Measurements of minimal lumen diameter (MLD) at each calcified plaque lesion of three main coronary arteries, left anterior descending (LAD), left circumflex (LCx) and right coronary artery (RCA) for the 200 datasets  by a single researcher (with experience of more than 20 years in CCTA image interpretation) for three times per lesion with average value taking as the final. The MLD was measured at the narrowest part of each coronary lumen  to determine the degree of stenosis on the original CCTA and Real-ESRGAN-processed images with measurements on ICA as the reference to calculate the diagnostic value.Determination of blooming artifact reduction by using Formula (1) below.The finetuned Real-ESRGAN model was used to postprocess 50 CCTA datasets not involved in the finetuning process, i.e., to increase the image spatial resolution from 512 × 512 pixels  to 2048 × 2048 pixels (high-resolution) for calcium deblooming. Approximately 10 min was needed to postprocess each dataset (hundreds of images) with the use of the Kaggle platform. These 50 CCTA datasets with paired reference images (ICA datasets) were those used in our previous study for evaluating the performance of the ESRGAN model on blooming artifact suppression. Our previous approach to evaluate the ESRGAN model was also adopted in this study. Details of these datasets and evaluation strategy were available from our previous article. The following is the summary of our evaluation approach and p-value less than 0.05 indicated statistical significance.IBM SPSS Statistics 27  was used for statistical analysis. Mean ± standard deviation (SD) and percentages were used for presenting continuous and categorical variables, respectively. For the 50 original CCTA, 50 Real-ESRGAN-HR, 50 Real-ESRGAN-Average and 50 Real-ESRGAN-Median datasets, sensitivity, specificity, PPV, negative predictive value (NPV), positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were calculated and compared across these four groups with ICA as the reference. The ICA measurements were conducted in our previous study and their details were available from our recent paper . Diagnosp < 0.01), as shown in There were a total of 184 calcified plaques that were assessed in this study with the same plaque distribution at these three main coronary arteries as reported in our recent study . Comparer = 0.937–0.991, p < 0.001).We randomly selected 20 cases for testing intra-observer reproducibility of measurements among the original CCTA, Real-ESRGAN-processed images and ICA with an interval of 8 weeks between the first and second measurements. High correlation was achieved in MLD values by the same observer among all the measurements . For the non-assessable 228 segments, the cycle GAN model significantly improved the specificity and accuracy, compared to the original CCTA , along with significantly higher AUC . Similar to assessment of all segments, the PPV was similar between these two groups . Authors claimed that the use of cycle GAN model could avoid 4 out of 99 ICA examinations based on their study, with an estimated 747 ICA procedures to be avoided per year. Despite promising results achieved within that study, the PPV was low (<45%) with moderate specificity and very low specificity in all segments and non-assessable segments groups. In contrast, our findings showed much better results than those from Inage et al.’s study [Inage et al.  in their’s study . Both spWith further reduction in false positive rates leading to improved specificity and PPV with the use of finetuned Real-ESRGAN model compared to our previous report , the negOne of the important themes in modern health care is personalized medicine, which generally refers to tailoring service delivery based on patient’s conditions. Our finetuned Real-ESRGAN model significantly improves the diagnostic performance of CCTA in assessing calcified plaques, which is one of the causes of coronary artery stenosis. Hence, our work advances the development of personalized medicine by using the latest DL technology to provide a better diagnostic service to a specific group of patients with the coronary artery stenosis caused by the calcified plaques .Spatial resolution is one of the important elements for visualization of fine details in medical imaging, which is essential in accurate diagnosis of various pathological conditions. Hence, the use of Real-ESRGAN model can be extended to other related areas, for example, textual detail restoration for low dose CT images , visualiThis study has some limitations. First, although significant improvements in specificity and PPV were achieved over previous studies ,4,5,7,27In conclusion, we demonstrated significant improvements in the diagnostic performance of CCTA with the use of advanced DL approach, the finetuned Real-ESRGAN model for suppressing the blooming artifacts associated with severe calcification in the coronary arteries, with increased specificity and PPV than the previous studies. The Real-ESRGAN-Median images led to the higher diagnostic value with the specificity and PPV reaching 80% and 62%, respectively, at the RCA, although low to moderate diagnostic values at LAD and LCx. The false positive rates were significantly reduced when assessing calcified plaques at the three main coronary arteries, however, false negative findings should not be ignored. Further studies in large datasets are needed to validate our findings with potential clinical impact on economic benefits and patient management.


Present and future of EMDR” , this is a sequel to the same topic, and is a result of increasing interest in this pioneering psychotherapeutic approach.After the extraordinary success of the previous Frontiers Research Topic dedicated to “In total, 16 articles have been published and five co-editors, 77 authors, and a very large number of reviewers actively collaborated and contributed to this Research Topic on an almost daily basis.In particular, during the COVID-19 pandemic (a period characterized by a large number of people exposed to an ongoing trauma), many studies were carried out, which demonstrated the feasibility of EMDR therapy applied online  that document how EMDR therapy, applied during the Coronavirus period, helped to relieve stress in healthcare workers, patients, and their family members.Hence, in this special second Research Topic, readers can find a large number of innovative articles  .In this special Frontiers Research Topic, scholars will find and appreciate several interesting articles dedicated to the field of trauma and stress-related conditions; from child-birth to traffic accidents and domestic violence , clinicians and researchers are revising their diagnoses of PTSD, allowing them to better address the seriousness of any somatic or psychiatric condition in a safer manner  has greatly increased in recent years.Present and future of EMDR), two recent articles have gone a step further and are highly relevant to the field.As highlighted by Castelnuovo et al.  in theirNature by Baek et al. : it deeply investigates the PTSD mechanisms and hypothesizes how EMDR achieves trauma relief. The authors used a biologically inspired computational model, based on firing rate units, comparing data from patients treated with EMDR or prolonged exposure.Also in this Research Topic, readers will find a very engaging paper on the neurophysiological mechanisms implicated in EMDR (Hase and Brisch on EMDR and its therapeutic relationship. In this article, the two authors try to describe the therapeutic relationship in EMDR therapy from an attachment-based perspective. The authors conclude their article by describing EMDR therapy as a sensitive psychotherapy, discussing the implications for the treatment, training, and research.Finally, I would like discuss the innovative article by Frontiers in Psychology, completely dedicated to EMDR.Therefore, it is my absolute pleasure to present this second special Research Topic in The author confirms being the sole contributor of this work and has approved it for publication.


Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin–pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions.P1 is a model temperate myovirus. It infects different  The lytic development of bacteriophages in bacterial cells ends with cell lysis to release the progeny phages. The effectiveness of lysis and its precise control in time is crucial for the evolutionary fate of a given phage . PrematuTypically, double-stranded DNA phages encode two kinds of essential proteins to lyse cells at the release of phage progeny . One of Despite the different mechanisms of action, the triggering of lysis by either holins or pinholins is associated with the collapse of proton motive force (PMF) and cessation of macromolecular synthesis, leading to cell death ,23,24). The triggering of cell lysis occurs precisely when mature phages are ready to be released. It is associated with the achievement of a threshold concentration of holin/pinholin in the cytoplasmic membrane, leading to the formation of holes/pinholes and the release or activation of endolysin/SAR-endolysin ,25  and mlp (i-spanin) partially overlap and are located in yet another unlinked operon. They were identified based on the predicted features of their products, but their function has not been verified experimentally [The spanin genes of P1, mentally . In laboThe organization of the known and putative cell lysis-associated genes of P1 is conserved in a P1-related phage, P7 (GenBank acc. no. AF503408). The amino acid sequences of their products, except that of LydC, are identical or nearly identical in these two phages.E. coli and Enterobacter cloacae cells during lysis mediated by P1.The complexity of P1/P7 lytic functions poses a question about its biological sense. In this work, we dissect the functions of known and predicted P1 holins, show the different impacts of particular holins on the cell lysis of different P1 hosts, and the concerted contribution of all of them to efficient lysis in a programmed time. Additionally, we demonstrate that LydD is a pinholin, and that it has a dominant influence on the morphological transitions of lyz gene in a P1 variant encoding the thermosensitive phage repressor (C1-100) results in the inability of lysogens to lyse upon the thermal induction of P1 lytic development  under the control of an inducible promoter lysed faster and more efficiently upon the induction of this promoter than cells containing only the lyz gene in an isogenic plasmid  led to the formation of only a few small colonies of transformants. They either did not form colonies upon passaging or could be transferred to a liquid medium but stopped growing upon the induction of cloned gene expression. The growth cessation and lethal phenotypes of cells producing LydC or LydA observed here are consistent with the previously observed phenotypes of bacterial cells expressing cloned holin genes . TheE. coli cells, we introduced to the P1 genome, by recombinational replacements, the kanamycin resistance cassette or indels inactivating the lydA, lydC, or lydD gene. Additionally, P1 variants that carried the combinations of mutations in any two of these genes were constructed. The lydA mutant and the double lydA lydC mutant formed plaques on a layer of indicator strain cells (E. coli N99) when plates with infected cells were incubated at 42 °C (under conditions non-permissive for the formation of lysogens). In the case of the double lydA lydD mutant, no plaques could be observed. This shows that LydD alone is sufficient to cooperate with Lyz in cell lysis by P1, but LydC alone is not, at least under the conditions of our experiment.To test the contributions of LydA, LydC, and LydD to the P1-mediated lysis of E. coli cells, although with a delay. The delay differed slightly in the case of two different lydD mutants: lydD::kanR and lydDΔ16-81  and with various holin genes inactive were tested for the ability to lyse cells   , as was dD genes E,F resuldD genes E or in tdD genes . Taken tE. cloacae cells progressed through similar morphological changes to those that accompanied E. coli cell lysis: the cells shortened and thickened, converting into a more ball-shaped form, and then cell envelopes disintegrated more or less evenly  can digest cell wall peptidoglycan, while the other (holin or pinholin) can kill the cell by disrupting the transmembrane proton motive force and enabling or facilitating the access of endolysin/SAR-endolysin to the cell wall in an active form . TActinomyces naeslundii phage Av-1 is flanked by two genes that encode two functional holins of unrelated amino acid sequences [Bacillus phage SPP1, which also encodes two holin-like proteins, cell lysis could only be caused by the concerted action of the endolysin of this phage with both of these proteins [The redundancy of the genes that could encode proteins of predicted holin features is not unique for P1 and its relatives ,51,52,53equences . In the proteins ,53. The R and lydD genes cloned in separate plasmids under the control of different inducible promoters. Clearly, the inability of LydD to support the lysis of cells producing the canonical endolysin is not associated with the action of any P1 protein that could potentially inhibit the function or synthesis of LydD. Taken together, these results indicate that LydD is a pinholin, consistent with the location of the lydD gene in the same operon as lyz.Typically, in the lytic modules of phage genomes, genes for canonical endolysins adjoin the genes for holins, while genes for SAR-endolysins adjoin the genes for pinholins ,11,12,13E. coli cells containing wild-type P1 or its mutants during lysis conform to the functional assignment of LydD to pinholins, and LydA and LydC to canonical holins, and the concerted action of all of them in P1-mediated cell lysis. While the lysis of E. coli by wild-type P1 progressed through morphological changes characteristic of SAR-endolysin–pinholin-mediated lysis, lysis by the P1 mutant deprived of LydD progressed through changes characteristic of canonical endolysin–holin-mediated lysis.The morphological transitions of 2168 of lambdoid phage 21 indicate similarities as well as differences. Both, LydD and S2168 are in about 80% alpha-helical. They both contain two TMDs and positively charged C-terminus [2168 and LydD and identified with HHPred [2168 , while the 91-aa MrpF subunit of multisubunit Na+/H+ antiporter Mrp is the best matching structural homolog of LydD . MerF is also among the predicted structural homologs of LydD, but it is only in the fourth position among other homologs . However, while the 5-aa N-terminal region preceding the TMD1 of S2168 contains only one positively charged amino acid residue and the addition of two positively charged amino acid residues to it converts modified S2168 to a pinholin antagonist that inhibits lysis triggering [2168 as involved in interhelical interaction in the pathway of pinhole formation [2168 may differ as do the mechanisms of activation of LydD and S2168 cognate SAR-endolysins .E. coli, it causes cell lysis in the absence of holins, if it can be transferred to the periplasmic side of the CM with the help of SecA [lydA to retain some activity in E. coli cell lysis was explained previously as attributed to a slow, spontaneous release of the membrane-tethered Lyz. Our results indicate that it can be attributed, at least in part, to the activity of LydD. LydD alone could cooperate with Lyz in the lysis of E. coli cells with cloned lyz and lydD genes, and in the lysis of immobilized E. coli cells by P1. Apparently, under the latter conditions, the canonical Lyz/LydD pairing is more efficient in P1-mediated cell lysis than Lyz/LydA pairing.Lyz was shown previously to cooperate with LydA in cell lysis when expressed with LydA from a plasmid . However of SecA . Thus, tIn a liquid medium, LydD appears to have only an accessory role in P1-mediated lysis. LydA alone could pair with Lyz in lysis under these conditions, suggesting the sufficient production of Lyz in its active form. In its ability to cooperate with pinholin as well as with canonical holin, Lyz resembles the SAR endolysin of lambdoid phage 21, which normally cooperates with pinholin in cell lysis, but could also complement the lysis defect of an induced lambda lysogen encoding a canonical holin and depleted of its canonical endolysin gene .E. coli cells in a liquid culture with shaking depended mainly on LydA, but LydA was not as important for the lysis of E. cloacae cells under similar conditions. While P1 producing LydD or LydC could not cause the efficient lysis of E. coli cells if it did not produce LydA, it could cause the delayed but efficient lysis of E. cloacae cells. Additionally, while LydA appeared to be a dominant cytoplasmic membrane puncturing protein, which