In order to estimate the effects of high hydrostatic pressure treatment at 400 MPa for 0 min and 10 min  and high hydrostatic pressure in combination with sous-vide treatment  on the quality of largemouth bass stored at 4 °C for 30 days, the physicochemical changes were evaluated by microbiological determinations, pH, sensory evaluation and texture analysis, and the flavour changes were analysed by solid-phase microextraction–gas chromatography–mass spectrometry (SPME-GC-MS) and amino acid automatic analyser. The results show that HHP-0+SV and HHP-10+SV treatment effectively inhibited microbiological growth and attenuated physiochemical changes  of largemouth bass fillets. HHP+SV treatment prolonged the storage period of largemouth bass fillets for 24 days. The content of total free amino acids in control (CK) samples was high, but HHP+SV treatment caused the loss of free amino acid content. Especially when stored for 30 days, the total free amino acid content of HHP-0+SV and HHP-10+SV was only 14.67 mg/100 g and 18.98 mg/100 g, respectively. In addition, a total of 43 volatile compounds were detected and elucidated, among which hexanal, heptaldehyde, octanal and nonanal showed a decreasing tendency in HHP groups and an increasing trend in HHP+SV groups throughout the storage. In many places, especially in coastal areas, fish is considered to be an excellent source of nutrition, containing more proteins than other animals . LargemoSince consumers prefer ready-to-eat products that are as natural as possible and have sufficient shelf life, it is important to consider appropriate minimal processing without altering the content and bioavailability of major nutrients . In thisSV temperature cooking is a food preparation process of heating samples for a long time at a lower temperature . The fooTwo emerging technologies, HHP and SV are regarded as effective and economical methods. The utilization of HHP or SV alone to effectively control food spoilage has been widely reported. However, reports are rare exploring the effects of HHP combined with SV on largemouth bass as far as we know. In this study, the effects of HHP and HHP+SV on the quality of largemouth bass were evaluated by measuring the indexes of microorganism, pH value, sensory, colour, texture, GC-MS and FAA.Cultured largemouth bass (weight of 0.38 ± 0.07 kg) were originated from Wuhan, and subsequently transported to the slaughter room alive in plastic bags filled with crushed ice within an hour. After stunning, the scales were removed. The whole bass were immediately washed with sterile water, and then cut into similar-sized fillets (about 7 cm × 5 cm × 2 cm). Fillets were randomly divided into five groups, and then fillets were subjected to HHP and SV pretreatments. Then, the fish fillets were drained, packaged separately in high-density polyethylene bags and stored at 4 °C. Three samples of each group were selected randomly for detection and analysis at day 0, 6, 12, 18, 24 and 30 of the storage.The vacuum-packed fish fillets were immersed in a constant temperature water bath , which contained 1.3 L distilled water that was pre-heated to 90 °C. The cooking process was ended until the core temperature of samples reached 90 ± 5 °C. In the light of pre-experiments, the cooking time was 15 min. Then the samples were immediately cooled to 7 ± 2 °C in an ice water bath.The treatment of the packaged fish fillets was implemented using high hydrostatic pressure equipment . High hydrostatic pressure treatments were performed at 400 MPa for different times. High hydrostatic pressure treatment was to place the packaged bass slices in the high hydrostatic pressure chamber (1 L) with water as the medium (25 °C), and keep them for a certain time when the pressure reached 400 MPa (30 s). There were three replicates for each treatment, and 300 g bass slices were used in each replicate. After HHP treatment, SV treatment was carried out according to (1)High hydrostatic pressure treatment at 400 MPa for 0 min. (HHP-0)(2)High hydrostatic pressure treatment at 400 MPa for 10 min. (HHP-10)(3)High hydrostatic pressure treatment at 400 MPa for 0 min in combination with sous vide. (HHP-0+SV).(4)High hydrostatic pressure treatment at 400 MPa for 10 min in combination with sous vide. (HHP-10+SV).A total of 10 g sample was added into 90 mL 0.85% sterilized physiological solution and the mixture was homogenized for 60 s. The microbiological evaluations were performed based on the work of Rezaeigolestani et al. , where sThe pH value was recorded on a pH meter . Fish muscle (10 g) was thoroughly homogenized with 100 mL distilled water and soaked for 30 min, then filtered to determine the pH.According to the description of Liu et al.  with somTexture profile analysis was measured using a Texture Analyzer  equipped with a specific cylindrical probe placed horizontally on a heavy platform, according to the method reported by Yu et al. . The dev−1.According to Shi et al. . Solid-pm/z using positive ionization with an electron energy of 70 eV. Volatile compounds can be identified by comparing the mass spectrometry results of volatile compounds with data stored in the National Institute of Standards and Technology (NIST08) spectrum database. Area normalization was used to quantify volatile compounds.The temperature in the GC oven was held at 40 °C for 2 min, then increased at 2 °C/min to 90 °C, held for 5 min, then increased at 8 °C/min to a final temperature of 250 °C. The ion source was EI. The MS source and transfer line were kept at 230 °C and 280 °C, respectively. The mass spectrometry data were collected over the full scan range of 35 to 350 g for 15 min with 10 mL supernatant. Taking 5 mL supernatant, pH was adjusted to 2.0 with 6 mol/L NaOH, and then volume was adjusted with distilled water to 10 mL. The extraction was filtered using a 0.22 µm filter membrane and applied to an automatic amino acid analyser . Three parallels for each sample.FAAs were measured according to Yu et al. . Fish fip < 0.05 by DPS software analysis and Duncan’s multiple range test. All figures were obtained by Origin 2017 .All data are presented as mean ± standard deviation. The significant difference was at the level of p < 0.05). The higher initial TVC for fresh largemouth bass was 4.5 log CFU/g (p < 0.05). Ekonomou et al. [The main factor limiting the shelf life of largemouth bass fillets is the development of microbial groups during the preservation. Meat products are often easily degraded by microbial activities. og CFU/g . Shuai Zu et al.  found thu et al. . CompareThe changes in pH values for HHP, HHP+SV and the control are given in Sensory characteristics of largemouth bass are shown in p < 0.05). This showed a firm and elastic taste was obtained by SV pretreatment, which was popular with consumers. The initial hardness of the largemouth bass fillets from the CK group was 14,499 g. After treatment, the hardness showed a decreasing trend and gradually decreased as the storage time increased. The HHP+SV showed a lower elasticity value when compared with HHP. The springiness of bass fillets decreased with an increase in storage time and reached the minimum value at 30 days. Chewiness refers to the time required to chew food at a fixed speed to reduce it to a size suitable for swallowing [Texture analysis is displayed in allowing . CohesivThe flavour changes of the pretreated bass fillets under storage conditions were investigated by HS-SPME GC/MS. A total of 43 volatile compounds including alcohol (10), aldehyde (10), ketone (3) hydrocarbons (17) and others (3) were detected in the sample.As shown in Pseudomonas, Shewanella and Enterobacteriaceae) and chemical reactions mainly including the oxidation of polyunsaturated fatty acids [In the first group of compounds, hexanal, heptaldehyde, octanal and nonanal exhibited a tendency to reduce gradually in HHP groups throughout the whole storage, whereas they tended to increase in HHP+SV groups. The relative content of the second group of compounds in fish fillets was low. The species and peak areas of aldehydes were significantly reduced after HHP and UHP+SV treatment. Only the HHP-treated samples did not detect benzaldehyde, indicating that the HHP-only treatment caused the samples to lose their nutty aroma, while the HHP+SV treatment avoided this phenomenon and maintained the original flavour characteristics. After treatment and storage of the samples, trans-2-heptenal and 1-nonanol compounds disappeared. In some cases, metabolites produced during corruption may be further metabolized by microorganisms . These aty acids .p < 0.05). There were significant differences among CK, HHP and HHP+SV treatments. The total amount of FAA in CK samples was high. This indicates that HHP and HHP+SV treatment would lead to the loss of free amino acid content. The total FAA content of the HHP group was higher than that of the HHP+SV group at early storage (0 day) and late storage (30 day), which indicates that SV treatment can slow down the production of amino acid molecules. In the latter stage of storage (30 day), the total FAA in the HHP+SV group decreased more than that in HHP group. In the composition of FAA, alanine was the only amino acid with the highest content in untreated samples. Higher levels of specific amino acids in HHP and HHP+SV samples compared with untreated samples may influence the perception of sweet , bitter , umami (umami amino acids include glutamic and aspartic acid) and tasteless  attributes in comparison with untreated samples  on day 18 and 24 of storage, respectively. HHP-0+SV- and HHP-10+SV-treated fish fillets exceeded 7 log CFU/g on day 30 of storage. This showed that HHP+SV treatment can prolong the storage period of bass compared with HHP. The results of sensory evaluation, texture characteristics, GC-MS and FAA showed that HHP+SV had higher sensory and texture and good wind characteristics compared with HHP, but it would cause the loss of free amino acid content of bass. In general, HHP+SV processing is considered to be an effective technology, compared with HHP processing and SV processing alone.


