Glutamine (Gln) is an abundant nutrient used by cancer cells. Breast cancers cells and particularly triple-receptor negative breast cancer (TNBC) are reported to be dependent on Gln to produce the energy required for survival and proliferation. Despite intense research on the role of the intracellular Gln pathway, few reports have focussed on Gln transporters in breast cancer and TNBC.The role and localisation of the Gln transporter SLC38A2/SNAT2 in response to Gln deprivation or pharmacological stresses was examined in a panel of breast cancer cell lines. Subsequently, the effect of SLC38A2 knockdown in Gln-sensitive cell lines was analysed. The prognostic value of SLC38A2 in a cohort of breast cancer was determined by immunohistochemistry.SLC38A2 knockdown decreased Gln consumption, inhibited cell growth, induced autophagy and led to ROS production in a subgroup of Gln-sensitive cell lines. High expression of SLC38A2 protein was associated with poor breast cancer specific survival in a large cohort of patients (p = 0.004), particularly in TNBC (p = 0.02).SLC38A2 was identified as a strongly expressed amino acid transporter in six breast cancer cell lines. We confirmed an autophagic route of degradation for SLC38A2. These results position SLC38A2 as a selective target for inhibiting growth of Gln-dependent breast cancer cell lines. AA levels are critical for regulating the balance between the positive anabolic actions of the mammalian target of rapamycin complex 1 (mTORC1) microenvironmental sensor and the effects of inhibiting mTORC1, leading to autophagy and catabolism.2The significance of amino acid (AA) metabolism and glutamine  in tumour progression has been well recognised, leading to intense interest in therapeutic applications.3 Triple-negative breast cancer (TNBC), an aggressive form of breast cancer, is dependent on exogenous Gln for survival and growth5 and inhibitors of Gln transport and metabolism have been proposed as potential anti-tumour therapies.7Breast cancer is a heterogeneous disease characterised by different morphology, clinical outcome, and response to treatment. From a metabolic point of view, Gln-dependent mechanisms also can vary substantially among breast cancer subtypes.8 A recently proposed classification by Broer et al., suggested that AATs can be divided into loaders, such as SLC38A1/SNAT1, mediating the net uptake of specific AAs;9 AAT harmonisers, such as SLC1A5/ASCT2 and SLC7A5/LAT1, which rapidly exchange different AAs and ensure that all AAs are present in the cytosol; and rescue AATs, such as SLC38A2/SNAT2 and SLC1A4/ASCT1 that provide redundant capacity and are upregulated under stress conditions.11 The role of Gln metabolism and transporters, including SLC38A2, in mediating resistance to anti-endocrine therapies has been recently demonstrated.14 However, despite the high-dependency on Gln for TNBC growth, the role of Gln transporters in this subtype has been less investigated.15 As there are many Gln transporters, we investigated which were most abundant and their relationship to Gln dependence.Gln is transported into cells through many amino acid transporters (AATs).We found the Gln transporter SLC38A2/SNAT2 to have the highest expression level in many breast cancer cell lines. Consequently, we investigated and subsequently demonstrated its role in adaptation to Gln starvation in Gln-sensitive cell lines, including TNBC cells. High SLC38A2 levels were associated with poor clinical outcome in TNBC patients, suggesting a key metabolic role for this transporter.Cell lines were obtained from ATCC , and they were authenticated six months prior to the first submission of the manuscript. All cell lines were maintained in DMEM 25 mM glucose supplemented with 10% FBS and no antibiotics. All the experiments were performed in 5 mM glucose and 10% dialysed FBS was used. For spheroid culture, we plated 5000 cells into ultra-low–adherent round-bottom 96-well plates (VWR) with Matrigel, except for MDA-MB-231, which did not need Matrigel.5 cells per well) were seeded in triplicate on six-well plates. N-acetylcysteine was added at 10 mM and replaced every 2 days. Trypsinised cells were counted using the Auto T4 Cell Counter (Nexcelom Bioscience). We used α-(Methylamino) isobutyric acid (MeAIB)  at 10 mM concentration for 2D and spheroid culture. Treatment was started