could not be replaced by LydD, when bacteria grew with shaking in liquid culture, LydD was sufficient for the lysis of cells immobilized in a solid medium.Our results indicate that whether the Lyz–LydD or Lyz–LydA pairing predominate during lysis mediated by P1 depends on cell growth conditions and a host of P1. The P1-mediated lysis of lydB lysed cells prematurely [lyz, lydA and lydB genes cloned under the control of an inducible promoter started to lyse much later upon the induction of this promoter than cells containing the isogenic plasmid without lydB [Perhaps, whether LydA or LydD predominantly cooperate with Lyz under the given conditions of P1 development depends on the regulation of their activity or the regulation of their gene expression. One possible regulator is the LydB protein, which acts as an antiholin, antagonistic to LydA. Phages carrying an amber mutation in maturely . Cells wout lydB . How exalyz and lydD genes, and the lydA gene with its cognate antiholin gene lydB, are expressed from late P1 promoters, opposite to lydC, which is preceded by a predicted early promoter [E. coli cell lysis by P1 appears to be an accessory one independent of the bacterial growth conditions that were tested in this study. The P1 lydA lydD mutant could not form plaques. Additionally, the lack of LydC did not influence cell lysis in the absence of LydA. One cannot exclude that the level of early lydC expression may predetermine the ratio of LydA to LydD in P1-mediated lysis. A candidate for an additional regulator might be LydE, whose predicted gene partially overlaps the end of lydD, which could suggest antipinholin function. However, the removal of lydE from the lyz lydD lydE operon cloned under the inducible promoter in a plasmid did not influence lysis upon the induction of transcription from this promoter  [Enterobacter cloacae subsp. cloacae DSM6234 strains (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) as phage hosts. E. coli strain DJ125 , which is a recombination-deficient derivative of C600 [E. coli N99 strain. The bacteriophage P1 used in this study is a mutant (P1 c1-100 IS1::Tn9 mod749::IS5) that carries transposon Tn9 with a chloramphenicol resistance marker and the c1-100 mutation conferring thermosensitivity to the phage repressor protein [c1-100 Tn9 lysogens upon transfer from 30 to 42 °C. The derivatives of P1 carrying mutations in genes encoding known or predicted lytic functions and constructed in this study are listed in cI857, which was used as a control and as the source of R gene, was kindly provided by Anna Bębenek.Experiments to test the lytic phenotypes of bacteriophages or their mutant derivatives were performed with the use of K2, str)  and Ente of C600 ,75, was  protein . The theThe plasmids used are derivatives of pMB1, pACYC184, or pSC101, and are listed in 600). Bacteriophages were propagated upon thermal induction of lysogens. Briefly, cells lysogenized with λ857cI or P1 c1-100 IS1::Tn9 mod749::IS5 (or its mutant derivatives) were grown in LB or LB supplemented with chloramphenicol (12.5 μg/mL), respectively, overnight at 30 °C. The overnight cultures were diluted 50× in fresh LB with 0.2% maltose or 0.2% glucose and chloramphenicol, respectively, and grown at 30 °C until the optical density (OD600) reached 0.3–0.4. Prophages were thermally induced by rapid heating of cultures to 42 °C in a shaking water bath heated to about 60 °C, then the cultures were returned to 42 °C and grown with shaking for about 45–60 min until signs of lysis were noticed. The crude lysates were harvested when the OD600 fell to 0.15 and centrifuged at 29,030× g for 40 min at 4 °C. The remaining cells and cell debris were removed from the lysates by filtration through 0.22 μm syringe filter . The titer of phages in the lysates was assayed using the standard double-layer agar method, according to Adams (1959) [Bacteria were grown in Luria–Bertani liquid medium  or on LB medium solidified with 1.5% agar at 30, 37, or 42 °C, as indicated. When required, the medium was supplemented with glucose or maltose to a final concentration 0.2%, or with antibiotics: ampicillin (amp) , chloramphenicol (cm) (12.5 or 25 μg/mL), or kanamycin (km) (12.5 or 25 μg/mL). Incubations of liquid cultures were performed with shaking (200 rpm) overnight or until the desired optical density (ODs (1959) .E. coli cells were transformed with plasmids as described previously [E. coli DNA polymerase I , thermostable Ex Taq DNA polymerase , and DreamTaq DNA Polymerase  were used according to the supplier’s protocols.DNA amplification, plasmid isolation, and agarose gel electrophoresis were carried out using standard techniques . E. colieviously . Restric2 and 100 µL of 20 mM MgSO4 mixed with 100 µL of phage lysate (10−8–10−9 PFU/mL) and incubated at room temperature for 20 min to let the phages to adsorb the bacteria. Adsorption was stopped by the addition of 1 M sodium citrate (200 µL) and vigorous mixing, followed by the addition of 1 mL LB and incubation of the mixtures for 60 min at 30 °C to express the antibiotic-resistance phenotype. The phage–host mixture was then plated on solid LB medium supplemented with chloramphenicol and, when required, additional antibiotics, and incubated overnight at 30 °C to obtain colonies of lysogens. A portion of fresh overnight culture of bacteria (100 µL) was supplemented with 100 µL of 20 mM CaClc1-100 mod749::IS5 IS1::Tn9 mutants were constructed by RecA-mediated recombinational replacement in vivo using plasmids carrying relevant P1 genes with the desired mutations as DNA donors  and ampicillin (100 μg/mL), and, when required, kanamycin (25 μg/mL in the case of plasmids carrying P1 genes inactivated by the insertion of kanR cassette). They were purified through single colonies and used to thermally induce prophages, as described above. The obtained phages were used to lysogenize N99 cells. Lysogens expected to contain the insertions of the kanR cassette in the target prophage gene were selected on LB medium supplemented with chloramphenicol and kanamycin, and then screened for resistance to ampicillin by testing their ability to grow on LB medium supplemented with chloramphenicol, kanamycin, and ampicillin (50 μg/mL). Lysogens sensitive to ampicillin were used for further studies.All P1 nors see . Cells cLysogens expected to contain a mutation without an antibiotic resistance determinant in the target prophage gene were selected on LB medium supplemented with chloramphenicol (12.5 μg/mL) and ampicillin (50 μg/mL). To increase the probability of recombination between the phage and the donor DNA in the plasmid, each lysogen was used to inoculate 5 mL of LB medium supplemented with chloramphenicol (12.5 μg/mL) and ampicillin (50 μg/mL). The lysogen cultures were grown overnight with shaking at 30 °C and plated on LB solid medium supplemented with chloramphenicol (12.5 μg/mL) and ampicillin (500 μg/mL). Single colonies of lysogens able to grow on this medium were used for thermal induction of prophages. The obtained phages were used to lysogenize N99 cells, and the lysogens were selected on LB solid medium supplemented with chloramphenicol only (12.5 μg/mL). Lysogens were subsequently screened for the ability to grow on medium supplemented with chloramphenicol and ampicillin (50 μg/mL), to eliminate those that contained a donor plasmid either integrated with the prophage or in a circular form. Lysogens sensitive to ampicillin were tested for the presence of the desired mutation in the prophage by PCR amplification of the relevant DNA fragment and restriction digestion or sequencing of the obtained amplicon.c1-100 mod749::IS5 IS1::Tn9 or its mutant was used to inoculate LB medium supplemented with glucose and chloramphenicol to an optical density (OD600) of about 0.1. Cells were grown with shaking at 30 °C. When the OD600 reached 0.3–0.4, the prophage was induced by thermal induction, as described above, and further changes in the optical density of the culture were measured spectrophotometrically at indicated time intervals.A fresh overnight culture of cells lysogenized with P1 Morphological changes accompanying cell lysis upon the thermal induction of prophages in lysogens were monitored by the time-lapse microscopy of living cells. To immobilize cells in one focal plane, samples (10 µL) withdrawn from the cultures of induced lysogens before the expected lysis time were placed on a microscopic slide on the upper surface of an agarose microslab  and covered with a coverslip. The microslabs were prepared as described previously . Cells w