Giardia duodenalis remains a neglected tropical disease. A key feature of the sustained transmission of Giardia is the ability to form environmentally resistant cysts. For the last 38 years, proteomics has been utilised to study various aspects of the parasite across different life cycle stages. Thirty-one articles have been published in PubMed from 2012 to 2022 related to the proteomics of G. duodenalis. Currently, mass spectrometry with LC-MS/MS and MALDI-TOF/TOF has been commonly utilised in proteomic analyses of Giardia, which enables researchers to determine potential candidates for diagnostic biomarkers as well as vaccine and drug targets, in addition to allowing them to investigate the virulence of giardiasis, the pathogenicity mechanisms of G. duodenalis, and the post-translational modifications of Giardia proteins throughout encystation. Over the last decade, valuable information from proteomics analyses of G. duodenalis has been discovered in terms of the pathogenesis and virulence of Giardia, which may provide guidance for the development of better means with which to prevent and reduce the impacts of giardiasis. Nonetheless, there is room for improving proteomics analyses of G. duodenalis, since genomic sequences for additional assemblages of Giardia have uncovered previously unknown proteins associated with the Giardia proteome. Therefore, this paper aims to review the applications of proteomics for the characterisation of G. duodenalis pathogenicity and the discovery of novel vaccine as well as drug targets, in addition to proposing some general directions for future Giardia proteomic research. Giardia can cause a gastrointestinal disease known as giar-diasis, which occurs through the ingestion of cyst-contaminated food and water. It is estimated to cause 280 million cases of intestinal diseases annually worldwide, with a higher prevalence in areas with low sanitation and hygiene standards [Giardia comprises nine recognised species, with Giardia duodenalis  being the only species capable of infecting a broad range of hosts, while other species only infect specific hosts. G. duodenalis has eight classified assemblages (A–H) [G. duodenalis [Giardia spp. rely on the observation of cysts or trophozoites in stool samples, with limited options for immunological-based assays and molecular methods for the diagnosis of giardiasis [The protozoan parasite tandards ,2. Accores (A–H) . Among tes (A–H) . As a reodenalis . While podenalis , in rareardiasis .G. duodenalis has a simple life cycle, consisting of disease-causing trophozoites and infectious cysts. Infection begins when the host ingests the cysts, which then enter the digestive tract, where they ultimately develop into motile trophozoites in the small intestine through a process called excystation. The trophozoites can then transform back into their cystic form through a process called encystation. Giardia can be maintained in the laboratory and induced to complete its life cycle by using laboratory methods [ methods , which oG. duodenalis proteomics is not a new field and genomic information on the protein-coding genes of G. duodenalis has been published [G. duodenalis, complementary to genomic discoveries [Giardia’s simple life cycle, the binucleated trophozoite is able to divide without cytokinesis, producing highly infectious and adaptable cysts [G. duodenalis encystation as a focal point of research from both genomics and proteomics perspectives. In addition to expanding proteomics to address specific inquiries on infectivity and pathogenesis, there is increasing recognition that the available proteomic characterisation of Giardia is mainly derived from assemblage A, while proteomic data on assemblage B remain sparse, leading to a biased understanding of this parasite’s biology [G. duodenalis have been conducted to identify molecular targets with which to block Giardia transmission [Giardia genome [Giardia proteins, summarising key findings from studies on both assemblages A and B. We further identify important areas of research with which to inform the future direction of G. duodenalis proteomics.Although ublished , only a coveries ,11,12,13le cysts , thus re biology . In the smission , the dira genome , and pota genome  or specia genome ,20. TherCurrently, there is no vaccine available to prevent giardiasis in humans, while an unrefined veterinary vaccine only reduces the symptoms and duration of cyst shedding in cats and dogs . CommonlG. duodenalis alternates between cysts and trophozoites, it is crucial to compare the proteomes of trophozoite encystment at different stages. The change in morphology, followed by the modification of protein expression levels, is vital for encystment, thus implying that the proteins involved in this process may be key targets for vaccine and drug development. Several proteomic studies have aimed to investigate the biochemical aspects of the parasite , the sigG. duodenalis sporozoite encystation where the trophozoites and cysts were isolated from faeces. High-performance liquid chromatography (HPLC) was then used to fractionate the isobarically tagged proteins for further proteomic analyses using a database search [G. duodenalis, raising their potential as likely targets for the development of vaccines and chemotherapies that inhibit the transmission of G. duodenalis into the epithelial cells of hosts [One powerful proteomics tool with which to estimate stage-specific protein abundance is isobaric tags for relative and absolute quantitation (iTRAQ), an advanced multiplexing technique that assists in identifying and quantifying proteins simultaneously . Lingdane search . As a ree search ,31. In pof hosts .G. duodenalis trophozoites, Camerini and her co-researchers performed a bottom-up proteomic study using a combination of SDS-PAGE, Western blot, and a mass spectrometry analysis, and detected several fluorescent protein bands in NBDHEX-treated samples, with only one or two cysteines found to be specifically NDBHEX-modified in each protein. For instance, modified Cys137 and Cys140, discovered in thioredoxin reductase, gTrxR, and Cys347, of gα-TUB structure proteins, were covalently bound to NBDHEX, suggesting that the functions of many of these protein targets were inhibited when treated with NBDHEX. The study also found that NBDHEX killed G. duodenalis trophozoites at a dose five times lower than that of MTZ , thus supporting the idea that this drug agent could be a good option for treatment-refractory giardiasis in the future [Other studies have examined the potential of using repurposed drugs. Camerini et al. (2017) investigated the use of the anticancer drug 6- hexanol (NBDHEX) to find protein targets other than phosphate dehydrogenase . To idene future .G. duodenalis, which may inform vaccine development. A study by Langford et al. (2002) highlighted that surface proteins might be crucial targets of protective IgA responses, and they identified several biotin-labelled proteins from total cell lysates of G. duodenalis WB strain (assemblage A) trophozoites and G. duodenalis GS/M (assemblage B) trophozoites using mass spectrometry [Cell surface proteins have been identified to be the source of antigens in the intestinal lumen between two genetic assemblages (A and B) of trometry . Anotherd et al. 02 highlitrometry . A surfaGiardia strains and assemblages upon infection from a proteomics point of view. To the best of our knowledge, only two proteomics studies have been published that aimed to analyse the virulence proteins in G. duodenalis: one study investigated G. duodenalis in humans and cockatoos [Although genome sequences for assemblages A, B, and E have been published ,36,37, lockatoos , and theockatoos . G. duodenalis that may be associated with the virulence of giardiasis in mammals, namely BRIS/95/HEPU/20141 (B-2041) and BRIS/83/HEPU/106 (H-106) [Giardia independent of assemblage and geographical variation. They utilised label-free shotgun proteomics by using a gel-based platform (LC-MS/MS) combined with an in-solution platform  to assess the total protein abundance and proteome coverage. According to the study, 1376 proteins were identified in both strains, with a large core of 76.6% common proteins being shared between the two strains [Giardia antigens in B-2041 compared to H-106, concluding that the greater the antigenic variation between different strains at the intra-assemblage level, the more diverse the population of the parasite that is capable of evading the host immune responses [G. duodenalis becomes a key source of variability in the virulence of different strains of the same assemblage.The first study, by Emery et al. (2014), presented the findings of a comparative shotgun proteomic study between two different strains of assemblage A of  (H-106) . B-2041  (H-106) ,39, the  strains ,41. B-20esponses . Indeed,G. duodenalis using 2D electrophoresis [G. duodenalis, isolated from dog stool, and identified 187 proteins, 27 of which matched hypothetical proteins, while the remaining ones had been previously annotated. Among the 27 hypothetical proteins, there were 20 soluble proteins and 4 insoluble proteins, and another 3 were found in both soluble and insoluble proteins. From the remaining 160 annotated proteins, the numbers of soluble and insoluble proteins found were 79 and 53, respectively, while another 20 were identified in both proteins. Most of the identified proteins were involved in metabolic processes, catalytic activity, nucleic acid binding, hydrolases, and oxidoreductases [Candida albicans [Pseudomonas sp. [Shigella flexneri [G. duodenalis with proteins of human G. duodenalis may lead to a better understanding of the biology of this parasite as well as of the virulence of giardiasis in different species, which can aid efforts to control zoonotic giardiasis. Notably, despite reports of high rates of G. duodenalis infection in domestic dogs in several countries, far fewer studies have been performed on canine isolates than on human isolates of G. duodenalis, highlighting a significant gap in the understanding of the risk of the transmission of giardiasis from dogs to humans [Another study, by Coelho et al. (2016), analysed the proteomic mapping of soluble and insoluble protein fractions of trophozoites in canine phoresis . The groductases . Additioalbicans , Pseudomonas sp. , and Shiflexneri . A compao humans ,46.G. duodenalis across in vitro encystation to understand the pathogenicity mechanism of Giardia [G. duodenalis from its in vitro culture into peptides. The peptides were then quantified and characterized by using LC/MS/MS followed by a database search. By comparing the proteins extracted from trophozoites, low-bile primed (LB) trophozoites, and the 16 h post-induction of encystation and mature cysts, the authors identified a total of 3863 proteins across all stages [G. duodenalis has a significant shift in overall protein expression across encystation. For example, proteomic changes during encystation include the downregulation of cell adhesion proteins, which is linked to changes in the cytoskeleton that cause the ventral disc and flagella to disappear [Several studies have focused on comparing the proteome changes of  Giardia ,22. A re Giardia . In theil stages ,13,47,48l stages ,16. Theyisappear .G. duodenalis, as secretory proteins have recently been shown to play vital roles in the cross-talk between cells [Giardia assemblages A and B to quantify secreted proteins, which may act as the main mediators of giardiasis pathology. In their study, the soluble and cytosolic fractions of the Giardia proteins were extracted from in vitro cultures of two different strains, namely the WB strain (assemblage A) and the GS strain (assemblage B). Two MS techniques were used, Q-Exactive and Orbitrap MS, to identify the proteins. The