on day 3 and renewed every 2–3 days. Images were captured every 2–3 days.Cells  and β-actin as housekeeping genes. Each PCR reaction was performed in triplicate. Primer sequences are reported in Table Cells were lysed in Trizol reagent (Invitrogen) and RNA was extracted using ethanol precipitation. The RNA quality and quantity were confirmed using the NanoDrop ND-1000 spectrophotometer. Reverse transcription (RT) was performed using the High Capacity cDNA RT kit (Applied Biosystems). qRT-PCR was performed using the SensiFAST SYBR No-ROX Kit (Bioline). Raw data were analysed using Reverse transfection of siRNA duplexes (20 nM) was undertaken in Optimem using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. The siRNA duplexes targeting SLC38A2  or scrambled control were used.12 Samples were incubated overnight with SLC38A2 , LAMP1 (CST9091), LAMP2 (CST49067), proteasome 20S LMP2 (ab3328), Rab5 (CST3547), TGN46 (AHP500G) primary antibodies and then incubated with fluorescently conjugated secondary antibodies or Alexa Fluor® 594 phalloidin (A12381) and DAPI for 1 h at 37 °C. The samples were imaged on a Zeiss LSM 680 or 780 confocal microscope.Immunostaining was performed as previously reported.4/well) in a 24-well plate were cultured for 5 days, the medium was collected, and cells were lysed with RIPA buffer (Sigma-Aldrich). Concentrations of Gln in the medium and in the cell lysate were determined with the Gln Detection Assay Kit  following manufacturer’s guidelines. Gln consumption was calculated as  and normalised to the protein level.Cells (1 × 10Reactive oxygen species (ROS) after SLC38A2 knockdown in normal medium (4 mM) or low-Gln medium (1 mM) was measured after 5 days using the Cellular ROS Assay Kit . The procedure was performed following the manufacturer’s protocol and fluorescence was measured at 590 nm emission.Protein concentrations were quantified using a BCA protein assay kit (ThermoFisher Scientific). Samples containing 35 μg of protein in NuPAGE™ LDS Sample Buffer (4X) were separated using 8–12% pre-cast SDS-PAGE gels (BioRad). Proteins were transferred to PVDF membranes and blocked for 1 h in 5% TBST/milk. Primary antibodies were all used at 1:1000. Rabbit anti- SLC38A2 (BMP081), SLC7A5/LAT1 (CST5347), SLC1A5/ASCT2 (CST8057), SLC38A1/SNAT1 (CST36057), xCT/SLC7A11 (CST12691), LAMP1 (CST9091), LAMP2 (CST49067), AMPKα (CST5832), Phospho-Thr172-AMPKα (CST2535), Acetyl-CoA Carboxylase , Phospho-Ser79-ACC (CST11818), phospho-Ser2448-mTOR (CST2971), Anti-SQSTM1/p62 (ab56416), ULK1 (CST8054), phospho-Ser555-ULK1 (CST5869). Appropriate secondary horseradish peroxidase–linked antibodies were used (Dako). Blots were developed on ImageQuant LAS4000 using ECL plus reagent .n = 1685) was arrayed as previously described.16 Immunohistochemical (IHC) staining was performed on 4 µm tissue microarrays (TMA) sections using the Novolink polymer detection system (Leica Biosystems) as previously described.17 Briefly, tissue sections were incubated 1 h at room temperature with the primary SLC38A2 antibody (1:25). Slides were washed and incubated with post-primary block for 30 min. Novolink polymer was applied for 30 min: 3,3-diaminobenzidine chromogen was applied for 5 min. Slides were then counterstained with haematoxylin for 6 min.The set (http://tissuearray.org).Stained TMA sections were scored using high-resolution digital images , at ×20 magnification. Evaluation of staining for SLC38A2 was based on a semi-quantitative assessment of digital images of the cores using a modified histochemical score (H-score) which included assessment of both the intensity and the percentage of stained cells. Staining intensity was assessed as follows: 0, negative; 1, weak; 2, medium; 3, strong and the percentage of the positively stained tumour cells was estimated subjectively. The final H-score was calculated by multiplying the percentage of positive cells (0–100) by the intensity (0–3). Dichotomisation of protein expression in predicting breast cancer specific survival (BCSS) was determined using x-tile software (18 ER and progesterone receptor (PgR) positivity was defined as ≥1% staining. Immunoreactivity of HER2 was scored using standard HercepTest guidelines (Dako) and by chromogenic in situ hybridisation in borderline cases. Breast cancer molecular subtypes were defined based on tumour IHC profile