Anthocyanins are an important pharmaceutical ingredient possessing diet regulatory, antioxidant, anticancer, antidiabetic, anti-obesity, antimicrobial, and anti-inflammatory properties. Pelargonidin is an important anthocyanin-based orange-red flavonoid compound used in drugs for treating hypoglycemia, retinopathy, skeletal myopathy, etc. The main sources of pelargonidin are strawberries and food products with red pigmentation. There is a lack of evidence for supporting its use as an independent supplement. In the present study, pelargonidin and pelargonidin-3-O-glucoside are studied for their structural properties using quantum chemical calculations based on density functional theory. The results confirmed that the parent compound and its glycosylated derivative acted as good electron donors. Electrostatic potential, frontier molecular orbitals, and molecular descriptor analyses also substantiated their electron donating properties. Furthermore, based on the probability, a target prediction was performed for pelargonidin and pelargonidin-3-O-glucoside. Hydroxyacyl-coenzyme A dehydrogenase was chosen as an enzymatic target of interest, since the presence work focuses on glucuronidated compounds and their efficacy over diabetes. Possible interactions between these compounds and a target with nominable binding energies were also evaluated. Further, the structural stability of these two compounds were also analyzed using a molecular dynamics simulation. Raphanus sativus), black raspberries , and strawberries (Fragaria × ananassa). Elderberries (Sambucus), sour cherries (Prunus cerasus), and red raspberries (Rubus idaeus) contain lower concentrations of 7-hydroxyflavonoids. The other food sources reported as containing pelargonidin include cereals, gigantic butterburs (Petasites japonicus), goji, cherry tomatoes (Solanum lycopersicum var. cerasiforme), and common corn salad  [Secondary metabolites derived from fruits, especially berries, are common in daily human diet. In particular, dark red and blue colored berry fruits contain high anthocyanins and glucosyl flavonoids content. These compounds protect human health against oxidative stress, cell damage, and cardiac difficulties ,2,3. Pellocusta) ,5,6,7,8.Numerous cell culture studies were conducted for finding the effect of anthocyanins on cytokine production and other aspects of immune function ,10,11,1215H11O5+) and pelargonidin 3-O-𝛽-glucopyranoside or pelargonidin-3-O-glucoside—(C21H21O10+), have been used as a replacement for food additives such as alloxan, monosodium glutamate, aspartame, etc. These additives are known to pose serious threat to human health as they cause DNA damage, chromosomal aberration and diabetes [Because of their antioxidant property, pelargonidin (2-(4-hydroxyphenyl)chromenylium-3,5,7-triol)— were used for simulations. The Auto Dock program and Lamarckian genetic algorithm were used for molecular docking studies. The electron donating properties of pelargonidin and pelargonidin-3-O-glucoside were assessed using electrostatic potential, frontier molecular orbitals (FMO) and molecular descriptor analyses.In order to study the molecular properties of flavonoids, their ground state energies must be known. Optimization of structural features was performed using density functional theory. The initial geometries of the molecules are obtained from PUBCHEM-NCBI database and used for optimization. The structure of optimized conformers of pelargonidin and pelargonidin-3-O glucoside are shown in The electrophilic and nucleophilic properties of the molecular systems through interaction between the occupied and unoccupied molecular orbitals or energy levels are analyzed with the help of FMO models . Figure The chemical reactivity of pelargonidin and pelargonidin-3-O-glucoside can be well understood with the help of surface electrostatic potential . From FiThe molecular descriptive parameters estimated for pelargonidin and pelargonidin-3-O-glucoside are provided in The Brain Or IntestinaL Estimated permeation technique (BOILED-Egg) is a precise prediction model that operates by calculating the polarity and lipophilicity of tiny compounds. Due to the model’s speed, accuracy, conceptual simplicity, and clear graphical output, concurrent predictions for brain and intestine permeation are produced from the same two physicochemical descriptors and directly translated into molecular design. The BOILED-Egg may be used in a range of contexts, from the screening of chemical libraries during the preliminary stages of drug discovery to the assessment of potential therapeutic candidates . From thPredictions of bio molecular interaction of small ligands such as flavonoids are important for prediction of their potential side effects. Swissadme is an online database that provides large set of data on structural properties of proteins and their interactions. Homosapien class of targets were chosen and the binding probability for pelargonidin and pelargonidin-3-O-glucoside were analyzed and depicted in The interactions between pelargonidin and pelargonidin-3-O-glucoside have been examined using the molecular docking method. To compare the differences between these compounds at the molecular level, a 20 ns MD simulation approach was employed. Using the GROMACS program, the stability and degree of interactions between HAD and pelargonidin and pelargonidin-3-O-glucoside were further explained. The trajectory data from the 20 ns MD simulations were then analyzed in order to determine the dynamic parameters of the two compounds. In the molecular dynamics research, each system was assessed using the root mean square deviation (RMSD), radius of gyration (Rg), solvent accessible surface area (SASA), and root mean square fluctuation (RMSF). The obtained results are displayed in RMSD is used to assess if the complex system has attained a stable state by calculating the average deviation between the complex’s current conformation and its actual conformation at a particular time. The RMSD value of the pelargonidin-HAD complex configuration remained rising throughout the simulation, as shown in The time evolution of the radius of gyration (Rg) is useful for evaluating the kinetics of protein collapse. In order to verify the protein compactness, the radius of gyration of pelargonidin and pelargonidin-3-O-glucoside was quantified. Furthermore, the relationship between the radius of gyration and the simulation time was plotted b. The RgAs shown in In the present investigation, the naturally occurring flavonoids pelargonidin and pelargonidin-3-O-glucoside were theoretically studied for their structural activity through various analyses such as frontier molecular orbital analysis, molecular electrostatic potential analysis, and molecular descriptive parameter. The obtained results are as follows. The initial step of this investigation was geometry optimization. With the help of density functional theory, using exchange correlation functional B3LYP and basis set 6-311G , ground state energies for the two flavonoids were obtained. Geometry corresponding to ground state minima was also simulated. Frontier molecular orbital analysis nominates pelargonidin to be more favorable for the radical scavenging process as it possesses the majority of electron localization over the B-ring and A-ring where –OH units are present. The energy gap for pelargonidin is 1.85 eV and for pelargonidin-3-O-glucoside it is about 1.91 eV, which again makes pelargonidin a nominal antioxidant. The electropositive region in the MEP surface of these compounds confirmed their responsivity towards the free radical scavenging action. The glucosyl unit present in pelargonidin-3-O-glucoside seems to be highly delocalized and suppresses the activity for its host. Molecular descriptive parameter values clearly stated that two flavonoids are better electron donors rather than electron acceptors. BOILED-egg plot analysis suggests that pelargonidin can be absorbed by intestine whereas pelargonidin-3-O-glucoised does not choose any absorption pathway. Target prediction reports showed that the flavonoid pelargonidin prefers enzymatic receptor-based targets followed by a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, whereas pelargonidin-3-O-glucoside prefers enzymatic targets and membrane receptor; hence it finds major applicability in cancer treatment. By keeping the enzymatic target as common interest for both the compounds, molecular docking and MD simulations were performed with HAD and it was found that pelargonidin binds with the target effectively, thus favors inhibition easier. On the basis of stability data attained from MD simulations, pelargonidin-3-O-glucoside seems to be more stable than pelargonidin. Overall, these findings suggest that the structural activity and HAD target inhibition potency of pelargonidin was better than its derivative pelargonidin-3-O-glucoside.