proteins were then quantified by using intensity-based absolute quantification (iBAQ). A total of 1542 GS proteins and 1641 WB proteins were identified by using Q-Exactive [Giardia are cathepsin B cysteine protease and other members of the Giardia family of cysteine-rich proteins. In addition, Duoborg et al. discovered a new virulence factor, Giardia tenascin, which contributes to a novel mechanism of Giardia pathogenesis and was found to be highly abundant in the whole secretome [Increasing interest in host–parasite interactions in pathogenesis has led to the introduction of secretomic studies in en cells . Mass spen cells ,13,50. DExactive . The autecretome . G. duodenalis during the colonisation of intestinal epithelium cells (IECs) [Giardia interact with the IECs of a host. These results align with findings by Ringqvist et al. (2008) that Giardia releases glycolytic enzymes when it infects a host [Similarly, other studies sought to uncover changes in the upregulation and downregulation of functional proteins, particularly in host–pathogen interactions ,26,51. As (IECs) . This sts a host .G. duodenalis is unable to perform de novo pyrimidine or lipid synthesis, relying solely on nutrients from the host [G. duodenalis trophozoites initiate cytoskeletal changes as the parasite attaches to IECs very strongly, leaving marks on the cell surface and hence disturbing the arrangement of the actin cytoskeleton [Giardia infection, severing its protective role from the actin cytoskeleton [Giardia pathogenesis.In addition, Ma’ayeh et al. (2017) noted competition in obtaining nutrients between the parasite and host cells, given the similarity of the metabolic proteins released by both the parasite and host . For insthe host ,53. A fuskeleton ,55,56, eskeleton ,57. CollGiardia encystation, namely deacetylation [G. duodenalis [G. duodenalis DHHC proteins in protein S-palmitoylation during Giardia encystation [G. duodenalis, and concluded that the presence of DDHC proteins in the encysting parasites indicates that the protein S-palmitoylation is maintained and involved in cell signalling, protein-sorting, and protein exporting throughout encystation. However, these proteins showed variation in intracellular localization in trophozoites and patterns of cyst wall expression, suggesting that differentially regulated palmitoylation in Giardia encystation enables the parasite to adapt to various environments [Several post-translational modifications (PTMs) of proteins are reportedly involved in tylation  and phostylation . Consequodenalis ,60,61,62Giardia proteins also cause antigenic variation, as seen in the presence of VSP subpopulations across different Giardia assemblages or different strains of the same assemblage [G. duodenalis, namely WBC6 and WBA1 (both representing assemblage A), as well as GS/M-83-H7 (classified as assemblage B), showed that VSP5 (GL50803_113793) and VSP44 (GL50803_113450) were identified in strain WBC6, VSPH7 (GSB150963) was identified in strain GS/M-83-H7, and VSPA6 (GL50803_221693), a hypothetical protein, was identified in strain WBA1 [PTMs of semblage . Müller 7 GSB1509 was idenGiardia that have been identified thus far [Giardia [Recently, Emery-Corbin et al. (2021) utilised a chromatin proteomics analysis, using mass spectrometry for histone identification and MaxQuant software for PTM mapping, to generate a molecular map of histone methylation, acetylation, and phosphorylation modifications in this parasite core histone . The grothus far ,65. Addi[Giardia .G. duodenalis under stress revealed a total of 2999 lysine acetylation (Kac) sites on 956 proteins and 8877 2-hydroxyisobutyrylation (Khib) sites on 1546 proteins when G. duodenalis was under sugar starvation [G. duodenalis was cultured under sugar starvation for 72 h, indicating their involvement in energy conversion metabolism. They concluded that the correlation of acetylation and 2-hydroxyisobutyrylation expressed proteins linked to amino acid metabolism, suggesting that Giardia’s regulatory mechanism involves dynamic changes in acetylation modification to supply energy in the absence of glucose [Another recent study, by Zhu et al. (2021), that utilised a global approach in investigating metabolic conversion mechanisms of arvation . The aut glucose . G. duodenalis have provided significant information with which to elucidate the differential proteins expressed across the encystation of G. duodenalis. With the aid of advanced tools, such as iTRAQ labelling, the proteome of G. duodenalis cysts has been successfully investigated [G. duodenalis are mainly focused on identifying novel vaccine and therapeutic targets, as well as on improving the understanding of G. duodenalis pathogenicity, including describing the PTMs of Giardia proteomes, as illustrated in In the last ten years, proteomics studies on stigated . In addiGiardia assemblages, beyond the human-infective assemblages A and B. Through these inquiries, new insights into inter- and intra-assemblage variations and their association with evolutionary origins, biology, and protein function can be gained. Nevertheless, genomic characterisation needs to be developed first to allow the future comparative proteomic analyses of these isolates to move forward [G. duodenalis may also focus on investigating the post-transcriptional regulation of this parasite, especially on translational inhibition relating to Giardia’s Pumilio homology proteins (Puf), which may have a crucial role in the encystation of this parasite [Giardia by Emery-Corbin et al. (2021) [In the future, proteomic research may focus on comparing soluble and insoluble proteins extracted from cysts and trophozoites, which may enable the discovery of common proteins in soluble and insoluble fractions of both cysts and trophozoites that could become key chemotherapy targets for giardiasis. Proteomics can be further utilised to identify different protein sites that could be targeted by newly discovered drug agents ,67 to de forward . Future parasite . With th. (2021) , chromat. (2021)  and cycl. (2021) , by usinGiardia proteins contribute significantly to the parasite’s strain virulence and its pathogenicity mechanism. The PTMs in the parasite expand the protein functionality and diversity, which allows G. duodenalis to acquire an elusive mechanism with which to bypass host defences and successfully complete its life cycle in a host. Currently, some transcriptomics studies on G. duodenalis have elucidated the expression of a few virulence factors during the interaction, such as high-cysteine membrane proteins [Giardia interaction, as these proteins may damage the epithelial cells of a host and contribute to an evasive bypass of a host’s immune system. Since proteomics enables the comparison of proteins between healthy individuals and those infected with Giardia, this approach also has significant potential to enlighten researchers about effective disease management for giardiasis. Although Giardia proteomics is currently limited to enhancing the basic knowledge of the parasite proteome and host–parasite interaction, extending proteomics for clinical applications may someday aid in the direct management of giardiasis.The PTMs of proteins  and pro-proteins . HencefoGiardia is a remaining paucity of information on assemblages that infect species other than humans. Nevertheless, advancements in the characterisation of proteomes of most assemblage A and B strains, and a few assemblage E strains, raises the potential to elucidate proteomes of other assemblages in the future, given that a strong fundamental proteomics workflow with which to investigate Giardia has now been established. However, proteomic studies on Giardia assemblages other than A, B, and E remain of limited interest, because these assemblages are not infective to humans, there is sparse genomic data for reference, and the cost of downstream processing is prohibitive. This lack of understanding of other assemblages constitutes a prevailing gap in the understanding of Giardia as a whole, which may have implications for current efforts to understand how the human-infective parasite interacts with hosts.A key limitation in proteomics research on G. duodenalis, and investigating the PTMs of Giardia proteins, findings from each of which inform the others. Current proteomics studies on Giardia are still limited because the genomic sequences are not well-established in all eight assemblages of the parasite. Even with this being the case, we can foresee that the integration of proteomics technology and transcriptomics will enable the identification of novel genes in the parasite that are overlooked by gene prediction programs. In the near future, advanced technologies such as MALDI-TOF, HPLC, and iTRAQ labelling techniques may be used more frequently. In conclusion, even with the limited and emerging published genomic information to date, the existing literature undoubtedly reveals the potential of proteomics to significantly enrich the understanding of the parasite and shine light on new avenues of research for the effective control of giardiasis.This review summarises the importance and applications of proteomic studies in studying giardiasis. Proteomics has been successfully applied to study the parasite across varying lines of inquiry, such as determining protein targets for vaccine and drug development, investigating the virulence of giardiasis, understanding the pathogenic mechanism of


Rearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance.To gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student’s paired t-test and statistical significance for the cell viability assay was a one-way ANOVA.GDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM.Outcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer.The online version contains supplementary material available at 10.1186/s12885-023-10559-1. Our results showed that Palbociclib inhibited cell proliferation after rGDNF treatment and dual treatment with Palbociclib and TAM in the presence of rGDNF resulted in greater inhibition compared to both individual treatments. Our findings support that GDNF-RET signaling indirectly transcribes CCND1 and subsequently promotes cell proliferation through the cyclin D1-CDK4/6 complex and that inhibition of GDNF-RET signaling resensitizes cells to TAM.Given our findings, one potential therapeutic option to treat GDNF-RET-EGR1 resistant BC cancer would be to inhibit both cyclin D1-CDK4/6 and ERα signaling. There are currently three CDK4/6 inhibitors available to treat advanced BC. Ibrance , Venzenio (Abemaciclib), and Kisqali (Ribociclib), which are all currently FDA approved for combination use with specific endocrine therapies –41. FurtOverall, we observed that GDNF-RET signaling forms a positive feedback loop with EGR1, and in turn, EGR1 upregulates CCND1 to induce cell proliferation, therefore promoting tamoxifen resistance. Inhibition of this signaling pathway, through Palbociclib treatment, inhibited cell proliferation and resensitized cells to tamoxifen, resulting in further cell inhibition. This data suggests that dual treatment of cyclin D1-CDK4/6 inhibitors and endocrine therapy could have a positive impact on treating advanced endocrine resistant breast cancer patients. Further studies are needed to determine the prognostic impact of these inhibitors in GDNF-RET-EGR1 expressing breast cancer and whether these inhibitors should be used alone or in combination with current endocrine therapies.Additional file 1: Table S1. sgRNA and primer sequences. Additional file 2: Figure S1. Cell viability of TamSEGR1-OE and TamSVc cells in the presence of 1uM TAM. Data are represented as mean ± SEM. Figure S2. Western blot from Fig. Figure S3. Cell viability assay in TamS cells showing the four treatment controls corresponding to Fig.