Allogeneic hematopoietic stem cell transplantation (HSCT) is an intensive curative treatment that increases family caregivers’ burden. The aim of this study was to explore the feasibility of remotely assessing and addressing family caregivers’ support needs in terms of demand and acceptability using the Carer Support Needs Assessment Tool Intervention (CSNAT-I) in the HSCT context.CSNAT-I consists of an evidence-based tool and a five-stage person-centred process. The intervention was performed remotely by two designated nurses from two HSCT centres, one before HSCT and the second 6 weeks after (November 2020 to March 2021). To capture the experiences of using CSNAT-I, interviews were conducted with family caregivers and reflections were gathered from the designated nurses.Of 34 eligible family caregivers, 27 participated, 70% were partners and the rest children, siblings or other relatives. The main support needs were knowing what to expect in the future and dealing with your feelings and worries. The most frequent support actions according to CSNAT-I were psychological support and medical information. Four categories summarised family caregivers and designated nurses’ experiences: CSNAT-I was relevant and became an eye opener; nurses’ experiences were important for enabling trustful CSNAT-I conversations; CSNAT-I provided family caregivers with support and a sense of security; and CSNAT-I gave family caregivers insight and enabled change.Both family caregivers and designated nurses experienced that using CSNAT-I in an HSCT context was feasible and had the potential to provide valuable support for most of the participating family caregivers. The rationale behind this study is the lack of feasible person-centred support interventions targeting family caregivers’ (FC) support needs in the context of allogeneic hematopoietic stem cell transplantation (HSCT). HSCT is an intensive curative treatment for hematological malignancies, such as leukaemia, with a high risk of relapse and severe complications for patients, such as graft versus host disease and complex infections –3. The lBased on interviews with FC, researchers in palliative care have developed The Carer Support Needs Assessment Tool Intervention (CSNAT-I). The intervention is specifically developed to directly assess and address practical, emotional, existential, and social support needs . ConversThere are both similarities and differences between a palliative care context and the HSCT context. The similarities are the complexity of the illness and associated complications, which require an advanced level of highly specialized care. The differences are that in palliative care the patient’s condition deteriorates and ends in death, while in HSCT care the treatment has a curative intent and patient’s medical and health status often change rapidly. There is a need for feasible person-centred support interventions targeting FC support needs in the context of HSCT. Therefore, the aim of this study was to explore the feasibility of remotely assessing and addressing family caregivers’ support needs in terms of demand and acceptability using the Carer Support Needs Assessment Tool Intervention (CSNAT-I) in the HSCT context.This feasibility study has a longitudinal design Fig. . The focAdult FC were consecutively included from two HSCT centres, namely Stockholm and Lund. Patients were asked by the HSCT coordinator to select a FC involved in their everyday life. With the patients’ agreement, the HSCT coordinator sent the study information to the FC, who were then contacted by telephone, given oral information and invited to participate in the study. If they agreed, they were included in the CSNAT-I. The inclusion period was from November 2020 to March 2021 and the inclusion criteria were being a FC to a patient undergoing HSCT and able to read and speak Swedish.One designated nurse from each HSCT centre performed the CSNAT-I. They had 14 and 23 years of nursing experience respectively, had specialist training in oncology and long experience of HSCT. Both were included in the planning phase of using the CSNAT-I in the HSCT context. Initially, the designated nurses completed training about how to use the CSNAT-I. A Swedish version of the original CSNAT instructions was used, including information and explanations about the domains and the five-stage person-centred approach. Instructions are based on the online toolkit that is available in English. The CSNAT-I conversations were carried out at two time points, before transplantation and 6 weeks later when patients are usually back home again after the intensive inpatient treatment. The decision to select these two time points was based on clinical experience within the research group and earlier studies showing that these time points are demanding for the FC with uncertainty and changes in everyday life including responsibilities that often involve the need for support , 8. For The Swedish CSNAT tool version 3 and its support plan were used , 15, 16.Semi-structured telephone interviews were conducted by two of the authors  after the participant had completed the second conversation. The interviews evaluated the participants’ experiences of the CSNAT-I, including whether participation had led to any changes and suggestions for improvements. The median duration of these interviews was 29 min (range 16–48). The interviews were recorded and closely followed by a written summary of the answers and field notes were made during and directly after the interviews.After each of the two conversations, the nurses filled in a standardized written documentation form including questions about their overall reflections on the conversation, their experience of using the CSNAT-I and suggestions for improvements.After all conversations were completed, the designated nurses reflected individually on their experiences of the conversations together with one of the authors (KB). These conversations were recorded with a written summary.Descriptive statistics were used to describe the characteristics of the participants. Inductive qualitative content analysis was applied to the exit interviews, the reflective conversations with the designated nurses and the memos. The written summaries were read through several times to get a full picture of the complete data . In the In the written and oral information, we emphasised the voluntary nature of participation, the right to withdraw from the study at any time, that data would be treated confidentially and that the identity of the participants would be protected. Ethical approval was obtained from the Regional Ethical Review Board in Stockholm, Sweden (No. 2017/1112–31/4).The findings are presented first with data about the participants, and then an overview of the support needs and support actions in the HSCT context. Thereafter, FC and designated nurses’ experiences of the CSNAT-I is presented in four categories: the CSNAT-I was relevant and became an eye opener; nurses’ experiences were of importance to enable trustful CSNAT-I conversations; the CSNAT-I provided FC with support and a sense of security; and the CSNAT-I gave FC insight, preparedness and enabled change. Quotations are presented to illustrate the family caregivers’ and nurses’ experiences in their own words. Out of 50 eligible patients planned for HSCT, 45 underwent transplantation, four were postponed and one died before transplantation. Among those 45 patients, six had no FC who understood Swedish, two did not allow us to invite their FC to participate, one had no FC, in one case the pre-transplantation process was too quick to enable us to ask her/him to participate and in another it was not possible to ask the patient for permission due to cognitive deficits. Thus, 34 FC were eligible for inclusion and informed about the study, of whom four declined participation and 30 accepted (88%). Of the 30 FC who accepted, three only participated in conversation 1, which resulted in a total of 27 (80%) FC, 19 from Stockholm and 8 from Lund. All conversations were conducted over the phone, except for one by video. Conversation 1 took 45–60 min and conversation 2 13–30 min. The median age of the participants was 55 years and 56% were women. Other characteristics are presented in Table n = 4), one (n = 6), two (n = 4), four (n = 4), five (n = 2) and more than six (n = 7). The most frequent support needs being present in the CSNAT tool were knowing what to expect in the future; dealing with your feelings and worries; knowing who to contact; and talking with your relative about their illness. In Fig. n = 14) or medical treatment for themselves (n = 2), medical information about the patient from a physician (n = 9) or patient organisation (n = 4), or how to arrange home care (n = 4).The number of support needs reported by FC in the CSNAT tool at conversation 1 varied widely: zero .All FC identified several factors of importance for creating trust in the nurses, i.e. the nurses’ high level of competence and extensive experience of HSCT, their ability to have these conversations, as well as their professional and personal qualities. The nurses were described as being easy to talk to and being professional yet personal. The designated nurses also highlighted the fact that their experience and knowledge of HSCT-nursing made them comfortable and confident to have these conversations. They emphasised the need to have a genuine interest in listening. They expressed that an active listening approach had developed from their knowledge, experience and conscious choice of a humble attitude towards the life situation of FC.Both FC and designated nurses reported that conducting the conversations over the phone was positive, worked well and enabled them to build a trustful relationship despite being unable to see each other. Several FC mentioned the advantage of not having to go to the hospital. However, those who had difficulties with the Swedish language would have preferred face-to-face conversations and several FC expressed a wish for video-based conversations. The designated nurses reported a disadvantage of being unable to read body language or interpret reactions and emotions during the conversations. This was especially problematic when a FC cried or shared something sensitive, as it was impossible to show care and provide comfort through their own body language.“You need to have experience, have worked with patients and know about treatments, how the patient will feel in the future, follow-up etc. Answering the CSNAT tool will give them a sense of security…, used to talking to people, listening, maybe not coming up with solutions so quickly, but asking what do you think, what do you want to do?” (Designated nurse).All the FC expressed and appreciated that the conversations had focused on their life situation and needs. The conversations were about how the FC was doing, her/his thoughts and worries. Many of the FC expressed that they had initially believed that they did not need support, but that during the conversations it became obvious that they felt worse than they had thought. They experienced that the conversations were supportive and derived great benefit from them. The FC expressed that the conversations fulfilled another function compared to the information from doctors, i.e. focus on them as individuals. The FC felt relieved and more at peace after the conversations. For many, this was the first time that focus was exclusively on their life situation and needs, which they expressed as valuable and a good feeling, adding that they appreciated being listened to. The conversations gave them opportunities to ask questions, receive information and reflect and discuss situations in life, with a focus on their needs as a FC, while the patient was not present. The support from the nurses during the conversations also included practical aspects and advice, such as information about support available for FC, for example sick leave, and what to think about during the rehabilitation when the patient returned home. However, some of the FC expressed that they had no need for these conversations. Nevertheless, both designated nurses felt that most of the FC were satisfied after the conversations. Even the FC who did not have any support needs expressed that the conversations per se had been rewarding.“Very valuable to talk to and get support from someone with expertise in illness and treatments, without medical focus, and with warmth with a focus on me as a relative.” .When the FC recalled the CSNAT-I during the exit-interviews, they realised that they had gained new insights. They had learned about how they were actually feeling and what support they needed, e.g. the need to take care of oneself and ask for help. They had also gained new knowledge about the transplantation process and the patient’s situation, which led to reduced or more balanced worries about things that might happen later. The intervention also made it possible for them to focus on non-medical aspects, such as how to relate to the situation and that they as FC are important for the patient. They described feeling more prepared for the transplantation process with a sense of participating in the patient’s care. The feeling of preparedness mainly concerned being mentally prepared. For some, the CSNAT-I had led to a personal change in their life situation, and they understood that they needed help. These changes involved dealing with grief, contacting a social worker for psychological support and prioritizing their own needs, such as time for themselves, physical exercise and sufficient sleep during the night. However, a few did not make any changes after the conversations, despite the fact that most appreciated the conversations focusing on them as FC.“I got an insight into what I needed. I need and it’s okay that I’m sad and grieving and that I need support, I also gained insight into how I can be an even better support for him. It is important that I understand that I have to take care of myself as well because it then helps him. I did not realize that until then, you cannot just move on forward. It was the big win to get this win. Plus, how I handle, got better at handling my grief.” .This feasibility study conducted in an HSCT context shows that the demand from FC to participate in CSNAT-I was high. Furthermore, it demonstrated a high acceptability both among designated nurses and participating FC. Participants described not only appreciating the conversations, but also being provided with support and a sense of security. Several FC experienced gaining insights, preparedness and in some cases the intervention enabled them to make changes in their life. In summary, implementing the CSNAT-I in HSCT general practice is clearly acceptable.Almost 80% of eligible FC participated in the intervention. Including FC in interventions during the patient’s transplantation process  has beenIn the present study, all FC and designated nurses were satisfied with the CSNAT-I, including the remotely conducted conversations and the chosen time points for them. The FC expressed satisfaction with the conversations, according to given circumstances, and how they were conducted, i.e. by telphone, the support given by the nurses, with focus on the situation of the FC. However, the FC and the nurses reflected on possible improvements in the intervention, e.g. with converstions face-to-face it would be possible to read and show body language. In general, the first conversation was longer than the second one, which is in line with the developers’ intention , 15, as The results show that the designated nurses’ extensive experience of HSCT care and their ability to provide psychosocial support was highly appreciated by FC. The importance of the abilities of the healthcare professional conducting the conversations is also highlighted in another CSNAT-I feasibility study among FC of patients with chronic obstructive pulmonary disease, who stated that the interventionists’ demeanor, relational skills and knowledge were more important than her/his profession . In the In our context, FC described that participating in the CSNAT-I gave them a sense of security. The support plans of more than half of the FC included a need for more psychological support for themselves. This is related to the fact that in the HSCT context the greatest problem for FC is the sense of uncertainty, especially due to the unknown prognosis of the transplantation  and thatA strength of this feasibility study is that it was based on quite a large sample, was undertaken in two HSCT centres at two different geographical locations and that the two designated nurses were involved in the planning process of delivering the CSNAT-I in the HSCT context. However, having such dedicated nurses delivering the intervention makes it unique and a possible limitation is that the nurses might have been evaluated rather than the effects of the intervention itself. All the authors have vast experience in haematology, oncology and HSCT care, and one of the authors (AA) has high competence in palliative care. However, none of the authors were involved in the care of the recipients related to the family caregivers in the study. Although the authors’ pre-understanding could have influenced the data analyses and interpretation of the data, awareness of this risk and the continuous dialogue between the authors helped to minimize potential misinterpretations.In conclusion, both FC and designated nurses experienced that using CSNAT-I in the HSCT context was feasible. The intervention had the potential to provide most of the FC with both direct and proactive support. This study also highlights a need for further psychosocial support among FC.