Modern agri-food systems generate large amounts of crop-based biomass that are unfit for direct human consumption but potentially suitable for livestock feeding in production of meats, milk, and eggs. This study aims to develop novel feeds for cattle from some of those biomass materials through the natural microbial-driven processes of ensiling. Fruit and vegetables resembling supermarket discards were ensiled alone or co-ensiled with corn crop residues, mushroom wastes, etc. via laboratory experiments. Longitudinal sample analyses showed that (co-)ensiling was successful, with pH and fermentation acids changing rapidly into desirable ranges . The (co-)ensiled products had key nutritional parameters comparable to those of good quality forages commonly used on dairy farms. Additionally, in vitro incubation experiments indicated that the ensiled products could substitute certain conventional feeds while maintaining diet digestibility. Findings from this pilot study provide a proof of principle that quality novel feeds for cattle can be generated by co-ensiling food discards and low-value crop residues. Future research and animal feeding trials to demonstrate the utility of this approach can help societies more effectively utilize untapped biomass resources, strengthening the regenerative capacity of agri-food systems towards a more sustainable food future. In this context, strategies that promote circularity and expand the regenerative capacity of the agri-food systems are of paramount importance7. Toward this end, recovery and re-use of biomass materials that are already produced in primary production but ‘lost’ from the linear food supply chain present a unique opportunity. These biomass materials exit the food supply chain mainly as indigestible, unpalatable, or undesirable biomass (IUUB) typically unfit for direct human consumption. Oftentimes, these materials are still rich in carbohydrates, proteins, and other macro- or micro-nutrients9. These nutrients can be upcycled via livestock feeding, because of farm animals’ innate capability to digest a wide variety of biomass. Therefore, optimizing the use of IUUB materials through livestock presents a viable solution for producing more food with less resource-, environment-, and climate-burdens.The global food system has and will continue to weather unprecedented challenges in multiple dimensions. At the core are fundamental issues of meeting ever-growing demand in terms of food availability and equity10: (i) crop residues upon removal of human-edible parts, typically inedible/indigestible; (ii) processing byproducts (or co-products) consisting of the remaining residues from food/beverage processing industries, generally unpalatable; (iii) food waste/discards from various stages of the food supply chain, usually undesirable to humans. Tremendous amounts of IUUB materials are routinely used as feedstuffs, totaling 1140 million metric tons (MT) of crop residues and 600 MT processing byproducts annually worldwide11. Still, very large amounts remain untapped resources for livestock-based upcycling. This is particularly true for food waste/discards, which is estimated to be 1300–1600 MT globally13 and projected to increase in coming decades14.There are three broad categories of IUUB materials15. Among the reasons are the economics of precision feeding16, the inherent variability in nutritional attributes of food wastes from diverse sources17, concerns over disease transmission 18, and undervaluation of impacts of food waste on society and sustainability19. With the pressing challenges of sustainable food security amid climate change, there have been renewed interests in transforming food waste into feeds for livestock21. It has been demonstrated that proper thermal treatment can render post-consumer food waste (from restaurants and homes) safe for monogastric species; risk of transmission of pathogens and parasites is minimal18. Japan and South Korea reportedly convert and utilize 36% and 45% of such food waste for animal feeding24. Notably, food waste is not created equal20. Pre-consumer plant-based food discards would be more suitable for feeding ruminants. For example, feeding studies have demonstrated that marketplace fruit and vegetable discards could replace up to 50% of concentrate feeds in the control diet to support satisfactory steer growth25, or substitute 6–18% of concentrate feeds to support milk production (25 kg day−1) of Holstein cows26. Interestingly, there remains a huge opportunity as wasted fruit and vegetables constitute the single largest food waste stream globally and nationally in the United States29. To exploit the opportunity requires scientific and management intervention because fresh fruit and vegetable discards are prone to spoilage, which can reduce palatability and nutritional value and increase risks of microbial contamination30. Therefore, finding ways to extend the storage life and conserve the nutrients for safe feeding is essential.Feeding food waste to animals has had a long history, but the practice has become less common in modern production systemsHere, we report the efficacy of preserving fruit and vegetable discards via ensiling. Ensiling is a microbial-driven process commonly used on dairy farms for the very purpose of preserving freshly harvested feed crops (around 35% DM) for prolonged storage and feeding. But data are scarce on the utility and robustness of ensiling fruit and vegetable discards, which are much wetter . We hypothesized that fresh fruit and vegetables can be co-ensiled with crop residue biomass to produce high quality feeds for cattle. To test the hypothesis, we conducted a series of laboratory experiments in which fresh fruit and vegetables were ensiled alone or in combination (i.e. co-ensiling) with plant biomass such as corn crop residues or spent mushroom compost (see Methods for details). The overarching goal of our research is to develop viable solutions for optimal utilization of IUUB materials, contributing to sustainable livestock production toward enhanced regenerative agri-food systems. Specific objectives included: (i) assess the feasibility of ensiling fresh fruit and vegetables (FFV) alone, or co-ensiling with various biomass substrates via longitudinal studies, (ii) determine key nutritional characteristics of ensiled products, and (iii) evaluate the digestibility of ensiled products via in vitro incubation experiments.32. Not surprisingly, a considerable amount of liquid accumulated at the bottom of the ensiling vessel; about 110–150 mL per vessel (or 160–210 mL per kg of ensiling substrate) were collected on day 42  by gravity drainage. The liquid samples contained 7% DM, with soluble carbohydrates comprising 39.5% of DM. The samples were also enriched in P and Na concentrations but had lower pH, compared to the liquid sample that seeped from the bulk materials of diced FFV during ensiling preparation . Clearly, proper effluent management is required if FFV is to be ensiled alone on farms.Three ensiling trials were conducted (see Methods and Supplementary Tables Co-ensiling FFV with corn cobs (CC) or spent mushroom compost (SMC), as part of Trial 1, had key parameters falling within the desirable ranges as well , mushroom stems (MS), wet brewers’ grains (WBG), and SMC, all originated from local sources  in some samples, mostly the co-ensiling in Trial 2. Butyric acid or isobutyric acid were not detected in any of the samples.Lactic acid, as the most desirable acid among the various fermentation acids generated from the ensiling process, was produced in the greatest quantities throughout the experiment in all cases Fig. c. The en34. However, high concentrations of acetic acid produced during ensiling generally indicates suboptimal fermentation conditions35. Nevertheless, when consumed by ruminants, acetic acid in silage can be absorbed from the rumen and utilized for energy by cows34.Trial 3 was conducted to generate ensiled products for use in subsequent in vitro digestibility experiment  falling within the ranges of desirable values, except for FFV + CS + WBG with acetic and propionic acids exceeding the references.The fundamental purpose of ensiling is to preserve the biomass that is otherwise susceptible to spoilage, whether it is fresh-cut forage crops or fruit and vegetable discards such as in our case. Listed in Table Clostridia growth. Clostridia bacteria are one of the most common undesirable bacteria that may persist in unstable silage, leading to higher dry matter loss and poor silage palatability35. In our study, the wet substrates . The rapid decrease in pH within the first few days of fermentation might have inhibited Clostridia growth, which requires pH 4.5 or above36. It is also possible that the substrates (FFV and the other biomass) were not laden with Clostridia bacteria, unlike forage crops harvested directly from agricultural fields that may be contaminated with soil.Conventional silage-making requires substrate dry matter to be > 30–32% to minimize the risks of Crude protein (CP) concentrations in ensiled products ranged from 6.9% to 18.1% DM Table . Compare38. In our study, ensiled FFV alone had lower ADF (13.6%) and NDF (15.9% DM) concentrations relative to conventional forages. Co-ensiled products had ADF concentrations in the range of 20.4–40.4% DM and NDF concentrations 32.4–57.8%, which are comparable to those found in grass or alfalfa hay (Table 35).Fibrous feedstuffs are indispensable in ruminant diets for maintaining normal rumen fermentation and rumination, lowering the risk for rumen acidosis and post-calving disorders 39. High quality forages such as alfalfa have TDN typically ranging from 50 to 60% DM while low quality forages range from 40 to 50% 40. Accordingly, ensiled products evaluated in our study had TDN concentrations ranging from 50 to 60% which are equivalent to TDN content commonly observed in medium–high quality forages  is the sum of digestible fiber, protein, lipid, and carbohydrate components of a feedstuff. It is the simplest form of energy evaluation of a feed, directly related to digestible energy of the feedes Table .10 CFU g−1) or relatively safe (< 6.0 log10 CFU g−1) for conventional feeds , except for FFV + CC. In the latter case, a crack in the lid for one of the three ensiling vessels was observed at the end of the experiment, indicating a probable breach of anaerobic conditions and thus resulting in greater mold growth . Higher than acceptable mold counts may lead to depressed digestibility, feed intake, and animal performance. Fusarium and Mucor species were the predominant mold species identified in the samples. Fusarium is a large genus of filamentous fungi widely distributed in soil and plants; some have been reported to produce mycotoxins41. Mucor does not produce mycotoxins42. Yeast concentrations in our samples were considered acceptable for well-preserved conventional silage , except for the ensiled product of Trial 2 (Table Ensiled products tended to be ‘cleaner’ (lower molds and yeast counts) than day 0 samples Table . Mold co−1), and changes in VFAs makeup, compared to 0 h , increases in ammonia-N concentration , as well as health indices. Studies with in-depth evaluation of rumen microbial responses can help shed light on whether novel feeds would modify methanogen profiles and potential implications regarding enteric methane emissions. Further, IUUB materials generated in agri-food systems are diverse and versatile. Studies expanding the scope to test various crop biomass as co-ensiling substrates with fresh produce materials could bring about new upcycling opportunities. For fresh produce discards, particularly unsellable fruit and vegetables from retail markets, understanding the variability  and how it may affect animal performance is very important. Additionally, logistics such as source distribution and transport distance will need to be considered for lifecycle-based assessment of carbon footprints. Comprehensive analyses to address multi-sustainability objectives, e.g. socioeconomic impact, climate change, land-, water-, and nutrient-footprints from novel feeds substituting conventional feed ingredients will be critical to demonstrate broader impacts and to inform resource- and climate-smart policymaking.At the farm level, feed