The innovative behavior of employees is the micro-foundation of enterprise innovation. The objective of this study was to assess the role of gender differences in the effect of interpersonal trust on employee innovation and the mediating roles of organizational commitment and knowledge-sharing. This study tested research hypotheses with a multi-group structural equation model, using data collected from 688 participants in Shanghai, China. The results showed that interpersonal trust had significant impacts on affective organizational commitment, knowledge-sharing and innovation behavior. Affective organizational commitment and knowledge-sharing mediated the effect of interpersonal trust on employee innovation. Furthermore, the direct impact of interpersonal trust on innovative behavior was significantly higher for women than for men, whereas males’ affective organizational commitment increased their knowledge-sharing behaviors. In addition, there were no significant gender differences in the effect of interpersonal trust on organizational commitment and in the effect of knowledge-sharing on innovative behavior. These results confirmed that interpersonal trust was more important for female knowledge-sharing and innovative behavior, and affective organizational commitment was more important for male knowledge-sharing. Employees’ innovation behavior (IB) is crucial for organizations to be competitive. It typically includes several types of behaviors, such as the creation, promotion and implementation of novel ideas in the workplace . In partFemale employees remain significantly underrepresented in the ICT industry. There are differences between the genders in professional research directions and career choices, which often result in males occupying dominant positions in science and technology innovation . In the However, the innovation of ICT companies requires the cooperation of male and female employees. Gender diversity in companies can promote the employment of more skilled employees and result in greater overall success . The incNevertheless, there has only been limited progress in understanding the gender differences and the social determinants of employee innovation. Therefore, in this study, we explored the gender differences in the relationship between ITW and IB, using data from a sample of 688 respondents at 40 ICT companies in Shanghai, China. This article is articulated as follows. IB refers to employees’ discovery and generation of new ideas and the proposal and implementation of solutions ,3. It usITW is derived from employees’ positive expectations that the others in the organizations will perform particular actions important to them under risk-prone conditions . In mostAOC is crucial to KS and employee innovation. AOC constitutes a necessary condition for employees to voluntarily share important knowledge such as competences, key information and personal experiences . AOC proITW includes vertical and horizontal trusts . VerticaIB is the process of reengineering knowledge and reusing resources. Interpersonal trust is conducive to resolving the conflicts among members of an innovation team and shaping organizational commitment. Stronger interpersonal trust can lead to more mutual supports, cooperation and coordination among employees and less opportunistic behaviors . ITW canHypothesis 1 (H1).ITW could have a positive impact on IB.Organizational commitment is an emotional state maintained between an employee and an organization, usually measured by three categories: affective commitment, continuance commitment and normative commitment . Among tHypothesis 2 (H2).ITW could have a positive impact on AOC.Another potential consequence of ITW is KS. KS requires the willingness of individuals to actively participate in the exchange and creation of knowledge and to collaborate together in the workplace . KS is sHowever, promoting KS is a major challenge, as many employees tend to hoard their own knowledge, rather than taking steps to convert their knowledge into a form that can be understood, absorbed, and used by others, and they are suspicious of the knowledge given by their colleagues . In an oWhy is ITW so important in determining KS? There are several reasons. First, when there are trust relationships, employees will be less concerned about the loss of KS and be more likely to provide useful knowledge . EmployeHypothesis 3 (H3).ITW could have a positive impact on KS.KS depends on the giver’s willingness to voluntarily share knowledge. A high level of AOC will result in voluntary cooperation, including the sharing of knowledge. Extensive research has confirmed that AOC is a significant determinant of KS, across a broad range of countries and occupations . AOC plaHypothesis 4 (H4).AOC could have a positive impact on KS.Similarly, AOC can also increase the effort and value coherence and the attachment to the organization, thus promoting IB . IB is dHypothesis 5 (H5).AOC could have a positive impact on IB.KS is part of knowledge management and has been a focus of both academia and business due to its relevance for innovative capabilities . KS not Hypothesis 6 (H6).KS could have a positive impact on IB.Although a number of studies have applied a gender-focused lens to innovation research in the last decade, the gender dynamics impacting employee innovation have not been fully discussed . EmployeIn contemporary ICT companies, many innovative positions require relational as well as technical skills. The building and application of social capital, either actual or technology-based, are important to IB and KS. There are significant gender differences in the approaches used in the social networks, contacts, and alliances of colleagues to access knowledge and acquire new ideas, such that females tend to converse with others while males may experiment alone, when adapting to new technology .The socialization process and gender role differences between genders can vary in terms of the construction and application of interpersonal trust. Interpersonal trust, as an element of social capital, is important for both male and female employees in sharing complete and true information and knowledge and taking risks to embrace IB . HoweverHypothesis 7 (H7).ITW could have a stronger positive impact on KS for females than for males.Hypothesis 8 (H8).ITW could have a stronger positive impact on IB for females than for males.Previous research has suggested that males and females may use different criteria in their assessment of organizations. Males tended to have higher levels of organizational commitment than females, suggesting that males were more likely than females to hold jobs with commitment-enhancing features . FemalesHypothesis 9 (H9).AOC could have a stronger positive impact on KS for males than for females.Hypothesis 10 (H10).AOC could have a stronger positive impact on IB for males than for females.https://www.wjx.cn/, accessed on 1 December 2021). The questionnaires include demographic questions and the questions addressing the constructs used in this study. A total of 686 respondents completed the online questionnaires. The response rate was 85.8%. Unfortunately, we did not have detailed information regarding the respondents who chose not to complete the questionnaires. This study analyzed data from an online survey conducted in 40 small and medium- sized ICT companies in Shanghai, China. Since we do not have the whole list of ICT companies in Shanghai, we selected the 40 companies using snow-ball sampling and then contacted the human resource managers at these companies to randomly select 20 respondents from each company. These human resource managers sent emails with links to the questionnaires to these anonymous respondents, using an online platform  models and structural equation models (SEM) with AMOS . To compare the gender differences, we further applied the multi-group structural equation models. We analyzed the fits of these models to the data. We chose several goodness-of-fit indices for the analyses: chi-squared, comparative fit index (CFI), goodness-of fit-index (GFI) and the root mean square error of approximation (RMSEA). We tested several multi-group models with different restrictions and achieved the best model with possible restrictions on causal relationships .We first evaluated the CFA models. As shown in The correlations among latent variables in the CFA model are shown in We conducted a structural equation model after the implementation of the CFA models for the purpose of examining the research model and the first six hypotheses. The fitting indices of the model were acceptable: CFI = 0.983, GFI = 0.961, and RMSEA = 0.046.To test the moderate effect of gender on the structural relationships, we divided the sample into two gender sub-samples, and then applied multi-group SEMs. The unrestricted multi-group model had good fitness: CFI = 0.979, GFI = 0.941, and RMSEA = 0.037. In this model, as reported in We further tested four models with different restrictions. Based on the revised model, we added restrictions on the structural weights one by one. The chi-squared test showed that the restrictions of the same structural weights from ITW to IB, from ITW to KS and from AOC to KS significantly increased the chi-square results, indicating important gender differences in these structural relationships. Adding the last three restrictions to the model did not significantly change the model’s fit.The objective of this study was to provide insights into the gender differences involved in ITW on IB, while considering AOC and KS as mediators. We applied CFA and multi-group SEMs and surveyed 688 respondents from 40 ICT companies in Shanghai, China. The results contributed to the literature regarding gender gaps as well as the causal relationship between ITW and IB.The results of the structural relationships based on the total sample confirmed the positive effect of ITW on IB, supporting the hypothesis H1. ITW was helpful for creating an inclusive innovation culture, improving employees’ enthusiasm for innovation, reducing opportunistic behavior, and providing emotional safety and psychological support for employees to stimulate their IBs in ICT companies. The results showed that increased ITW also encouraged more risk-taking behaviors and then improved the innovation performance of the ICT companies, consistent with the previous studies ,7.Previous studies demonstrated that ITW not only promotes employees’ creativity, but also increases AOC and KS ,18. ThisThis study revealed that the direct impact of ITW on IB was stronger for females than for males, supporting the hypothesis H8. Similarly, female employees were more likely to share knowledge with others if they trusted their colleagues, supporting the hypothesis H7. It verified that female employees were more reliant on social ties and preferred working with co-workers’ supports . Female In contrast, KS was more related to AOC among male employees than females, providing evidence for the hypothesis H9. Previous studies on the relationship between AOC and KS hardly addressed the issue of the gender difference. This study revealed that stronger AOC encouraged male employees to share knowledge, even though it might be costly and risky. Due to the consistent correlations of IB with KS and AOC across genders, the indirect effect of AOC on IB via KS was stronger for male employees. The reasons why the AOC of male employees is highly correlated with KS could be that they had been socialized to favor loyalty and righteousness to their organizations. Similarly, the reason why female KS was less correlated with AOC could be that they had been socialized to respect cooperation with their surroundings .This study further revealed that the influence of AOC on IB was almost unchangeable across genders, providing no evidence for the hypothesis H10. In addition, the influence of ITW on AOC and the influences of AOC and KS on IB are similar across genders. These findings confirmed that the path from ITW via AOC to IB was important for both male and female employees in ICT companies.Although social determinants of IB in the workplace have been extensively studied, not so many studies have addressed the gender differences in its relationships with ITW, AOC and KS. Previous research has revealed the mediating effect of KS between ITW and IB ,11. ThisBy distinguishing gender differences alone in the path from AOC to KS, the results provided the next step to advance the previous research in the field of knowledge management. This study extended the research model for the relationship between AOC and KS. Although the existing literature suggested that both AOC and ITW were important for KS ,37, thisThe main contributions of this study were related to gender differences in the influence of ITW on IB and the mediating roles of AOC and KS in ICT companies. The results highlighted the relevance of ITW, AOC and KS in the promotion of IB. Therefore, the managers of the ICT companies that encourage more IBs should increase KS through the development of an organizational environment based on ITW and AOC. Such results showed the managers the importance of encouraging a trustful environment for female employees in terms of KS and IB. In order to nurture a trust-based organizational environment, managers should design human resource practices related to ITW, such as developing team competency, providing group rewards, and establishing workload sharing . Such acThis study also addressed the importance of AOC for KS among male employees in ICT companies. KS and AOC were characteristics of the organizational culture and support organizational innovation. This study provided evidence regarding supporting KS to male employees. Hi-tech companies can benefit from stimulating KS among male employees as a result of their AOC. Managers should design team-building activities and other managerial practices related to organizational commitment, such as mentoring programs and employee orientation processes, according to the needs of male employees .Some limitations of this study were worth noting and suggested possible avenues for further research. First, the sample adopted in this study was limited to 688 respondents from 40 Chinese ICT companies and thus caused some concerns over the generalizability of the results. There was also an intention to test the gender differences in other organizations and cultural contexts. Second, this study used cross-sectional data that limited the conclusions about causality. Thirdly, future studies could also improve the explanatory power of the model proposed by adding more variables that could more comprehensively explain the mediating mechanisms through which organizational environments shaped KS and innovative behaviors. Moreover, expanding the gender differences in the influencing mechanisms of different types of organizational commitment might also theoretically further relevant studies in the future.This study assessed the role of gender differences in the effect of interpersonal trust on employee innovation and the mediating roles of organizational commitment and KS. The results from structural equation models showed that ITW had significant impacts on AOC, KS and IB. AOC and KS mediated the effect of ITW on IB. Furthermore, the direct impact of ITW on IB was significantly higher for females than for males, whereas male AOC increased KS. In addition, there were no significant gender differences on the effect of ITW on AOC and on the effect of KS on IB. The results confirmed that ITW was more important for female IB, and AOC was more important for male KS. This study provided a better understanding for managers regarding gender differences and encouraging employee innovation.