security with adequate and uninterrupted supply of quality feeds is central to sustain a given livestock operation. A farm’s feed security, or feed insecurity risks, are subject to various factors. Some risks are external and beyond the farmer’s control. Examples include feed supply and price volatility, competition from non-agriculture sectors, or increasing adverse weather events aggravated by climate change leading to crop failure or harvest loss. Developing low-impact non-competing novel feeds from wasted food as well as inexpensive, reliable, locally available crop biomass can help alleviate some of the uncertainties and mitigate relevant risks. This would help enhance farming resilience and benefit societies with nutrient-rich food produced more sustainably.Global demands for meats, milk, and eggs are expanding, stemming increasingly from developing economies. The livestock sector must strive to meet the growing demands meanwhile addressing sustainability challenges and lowering unintended consequences. Innovative strategies and practices that mitigate feed vs. food competition and leverage livestock to upcycle human-unfit biomass are essential. Findings in our pilot study can help advance the endeavor in developing viable solutions to support sustainable livestock production while strengthening the regenerative capacity of the agri-food system toward a more livable future.28. Together, they accounted for 55.3% of the total (by weight) of the national supermarket fruit and vegetable waste data27. Our formula consisted of: (i) watermelon, romaine lettuce, apple, potato, and tomato, each making up 14% by weight in the mix, plus (ii) orange, cantaloupe melon, onion, bell pepper, and banana, each 6%.Three ensiling trials were conducted for a period spanning over a year. For consistency as well as cross comparison, we created a formula to make fresh fruit and vegetable mixtures with items obtained from a discount produce market to be used in each trial. The formula included ten types of fruit and vegetables that topped the list of unsellable fresh produce in US supermarkets, as reported by Buzby et al.50. Both MS and SMC substrates were obtained from a local large-scale mushroom facility. For ensiling preparation, the MS specimen was brush-cleaned of clinging compost materials and the SMC specimen was air-dried and clumps broken down by hand to smaller pieces. The WBG originated from a local brewery and was kept under refrigeration until beginning the ensiling experiment. Analyses of physical, chemical, and nutritional parameters of the raw substrates are in Supplementary Table Substrates for co-ensiling trials included corn cobs (CC), corn stalks (CS), mushroom stumps (MS), spent mushroom compost (SMC), and wet brewers’ grains (WBG), in addition to FFV. The raw materials for CS were obtained from one southeast Pennsylvania dairy farm and CC from another. Corn cobs were sieved to pass 7 mm. Corn stalks were processed first through a silage chopper then ground in a cutting mill to pass 1 mm. Mushroom stumps are the lower part of the stem that is removed and discarded upon harvest and prior to processing and packaging. Spent mushroom compost is the growth media cleared out of the mushroom house after the growing cycles, which consists of remains of the original components such as wheat straw, corn stalks, peat moss, etc.Three ensiling trials were conducted. Trial 1 tested the ensiling of FFV alone, co-ensiling of FFV with CC, and co-ensiling of FFV with SMC. Trial 2 tested co-ensiling of FFV with CS, MS, SMC, and WBG. Trial 3 was conducted with the co-ensiling of FFV + CS and FFV + CS + WBG, respectively; the ensiled products were used in a subsequent in vitro incubation experiment. Ratios of substrates in the co-ensiling treatments are listed in Supplementary Table For each ensiling trial, a fresh batch of FFV was made according to the formula described earlier. Raw items were cut into 14 mm cubes using a commercial food processor . To avoid clogging the processing unit, hard stems from peppers and bananas as well as rinds from melons were manually cut to approximately 14 mm pieces then added to the mixture. Processed FFV was bulked in a plastic tub, the content was thoroughly mixed by hand upon ensiling preparation.Ensiling was conducted using 0.95 L polyethylene containers with snap-on lids. The containers and lids were wiped with 70% ethanol immediately prior to filling. Each co-ensiling treatment was prepared by weighing out the substrates into a tub and homogenizing manually. FFV alone or with co-ensiling materials was packed into the ensiling containers, tamped down to eliminate air pockets and filled to the top to limit air-filled head space. Lids were snapped on, with circumferences coated with waterproof silicone sealant to prevent air exchange. To permit gas release while maintaining anaerobic conditions in the vessel, a water-filled fermentation lock was inserted through a rubber grommet on the lid and sealed with silicone sealant. Vessels were placed on a laboratory bench under ambient light and temperature (approximately 20 °C). Preliminary trials indicated that the temperature inside the vessels fluctuated in a narrow range of 18–20 °C during ensiling; temperature was not monitored in subsequent trials.The longitudinal experiments were conducted for 42 days in Trials 1 and 2; sample collection took place on days 0, 3, 7, 14, 28 and 42. Trial 3 was conducted for 28 days, and samples were collected on day 28 to be used for the in vitro experiment. At each sampling time, three replicates of the vessels per treatment were removed and the ensiling process terminated; the content was emptied into a plastic tub and mixed thoroughly with a sterile plastic scoop for sampling and analysis.In vitro incubation was conducted to evaluate digestibility when the ensiled products were added (as novel feeds) to total mixed ration (TMR) made for lactating cows at the Marshak Dairy. The latter is a 180-cow research and teaching facility at the University of Pennsylvania, School of Veterinary Medicine. The TMR consisted of grass hay, corn silage, triticale, ground corn, proteins, byproducts, minerals and vitamins. Each of the ensiled products  was a composite made from equal aliquots of three replicates. All feed samples were oven-dried and ground to pass 2 mm in a high-speed spice grinder. The in vitro incubation experiment consisted of seven treatments, in triplicate, with inclusion rates of 5% and 10% per novel feed, plus ground corn and protein mixes (C + P) added to diets of 10% novel feeds for the purpose of balancing nutrients against cow requirements , each containing 0.75 g feed sample and 12 mL of MacDougall’s buffer. To add inoculum, each vial was purged with CO2 for 30 s, then 6 mL inoculum was pipetted in, and the vial was purged again for 30 s. Vials were sealed with rubber septa and metal lids and crimped. Once all 21 vials had been filled, 60 mL syringes were inserted into the top for collecting and recording gas production, and the vials were placed into the water bath with gentle agitation at 37 °C for 24 h. Upon completing the incubation, all vials were removed from the water bath, gas volumes were recorded, subsamples (~ 2–3 mL each) were taken to check pH, and the remaining contents in the vial were strained through 4 layers of cheesecloth to separate the solid and liquid fractions. Approximately 500 mg of the solid fraction and 0.75 mL of the liquid fraction from each vial were placed into 2-mL Eppendorf tubes (in duplicate) and stored at − 80 °C until extraction for DNA. Additionally, to prepare samples for VFAs/ammonia analysis, 5 mL of the liquid fraction was spun at 10,000 × g for 10 min. Four mL of the supernatant was transferred to a new tube and 800 µL of 36% metaphosphoric acid was added, and the tube was spun at 15,000 × g for 20 min. The remaining supernatant was removed and stored in a -20 °C freezer until sending to a certified service laboratory  for analysis.Rumen fluid was obtained via stomach tubingThe same steps were repeated for 21 control vials with 0 h incubation. After inoculum was added, vials were gently agitated and then immediately processed for sampling following the same procedure described above.For samples collected during the ensiling experiment, a subsample of approximately 75 g was used for gravimetric DM determination using a forced-draft oven (80 ˚C 24 h). Another subsample, 50 g, was used for pH determination (1:1 ratio in deionized water). A third subsample, roughly 400 g, was sent to the same certified laboratory (above) for analyses. The remaining materials were archived in a − 20 °C freezer.https://www.foragelab.com/Services/Forage-and-Feed/Chemistry). The nutritional indices included all macro- and micro-nutrients as well as fiber profiles. The selected samples included those ensiled products i.e. at the end of ensiling trial (day 42 or day 28), and in some cases samples obtained at the beginning of experiments (day 0 or day 3). Furthermore, selected samples were analyzed for yeast and mold counts with mold identification. Additionally, liquid effluent from the ensiling of FFV alone was obtained by gravity drainage and analyzed for dry matter, water-soluble carbohydrates and minerals.Analyses of ensiling process parameters included concentrations of lactic, acetic, propionic, butyric, and iso-butyric acids plus 1, 2 propanediol, in addition to pH and DM. These analyses were conducted for longitudinal samples collected during the course of the ensiling experiments. Additionally, selected samples were analyzed for a suite of nutritional indices  and R338 (5′-TGCTGCCTCCCGTAGGAGT-3′) barcoded with a unique 12-base error-correcting Golay code for multiplexing as described in Song et al.53. Polymerase chain reaction was performed using the Accuprime Taq DNA Polymerase System . Thermal cycling conditions involved an initial denaturing step at 95 °C for 5 min followed by 20 cycles  and a final extension step at 72 °C for 8 min. Amplicons from each sample were combined and each library was added to a pool in equimolar concentration. The final pool was bead-purified using Agencourt AMPure XP Beads . Sequencing was performed at the PennCHOP Microbiome Core using the Illumina MiSeq platform.For samples obtained from the in vitro incubation, a portion of the liquid fraction from each vial was analyzed at the certified service laboratory (above) for VFAs  in addition to ammonia. Genomic DNA was extracted from 250 µL of the liquid fraction and 250 mg of the solid fraction of each incubation vial using the repeated bead beating and column (RBB + C) method followed by extraction with a commercial kit  as described in Yu and Morrison54 with mean separation by Fisher’s protected least significant difference test at a probability level of 0.05. Pairwise comparisons of in-vitro fermentation parameters at initial conditions vs. 24 h incubation were by one-sided t-test in SAS.Ensiled sample results for nutritional, ensiling process, and mold/yeast evaluation parameters reported by the certified laboratories were entered in a Microsoft Excel spreadsheet, means and standard deviations calculated. Graphical presentation of results was developed in Excel. In vitro fermentation parameters analysis of variance was conducted using SAS General Linear Models55. Briefly, paired end sequence data was de-multiplexed and amplicon sequence variants (ASV) were assigned using the DADA2 plugin56. A phylogenetic tree was constructed using FastTree 257. Taxonomy was assigned based on a pre-trained Naive Bayes classifier trained on the Greengenes database (v13.8) for the 16S rRNA gene spanning the V1-V2 region58. The between sample diversity (weighted and unweighted UniFrac distances) were computed using the ‘qiime diversity’ plugin.The raw 16S-rRNA amplicon sequencing data was processed through the QIIME2 (2020.6) pipeline59 implemented in the vegan package for R was used for beta diversity matrices. Pairwise Wilcoxon Rank Sum Test was used to determine differences in bacterial genera between treatment groups. The P values were adjusted using the Bonferroni correction method. A P value of 0.05 was used to define significance.A nonparametric permutational multivariate ANOVA (PERMANOVA) testSupplementary Information.


Background: The epidemiology and prevalence of the Human T-cell leukemia virus type-1 (HTLV-1) infection represent a recommended priority by global health agencies. An in-depth revision to update the status of this infection in countries including those of the Eastern Mediterranean Regional Office (EMRO) of the World Health Organization is hence required. Methods: Ninety-seven studies evaluating the HTLV-1 infection in low- and high-risk populations in EMRO countries were retrieved from the international electronic databases and were used to assess the epidemiological status of the infection in these countries. Results: Most epidemiologic reports were published from Iran, with more than 50% of Iranian prisoners and around 4% of healthy individuals reported to have the infection. In Egypt, a considerable prevalence of the virus spans around 1.11% of blood donors. Foci of HTLV-1 infection are also present in some countries and require a careful epidemiological evaluation. In the other EMRO countries, a lower prevalence that does not exceed 1% was reported. Conclusion: The epidemiology and prevalence of HTLV-1 in the EMRO countries require a tight revision and update. Published studies reveal a scarce distribution of the virus in the African countries of EMRO, while a lower prevalence is denoted in the Asian countries of EMRO, except in Iran, where the prevalence is high. Human T-cell leukemia virus (HTLV) types 1 and 2 are the first identified human retroviruses. HTLV-1 was isolated from cultured lymphocytes of a patient with cutaneous lymphoma in 1979 . HoweverMycobacterium tuberculosis and human immunodeficiency virus (HIV) was linked with a worse clinical course [HTLV-1 can be transmitted vertically from a mother to her child primarily by prolonged breastfeeding , throughl course ,16. Likel course . To datel course . The mosl course . The preInternational electronic databases, including PubMed, Scopus, Web of Science, Index Medicus for the Eastern Mediterranean Region, and the Islamic World Science Citation Index, were searched until August 2021. The search strategy was as follows:  AND (“Eastern Mediterranean” OR “Middle East” OR “North Africa” OR the names of each of the countries in the region) with no limitation to a specified field. Studies investigating HTLV-1 prevalence among populations at low and high risk of exposure to HTLV-1 were selected. Low-risk populations included healthy people, blood donors, pregnant women, and household-based survey participants. High-risk groups consisted of multi-transfused patients , people who inject drugs (PWID), prisoners, and patients with HIV or sexually-transmitted diseases. The studies were selected based on HTLV-I detection using serological screening tests such as enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay, with or without confirmation by complementary techniques such as immunoblotting or polymerase chain reaction (PCR).p-value less than 0.05 was considered statistically significant.The Eastern Mediterranean Region (EMR) comprises 15 Asian countries  and 7 African countries  with a population of nearly 679 million individuals . StudiesA total number of 1597 citations were identified in the five electronic databases. By screening the titles and abstracts, 190 non-duplicated citations discussing HTLV-1 prevalence among low- and high-risk populations in the EMRO region were identified. Forty-three articles published before 2001 were excluded, and the full texts of 147 remaining documents were reviewed. Forty-one papers were not included in this review. The reasons for excluding showed in The highest number of published reports on HTLV-1 infection in Southwest Asia was from Iran. However, general population-based reports of HTLV-1 infection were only identified from three among 31 provinces, including Razavi Khorasan, South Khorasan, and Golestan in the north and east parts of the country  19,20,2,22,23. MRecent phylogenetic and phylodynamic analysis of the long terminal repeat region of HTLV-1 in blood samples of 100 seropositive individuals from the north and south parts of Iran revealed that Iranian isolates belonged to the globally-distributed HTLV-1 Cosmopolitan subtype A . These rIn order to check the published literature related to HTLV-1 infection prevalence among Iranian blood donors, the studies published until August 2011 were previously reviewed, and the prevalence of infection was estimated as low as 0.12% (95% CI: 0.05–0.29%); it ranged from 0.015% in the southwest to 0.38% in the northeast of Iran . A meta-In terms of clinical findings among HTLV-1 infected individuals in Iran, most studies focused on the neurologic manifestations of the disease. Shoeibi et al. reported that gait disturbances followed by sensory and urinary complaints are the most common manifestation in HAM/TSP patients in Mashhad, Iran. In contrast, other clinical symptoms, including back pain, constipation, and erectile dysfunction, are not commonly reported. The hypertonicity is mainly reported in lower limbs, and most of the patients develop brisk tendon reflexes in all limbs. In terms of sensory findings, foot paresthesia was a common compliment, and impairment of vibration sensation was more prominent in lower limbs . While cHTLV-1 prevalence was also investigated among high-risk Iranian populations, including patients with thalassemia or hemophilia, those undergoing hemodialysis, HIV-positive individuals, PWID, and prisoners. Significant high rates of HTLV-1 infection  were previously estimated among Iranian multi-transfused patients . CurrentScreening 1654 serum samples from the general population of Mashhad did not demonstrate any HTLV-2 positive cases . This waIn countries of Southwest Asia other than Iran, HTLV-1 infection seems non-endemic . In a suIn a cross-sectional survey performed in Qatar, 0.18% of nearly 200,000 blood samples donated from 2013 to 2017 showed HTLV-1/2 seroreactivity in enzyme-linked and line immunoassays, and the trend for this infection was increasing from 0.08% to 0.23% . LikewisHTLV-1 infection rates were associated with the nationality of participants in most Arab countries. In Saudi Arabia, slightly higher infection rates were reported among non-Saudi blood donors 0.074–0.113%) as compared to Saudi donors (0.046–0.055%) ,113. Nev% as compNot a single report documented the prevalence of HTLV-1 infection in other Asian countries, including Bahrain, Oman, Palestine, Syria, United Arab Emirates, and Yemen of EMRO . HoweverAs with Iran, the limited genotype data indicated that the most frequent subtype of HTLV-1 is the “transcontinental” subgroup of cosmopolitan (A) subtype over the EMRO region .Only one epidemiologic survey in Egypt was conducted and reported one HTLV-1-positive case among 90 Egyptian blood donors during 2005–2006 . UnfortuIn the present study, we skimmed the known literature to evaluate the epidemiological status of HTLV-1 infection in the countries of the EMRO region. We relied on the scientific reports indexed in specific electronic databases. We also tried to include common international and regional databanks. Therefore, some reports about the prevalence of HLTV-1 infection may be lacking. These include indexed reports in other sources or data not included in any database but presented in conference proceedings, dissertations, organizational reports, and magazines. Reports considering HAM or ATL published from the EMRO region were also excluded.Moreover, the heterogeneous studied population of the surveys included in the present review may make the comparison of the results difficult. In addition, confirmatory laboratory tests were not performed in some studies or were not the same across all the studies. The present review included the studies with even small sample sizes to cover the entire EMRO population as possible.The epidemiologic data on HTLV-1 infection in most parts of the Eastern Mediterranean region are limited or lacking. The available published literature reveals that the prevalence of HTLV-1 infection in part of the Eastern Mediterranean region, except Iran, is low. The evidence on HTLV-1 prevalence in the African part of EMRO is scarce, and the epidemiological data in endemic regions are not updated. A more systematic and more frequent analysis of the status of HTLV-1 infection in EMRO countries is required, especially in countries such as Iraq and Morocco, where the HTLV-1 infection was identified and reflected the presence of foci in these countries. Finally, the longstanding infection control strategies, including the HTLV-1 screening of blood donors in most endemic regions, requires future validation by complementary techniques, such as PCR, to increase the specificity of the infection diagnosis.