However, sample preparation and data collection methods generate biases and noise which confound molecular diversity estimates and require special attention. Here, we examine how technical biases and noise that are introduced into multivariate molecular data affect the estimation of the components of diversity . We then explore under which conditions these biases affect the measurement of ɑ- and β-diversity and highlight how novel methods commonly used in community ecology can be adopted to improve the interpretation and integration of multivariate molecular data.The measurement of uncharacterized pools of biological molecules through techniques such as metabarcoding, metagenomics, metatranscriptomics, metabolomics, and metaproteomics produces large, multivariate datasets. Analyses of these datasets have successfully been borrowed from community ecology to characterize the molecular diversity of samples (Video AbstractSupplementary information accompanies this paper at 10.1186/s40168-022-01423-8. One of the most fundamental patterns of scientific discovery is the revolution in thought that accompanies a new body of data [ of data .The direct characterization and analysis of sampled pools of biomolecules, particularly DNA, RNA, proteins, and metabolites, has fundamentally altered the life sciences and specifically microbiology. Metagenomics and metabarcoding can identify organisms in a sample and detect inter- and intraspecific diversity, while (meta-)transcriptomics, (meta-)proteomics, and metabolomics can characterize the functional responses of biological individuals, populations, or whole communities Table . Despite. Being generally untargeted, they require less a priori knowledge about the biomolecules measured than targeted assays. Interest in MME techniques has grown rapidly over the past two decades, and their application has informed ecological and evolutionary theory. For example, metabarcoding has been used to show how dormancy affects the spatial distribution of microbes [MME techniques measure a wide range of molecular entities within a selected class of moleculesmicrobes , and metmicrobes . Similarmicrobes .sample × molecule matrices produced by MME techniques may be viewed as analogous to the plot × species matrices produced by community ecologists. Like in community ecology, studies in molecular and microbial ecology are often less concerned with the response of single molecular entities, especially when the identity and function of specific molecular entities are unknown. Rather, they focus on differences or changes in a whole profile or on emergent properties of the community . Similar to CE studies, MME data are often used to characterize molecular diversity and the relative abundance of molecular entities within samples and across space, time, or experimental treatments.MME data and conventional community ecology (CE) data are both multivariate and the ɑ-and β-diversity metrics, which describe species diversity in a sample and the difference in diversity between samples, respectively, were developed by CE and are often used to describe changes in the molecular profiles of samples . In CE measures are affected by biases that are introduced during the molecular sample preparation, molecular analyses, data collection, and processing steps that generate MME data. The techniques that produce MME data are relatively new, and while much work has been devoted to identifying their biases and limitations, how these affect the applicability of CE measures to assess patterns of molecular diversity has received less attention. However, within the context of ecology, the biases and limitations of diversity data have been extensively discussed, resulting in various approaches to overcome them. For example, issues related to detection limits given finite sample sizes are potentially the oldest analytical research topic in ecology  and have Here, we explore how detection biases and technical limitations associated with MME data affect the calculation of N, S, and SAD, and how this, in turn, affects the determination of ɑ- and β-diversity. Finally, drawing parallels from other areas of community ecology, we indicate analytical approaches that allow for the robust estimation of diversity metrics for biased or limited MME data.All components of biodiversity—and the patterns emerging from them—depend on the sampling scale , 28. TheThe spatial scaling of diversity has been explored in metabarcoding data –37, but In order to capture microbial diversity at larger scales and reduce the effect of small-scale spatial variability, pooling  is a common practice . HoweverWhile MME techniques often impose limits on the sampling scale, they generally require the researcher to explicitly select a definition of the units of diversity. These units can be species or molecular entities, such as ASVs or protein families. If the definition is not given , it is usually based on a threshold of molecular similarity of detected molecules to each other or to references. Definitions vary with e.g., 97 to 100% similarity for metabarcoding , 95% aveIn CE, for a given sampling scale, N measures the number of individuals in that space. However, MME techniques measure molecules rather than individuals. The two are not always related) e.g., because of differences in cell or body sizes, or in copy numbers of marker genes). Furthermore, the number of detected molecules often reflects a machine’s throughput, rather than biological reality . For exaThe decoupling of N and the abundance of molecules in situ creates two limitations. First, it precludes the estimation of the true abundances of molecular entities. Second, uneven observation depths make changes in the abundances of molecular entities  sensitive to the normalization method used . Severalad hoc, as discussed above. S can be influenced by the environment, dispersal, interactions among organisms, or changes in organisms producing molecules of interest (as in [CE’s species richness in an area (S) can be equated to the number of distinguishable molecular entities in a sample , which are often defined t (as in ). Assesst  describe how abundances vary across species in a community . SAD ofBecause SAD are distributions rather than a single metric, comparing SAD across multiple samples is not straightforward, as differences in the relative distribution of taxa  can result in different relationships between samples depending on N . This isWhen the number of observations is artificially determined by the technique , an increase in one species may result in an observed decrease of another, even if the absolute abundance of the other molecular entity is unchanged . This liFor many MME questions, rare molecular entities are uninformative, in addition to being more likely observed due to technical error, making them less quantifiable. MME fields have developed different approaches to remove such data. In untargeted metabolomics, molecular entities that are detected in blanks or exhibit high variation across technical replicates are removed from the metabolomic profile , 88. In How SAD curves are used in CE to derive multiple diversity estimates, including evenness and ɑ-diversity for any N, as well as to compare abundance profiles across gradients  is discuThe challenge of analyzing “imperfect” MME data is mirrored by similar issues that commonly arise in CE studies . For exaIssues related to detection limits given finite sample sizes are one of the oldest analytical research topics in ecology . Rarefaca posteriori modeling [In CE, detection errors that result in missing data are often estimated by revisiting a site , with marked capture-recapture experimental designs, or with modeling . Severalmodeling ), but sumodeling , targetemodeling , 107. Simodeling  derive a post hoc corrections to incomplete samples. However, this approach draws heavily on a priori understanding about species’ natural histories as well as historical data from previous studies, and may become more prominent as the molecular world is increasingly well characterized.Species occupancy models may be used to correct for false positive and false negative errors –112 and ɑ, can serve as a diversity metric that reflects the imbalance between few dominant species versus many rare species. For example, a community with a few highly abundant species and many rare ones will show a low ɑ value compared to a similarly rich community with more even abundances. Fisher’s ɑ is more sensitive to species of medium abundances and thus more helpful in the context of incomplete sampling [For the estimation of SAD, parametric curves  can imprsampling .Due to the compositional nature of MME data, less abundant or less detectable components can be pushed below detection thresholds limits which is akin to the classic ecological notion of “veil lines” . Post hoq (or order) of the Hill numbers. As q increases, the relative importance of abundant species increases, providing information on whether common or rare species contribute most to ɑ-diversity. Hill numbers have been shown to be more robust diversity estimators from molecular data, especially with q>1, as they are less sensitive to rare species and sparse datasets [ɑ-diversity regardless of the researcher’s technical decision-making [q>1 is particularly useful for datasets in which there is a high variation in S across samples  and in which the community is not fully characterized [Because of the long tail of rare molecular entities and the variable, uninformative N, computing a spectrum of diversity indices can provide a more complete picture of the diversity profile of a sample , 119. Indatasets . Importan-making . An adden-making . Leveragβ-diversity metrics are commonly used across MME data to quantify the extent to which the molecular entities observed differ between samples. In general, β-diversity is assessed by using similarity or dissimilarity metrics, and the choice of metric depends on the data’s limitations as well as the research question of interest. For example, in the commonly used metric Euclidean distances , species absences are equally as informative as presences, making them unsuitable for characteristically sparse MME datasets [datasets . In contβ-diversity in molecular data. For example, in metabarcoding, the practice of removing rare species has recently been shown to artificially decrease α- and increase β-diversity, while reducing the discriminatory power of β-diversity [If the emphasis is on the presence and absence of molecular entities rather than on their relative abundances, MME data can be converted into incidence data, and Sorensen dissimilarities can be calculated . Importaiversity .α-diversity, β-diversity indices based on Hill numbers have been developed [q parameter. For example, when comparing the similarity between two communities, it is possible to give more weight to shared abundant species than to shared rare species. By varying the value of q, these β-diversity assessments can draw continuous diversity profiles. As q increases, the removal of rare species becomes less central to the calculation of β-diversity.Similar to eveloped , 130, anβ-diversity metrics can be decomposed into nestedness, which quantifies the extent to which samples with smaller numbers of species are subsets of more species-rich samples, and turnover, which quantifies the replacement, or difference of species between samples [β-diversity changes can shed light into the ecological processes driving molecular diversity .β-diversity indices can be used to estimate how the relatedness among molecular entities affects observed community changes [β-diversity metrics can shed light into ecological processes . Null mccession ).β-diversity. For example, the analysis of functional diversity focuses on the diversity of characteristics or functional traits of diversity components [Considering similarities between MME and CE data can provide further avenues of innovation for analysis of mponents . Functiomponents , providimponents , 130. Fomponents .The future of microbiome research will likely involve combining various MME techniques  to determine “who is doing what” . Multi-oCombining omics with other techniques can increase the specificity of the results and address complex ecological questions, providing new insights into host-microbiome interactions, revealing the trophic structure of a community, and shedding light on the metabolic pathways linking community members. For example, by combining stable isotope fingerprinting (SIF) with classic metaproteomics, direct protein-SIF, allows for the study of the individual physiology and metabolism of microbes within a community . In one The greatest challenge to the future of multi-omics is arguably data interpretation , as the From an ecological standpoint, integrating MME data begins by studying whether different MME data behave similarly across an ecological gradient of interest. As MME data interpretation becomes more advanced and ecological questions become more sophisticated, the joint analysis of multiple MME data matrices will require more advanced statistical methods. Here, statistical advances related to the fourth corner problem, which refers to the difficulty of inferring trait-environment relationships directly from environmental, species abundance, and trait data, may become instrumental , 158. As