Insulin receptor substrates (IRSs) are proteins that are involved in signaling through the insulin receptor (IR) and insulin-like growth factor (IGFR). They can also interact with other receptors including growth factor receptors. Thus, they represent a critical node for the transduction and regulation of multiple signaling pathways in response to extracellular stimuli. In addition, IRSs play a central role in processes such as inflammation, growth, metabolism, and proliferation. Previous studies have highlighted the role of IRS proteins in lung diseases, in particular asthma. Further, the members of the IRS family are the common proteins of the insulin growth factor signaling cascade involved in lung development and disrupted in bronchopulmonary dysplasia (BPD). However, there is no study focusing on the relationship between IRS proteins and BPD yet. Unfortunately, there is still a significant gap in knowledge in this field. Thus, in this review, we aimed to summarize the current knowledge with the major goal of exploring the possible roles of IRS in BPD and asthma to foster new perspectives for further investigations. Insulin receptor (IR) and insulin-like growth factor (IGF) receptor (IGFR) signaling regulate a variety of cellular processes including glucose metabolism, differentiation, and cell growth . AlthougThe insulin receptor substrate family consists of six members, IRS1–6 . All famIn detail, the IRS members IRS1, IRS2, IRS5, and IRS6, are widely expressed in human tissues, whereas IRS4 presents a tissue-restricted pattern including the brain, thymus, and embryonic tissues. IRS3 acts as a pseudogene and is not expressed in humans ,19,20,21The IRS signaling pathway starts with the binding of ligands such as insulin and the insulin-like growth factor (IGF) to their corresponding receptors. This leads to autophosphorylation of the receptor via tyrosine residues  22]. IR. IR22]. In detail, the PI3K-AKT pathway is a critical node for cell signaling pathways . It reguIn addition to the PI3K-AKT pathway, upon activation of the IRS proteins, Grb2 binds to the YVNI motifs of the IRS proteins and triggers the activation of RAS-RAF-ERK1/2 signaling cascades  that regIRS proteins contain more than 50 S/T phosphorylation sites for the several IRS kinases at their COOH terminals ,29. In cAs a chronic inflammatory disease, asthma plays a special role among lung disorders. The exact etiology of asthma remains not fully understood, but it has a multifactorial and heterogeneous background where genetic and environmental factors have pivotal roles . In rece+) through major histocompatibility complex type II (MHC-II) in conjunction with costimulatory molecules such as CD80, CD86, and OX40L. Together with IL-4, this process triggers the differentiation of CD4+ to Th2 and T follicular helper cells (Tfh), which favors the humoral antibody production of IgE by inducing B cell isotypic commutation and plasma cell differentiation [In detail, during the sensitization phase, antigen-presenting cells (APCs), mainly dendritic cells (DCs), internalize allergens that have passed the epithelial barrier and migrate toward the draining lymph nodes ,44,45,46ntiation ,48. Th2 ntiation ,46,47.In the late phase, locally produced chemokines may cause the recruitment of macrophages, eosinophils, neutrophils, and Th2 lymphocytes ,42,44. TUnderstanding the precise contribution of these signaling pathways to M2 macrophage polarization is a key factor in the search for potential therapeutic strategies to disrupt the proinflammatory signaling pathways in severe asthma and allergic diseases in the future ,52,53.IL-4 and IL-13 have multiple important functions including the regulation of allergic responses . As statRecently, IRS2 has received increasing attention due to its role in the signaling cascade of type 2 cytokines, such as IL-4 and IL-13 ,52. IRS2During IL-4 and IL-13 signal transductions, IL-4 binds to IL4Rα and induces the dimerization with the IL-4 γchain or IL13Rα1, while IL-13 interacts with IL13Rα1 and leads to the ‘heterodimerization’ of IL4R-α . These cOne of the downstream effectors of IRS2 is the p85α subunit of phosphatidylinositol 3-kinase (PI3K), which in turn activates several downstream signaling pathways and regulates epithelial cell migration, suggesting that IL-4 may have a beneficial effect on airway epithelial cell repair via recruitment of the IRS2 pathway . InteresWhile tyrosine phosphorylation appears to activate IRS2 signaling, serine phosphorylation does the opposite by inhibiting p85α binding and PI3K activation and promoting IRS2 degradation ,62. The Bronchopulmonary dysplasia is the most common pulmonary complication in infants born before 30 weeks of gestational age and contributes to long-term morbidity and mortality . AccordiHyperoxic injury is one of the main risk factors in BPD pathogenesis, which is thought to disrupt critical signaling pathways that induce lung development, including branching and septation . SeveralIRS proteins are the main adaptor molecules of IGF signaling ,82. One IGF signaling is important for normal lung development. Thus, in in vivo experiments, components of IGF signaling were shown to have different expression patterns in different compartments during embryogenesis .Serum IGF-1 levels are significantly reduced in patients with BPD . HoweverFgf10 expression during the development of BPD [It has been shown in previous studies that the downstream targets of IGF signaling play a role in the development of BPD . Howevert of BPD ,95,96.It is also known that the activation of PI3K/AKT via hydrogen has a protective effect on alveolar epithelial cells type 2 (AEC2) during hyperoxia treatment in animal experiments .The mammalian target of rapamycin (mTOR) is a part of the PI3K/AKT pathway and can be induced by hyperoxia ,100,101.ERK1/2 is the other main downstream target of IGF signaling . It has Fgf10 is one of the most significant developmental genes expressed in the submesothelial mesenchyme of the developing lung [Fgf10 encodes a secreted diffusible protein that acts in a paracrine manner through the epithelial receptor Fgfr2b [ing lung ,107. Fgfr Fgfr2b ,109. Mosr Fgfr2b . There ir Fgfr2b ,111,112.r Fgfr2b . Althougr Fgfr2b . Other sr Fgfr2b ,116. In r Fgfr2b . In a str Fgfr2b . TGF-β takes a central role in postnatal lung development and alveologenesis. TGF-β signaling is activated by the binding of TGF-β to the type II TGF-β receptor (TβRII) . This coAs increased pulmonary TGF-β expression precedes most BPD-related pathophysiological manifestations and infiltration of neutrophils and monocytes into the lungs occurs during disease development ,128,129.Although IRS family members are the common proteins for the IGF signaling cascade, there is no study that focuses on the relationship between IRS proteins and BPD. Previous studies showed that ERK1/2, AKT, and mTOR phosphorylate IRS proteins at Ser/Thr motifs and negatively regulate the IRS protein’s actions . TherefoIt has been extensively shown that IRS proteins are major players involved in many processes such as inflammation, growth, metabolism, and proliferation. However, their roles in lung disorders are insufficiently understood. Here, we have comprehensively summarized the current knowledge on how IRS might be possibly involved in the pathogenesis of asthma and BPD, focusing on the signaling pathways of IGF, IL-4, FGF, and TGF- β receptors. Even though some of the evidence is still weak and hypothetical, it allows new perspectives for further investigations in order to shed more light on the role of IRS in asthma, BPD, and other pediatric lung diseases.


What are the fertility and family planning concerns of female physicians, and how do they affect women’s career trajectory in medicine?This qualitative study with structured 1:1 qualitative interviews with 16 female physicians identified fertility and family-building concerns that were used to develop and pilot test a survey for women in medicine. Results from the pilot survey of 24 female physicians found that 71% had delayed childbearing and 67% had altered their career for family-building reasons.These findings suggest that female physicians may delay childbearing and make substantial accommodations in their careers to support family building. This qualitative study assesses fertility knowledge and concerns and evaluates barriers to family building and impact on academic attrition reported by female physicians. Pervasive gender disparities exist in medicine regarding promotion, achievement of academic rank, and appointment to leadership positions. Fertility and childbearing concerns may contribute to these disparities.To assess fertility knowledge and concerns and evaluate barriers to family building and impact on academic attrition reported by female physicians.This qualitative study used mixed methods; first, structured 1:1 interviews exploring fertility knowledge and family-building concerns were conducted among 16 female physicians between November 2019 and May 2020. Transcripts were coded in Dedoose and used to develop a survey instrument with subsequent pilot testing conducted among 24 female physicians between April 2020 and September 2020. Data analysis was performed from January 2021 to March 2021.Fertility knowledge, perceptions of peer and institutional support surrounding childbearing, factors contributing to delayed childbearing, and impact of family planning on career decisions.Among 16 women who completed qualitative interviews, 4 (25%) were Asian, 1 (6%) was Black, 1 (6%) was multiracial, and 10 (63%) were White; mean (SD) age was 34.9 (4.0) years. Evaluation of fertility knowledge revealed 3 notable themes: (1) inadequate formal fertility education, (2) informal learning through infertility experiences of patients, peers, or personal struggles, and (3) desire to improve medical education through early introduction and transparent discussions about infertility. Exploration of childbearing concerns similarly revealed several salient themes: (1) high incidence of delayed childbearing, (2) perceived lack of peer and administrative support, and (3) impact of family building on career trajectory. These themes were borne out in pilot testing of the survey instrument: of 24 female physicians , 17 (71%) had delayed childbearing and 16 (67%) had altered their career for family-building reasons.Qualitative interviews identified fertility and family building concerns among female physicians and were used to develop a tailored survey for women in medicine. These findings suggest that female physicians may delay childbearing and make substantial accommodations in their careers to support family building. A large-scale national survey is needed to better characterize the unique fertility, childbearing, and parenting needs of women in academic medicine to better understand how these concerns may contribute to academic attrition. Studies have reported low fertility knowledge among US women and medical trainees.15 Despite this knowledge gap, little research has been done on the role of medical education in providing trainees with information about age-related fertility decline and fertility preservation.Although the reasons underlying these disparities are complex, fertility and family-building concerns among women in medicine may be contributing to academic attrition. Female physicians are more likely to delay childbearing compared with nonphysicians,22 This perceived lack of support may cause women to alter their careers to accommodate childbearing. In a survey of 600 female physicians, more than 70% reported that having children had influenced their career.9 These findings highlight a need to better understand how experiences of family building among women in medicine may influence career trajectory, and to explore the extent to which family-building concerns contribute to the persistent gender disparities within academic medicine.In addition to delaying childbearing, female physicians may face inadequate support when ready to grow their family.The objectives of this study were to (1) qualitatively assess fertility knowledge and family-building concerns among female physicians, and (2) develop a survey instrument to characterize the unique needs of women in medicine surrounding fertility and family planning, and how these needs may impact career trajectory.COREQ) reporting guideline.23This qualitative study was approved by the Northwestern University institutional review board and all participants provided written informed consent prior to participation. The study followed the Consolidated Criteria for Reporting Qualitative Research  medical education/awareness of the role of age in fertility, (2) financial cost and insurance coverage, (3) workplace support, (4) support outside of work, and (5) influence of fertility and family planning on career path  were recruited from the women in medicine conference, 13 (30.2%) were recruited from the Facebook Physician Moms Group, 16 (37.2%) were referred to participate in the study from a colleague/friend who heard about the study, and 10 (23.2%) saw the study shared on other social media platforms.Among 16 women who participated in qualitative interviews, 4 (25%) were Asian, 1 (6%) was Black, 1 (6%) was multiracial, and 10 (63%) were White; there were 13 attending physicians, 1 resident physician, 1 clinical fellow, and 1 pharmaceutical medical director; mean (SD) age was 34.9 (4.0) years. Most participants were heterosexual , married/partnered , and had children . No participants refused to participate or dropped out. Respondent demographics and characteristics are outlined in Interviews revealed 3 major subthemes regarding fertility knowledge: (1) inadequate formal education, (2) reliance on informal education, and (3) a desire for improved formal medical education . Participants also expressed that information they received in medical school was outdated or misleading. For example, one woman recalled being taught that fertility decline begins at age 35 and felt this led to a false sense of security among women in their early thirties. Others recalled that fertility was presented in a way that fostered fear, without education about fertility preservation.“Learning in medical school is what I would call outdated ... based pretty much on some older data and was always associated with fear and panic and you’re never going to be able to get pregnant …We certainly had no instruction whatsoever on family planning” (participant 1015).Citing inadequate formal education, participants reported learning about infertility informally through the experiences of patients, peers, or personal experiences. Notably, one participant described how she realized the high prevalence of infertility among female physicians through speaking with peers and cited these interactions as critical support as she faced her own infertility diagnosis.“I learned through talking to my coworkers … I went through a miscarriage and now infertility and I think that I was able to feel very supported in it because a lot of my colleagues are also very open about what they’ve been through with miscarriage, infertility, and pregnancy” (participant 1004).Overall, participants endorsed a need for improved formal fertility education. Many believed that information about age-related fertility decline and fertility preservation options should be presented early in medical training, with some specifying that this information be conveyed before medical students have selected their intended specialty to allow fertility considerations to play a role in specialty choice.“Medical school is when people are still considering different fields and telling us different fields may affect their fertility choices and options … so, before you’re in the time where you’re really thinking about starting a family, to have the information ahead of time would be good” (participant 1008).Participants supported facilitating open conversations among physicians about work-life balance, family building, and infertility, as well as incorporating dialogue with trusted colleagues and mentors, especially among those with similar career paths and goals.Interviews revealed 3 major subthemes surrounding barriers to family building, including (1) delayed childbearing, (2) insufficient resources and support, and (3) impact of childbearing on career trajectory. Exemplary quotes for each theme and subtheme are outlined in Although participants were aware of age-related fertility decline, 14 of 16 shared that they had delayed or were currently delaying childbearing. Ten cited barriers in training and/or work environment as the primary cause of this delay, whereas 4 cited a lack of a partner as the primary reason.Participants discussed that insufficient mechanisms of support, including workplace culture and policies, played an important role in their decision to delay childbearing. Without knowledge of certain benefits, or belief that these benefits were insufficient, women felt ill-equipped to start a family. They noted a lack of formal introduction to benefits, such as parental leave and childcare, and little guidance in navigating fertility preservation options. Consequently, many women felt their decisions surrounding childbearing were underinformed. To prevent these feelings of uncertainty, interviewees recommended having transparent discussions with trainees on fertility, family planning considerations, and institution-specific benefits .“I think that it would also be helpful for medical students and trainees to know what their options are, what insurance covers ... It wasn’t even touched at my orientation here as an attending” (participant 1001).Participants also emphasized the importance of changing the broader culture within medicine, citing a lack of support from physician peers and leadership. Two women perceived a gender difference in leadership support and stressed the importance of having women in leadership positions, especially when creating policies pertaining to pregnancy, parental leave, and parenthood. Interviews demonstrated that family planning affected career trajectory. Some women had specifically chosen their specialty based on flexibility for family planning. One woman had left academic medicine for private practice to increase autonomy over her schedule. Women who experienced stressors such as lack of schedule flexibility or social or institutional support were more likely to report symptoms of burnout and, in some cases, considered leaving medicine entirely. Thirteen women stated they would reduce their clinical time for additional flexibility for childbearing, and 2 women stated they would consider changing specialties or leaving medicine altogether to accommodate pregnancy and parenthood. One woman had left clinical medicine owing to a combination of these stressors.“It’s something I really love and enjoy doing but it’s not the only thing and having kids is something very important to me … if I felt like my current position was taking away from that I’d be happy to make accommodations or even leave medicine” (participant 1001).Among the 24 women who completed the pilot survey between January 2020 and February 2020  correctly identified that fertility declines most precipitously after the age of 35 years. Although 21 respondents (88%) correctly identified that fecundability among women aged 43 to 45 years is less than 5%, 15 (63%) overestimated the ability of ART to overcome age-related fertility decline within this age group. Using a 5-point Likert-type scale , participants rated the information sources they relied on to learn about the role of age and fertility. As seen in Seventeen respondents (71%) reported delaying childbearing due to medical training or choice of specialty. Again, using the same 5-point Likert-type scale, participants rated how influential a variety of factors were in influencing their decision to delay childbearing. As seen in There were 11 respondents (46%) who had personally experienced infertility, of which 7 (64%) had used IVF for conception. There were 10 respondents (42%) who had considered fertility preservation, of which 8 (80%) had previously sought consultation, including 5 respondents who had frozen eggs and 2 respondents who were in the process of freezing (or planning to freeze) their eggs. As seen in 15 our results suggest that women in medicine are aware of the impact of age on fertility. Despite this knowledge, most women in our study had delayed childbearing and a large proportion had personally experienced infertility, consistent with previous studies suggesting high rates of infertility among female physicians.9 Furthermore, women tend to overestimate the ability of ART to overcome age-related fertility decline. These data highlight the need for improvement in education about the limitations of currently available technologies to treat infertility and, more specifically, to overcome ovarian aging. Participants felt that formal training about the impact of age on fertility and the high rates of infertility among female physicians should occur early in trainees’ education to allow for informed decision-making in choice of specialty and family-building plans.Contrary to previous studies that found low fertility knowledge among medical trainees,27 In line with prior research,30 women in the present study demonstrated high interest in fertility preservation. Nevertheless, most women received information on fertility preservation from informal sources . Options for fertility preservation should accompany any discussion of fertility in the formal medical school curriculum. Furthermore, most pilot survey respondents did not know if egg freezing was covered by their health insurance plan. Cost is a well-documented concern of women contemplating elective egg freezing.31 Therefore, residency and fellowship training programs should provide their trainees with information on institution-specific insurance coverage and estimated costs of fertility preservation.32In recent years, oocyte and embryo cryopreservation have emerged as a means of increasing reproductive options.41 The marked prevalence of fertility and family-building concerns in the current study suggest that facilitating open communication and peer support between physicians may be an important step in this culture shift.In addition to highlighting a need to improve fertility education, our results suggest that women in medicine make substantial adjustments to their career trajectory to accommodate family building. Strikingly, 29% of survey respondents declined opportunities for career advancement, 21% chose a different specialty, and 17% changed their work setting from academic to private practice to accommodate having children. These results are alarming, particularly in light of known gender disparities that exist within academic medicine in time-to-promotion, achievement of academic rank, and appointment to leadership positions. It is, therefore, imperative to understand and ultimately address the family-building concerns specific to women in academic medicine. In the present study, commonly cited concerns pertained to unsupportive workplace policies and culture. Women cited a lack of support from physician peers and leadership, particularly related to taking time off for pregnancy, maternity leave, infertility treatments, or parenting obligations.Considering the current focus within academic medicine on wellness and female physician retention, our study addresses critical issues impacting women in medicine. The pilot survey is an initial effort to characterize the unique fertility needs and family planning concerns of women in medicine. Future directions include a larger national survey which will allow us to better describe factors that were not fully evaluated in this study due to its small sample size. National data will allow us to stratify findings based on level of training, setting of practice, and specialty to determine if substantial variations in experience exist based on these factors.This study had some strengths. The use of semistructured qualitative interviews in this study provided a narrative account of women’s experience with fertility in academic medicine and allowed for in-depth exploration of a nuanced topic. Questions in the pilot survey were based on themes that arose from the qualitative interviews and, therefore, addressed topics most pertinent to female physicians. Evaluation of the survey instrument by a panel of psychologists and reproductive endocrinologists ensured content validity, and subsequent evaluation among female physicians ensured appropriate response distribution, clarity, sensitivity, and depth.This study also had limitations. For example, it was limited by its potential for self-selection bias. Specifically, personal experiences with infertility or childbearing may have motivated participation in the qualitative interviews or survey. In addition, our study sample was primarily recruited from a women-in-medicine conference and social media via a Facebook Physician Moms group, which may not be a representative sample of all female physicians. Furthermore, our study sample primarily consisted of attending physicians, which precluded us from exploring differences based on career stage. Although conclusions from the pilot survey are limited by its small sample size, our preliminary data yielded striking results and has set the stage for distribution among a national sample to obtain more robust data about fertility and family-building concerns among female physicians.Persistent gender disparities exist in leadership and faculty rank in academic medicine despite equal matriculation into medical school. Results of this study suggest that women may alter their career trajectory to accommodate family building. A large-scale national survey is needed to better characterize the unique fertility, childbearing, and parenting needs among women in academic medicine to understand and, ultimately, address the gender gap.