and the Elston-Ellis mitotic score as: ER ± HER2- low proliferation (mitotic score 1), ER ± HER2- high proliferation (mitotic score 2 and 3); HER2-positive class: HER2 + regardless of ER status; triple negative: ER−, PgR− and HER2−.IHC staining and dichotomisation of the other biomarkers were as per previous publications.The Metabric breast cancer dataset was pre-processed, summarised and quantile-normalised from the raw expression files generated by Illumina BeadStudio (R packages: beadarray v2.4.2 and illuminaHuman v3.db_1.12.2). Raw files were downloaded from European genome-phenome archive (Study ID: EGAS00000000083). Correlation analysis and visualisations were performed in R statistical environment version 3.4.4.t-test was used for two sample analyses and normal distributions were assumed; otherwise, the non-parametric Mann–Whitney test was used. Analysis of variance was used for >2 sample analyses. The chi-square test was performed for categorical variables. No samples or experimental repeats were excluded from analyses.Statistical analysis and graphs were performed using GraphPad Prism v6.0. Results are plotted as mean values with standard deviation (SD). Statistical tests and the number of repeats are described in the figure legends. Student’s p-value < 0.05 was considered significant. The study endpoints were 10-year BCSS.Survival curves were analysed by the Kaplan–Meier and log-rank test. A SLC38A2 mRNA was the most abundant transcript in many cell lines , HER2+ (SKBR3), basal-like  and claudin-low  molecular subtypes of breast cancer Fig. . Differenes Fig. . Similar4We then cultured the same cell lines in a growth medium that contained high (4 mM) or lower levels of Gln (1 mM) and followed their growth for six days. The optimal growth of breast cancer cell lines MCF7, MDA-MB-231 and HCC1806 was dependent on Gln supplementation Fig. . In cont19 we investigated in more detail the role of SLC38A2 under glutamine starvation and endoplasmic reticulum (ER) stress in breast cancer cell lines.We then assessed whether the variable dependence of cancer cells on Gln could also be a consequence of altered expression of Gln transporter genes. A selected panel of cell lines having a higher sensitivity to Gln  and the less-sensitive MDA-MB-468 were exposed to low Gln medium for 24 h, and the Gln transporter levels were assessed by qRT-PCR and WB. In two of the sensitive cell lines (MCF7 and HCC1806), the system A transporters (SLC38A1 and SLC38A2) increased after Gln deprivation both at mRNA and protein level, but levels remained unchanged in the less Gln-sensitive MDA-MB-468 and in the Gln-sensitive MDA-MB-231 cell line Fig. . Due to 20 As different stresses are known to regulate protein localisation, and SLC38A2 has been demonstrated to undergo re-localisation from a cytosolic compartment towards the cell’s plasma membrane in anabolic conditions (such as insulin administration)21 and hyperosmotic stress,22 we investigated the localisation patterns of SLC38A2 in response to different stresses able to induce the ATF4 pathway, such as PP242 treatment (an ATP-competitive mTORC1/mTORC2 inhibitor),23 thapsigargin (TG) and energetic stress (24 h of AA starvation) in different breast cancer cell lines. Firstly, by two-colour super resolution-stimulated emission depletion (STED) microscopy we showed that SLC38A2 was located predominantly in the Trans–Golgi network (TGN) in MCF7 .CF7 Fig. , consistons Fig. . A specions Fig. .SLC38A2 mRNA and protein levels, particularly in the Gln-sensitive cell lines (MCF7 and HCC1806)  but not in SKBR3 and HCC1806 cell lines  Fig.  with no 06) Fig. . PP242 tnes Fig. . Moreovenes Fig. . SKBR3 cnes Fig. . Similarnes Fig. . InteresBR3 Fig. , but a dTGN Fig. . We confTGN Fig. .24Fig. 225 We found that SLC38A2 partially colocalised with these markers in normal conditions  and the proteasome marker LMP2, where SLC38A2 is degraded.ons Fig. . Howeverons Fig. . These d8A2 Fig. , diverse8A2 Fig. .25 However, it is known that long-lived transmembrane proteins more frequently undergo degradation through autophagy.26 Recent work in Hela cells has shown that SLC38A2’s adaptive response to nutrient starvation requires retromer-mediated endosomal sorting, which prevents SLC38A2 being sequestered in LAMP1-positive lysosomes.27SLC38A2 has been shown to undergo degradation through the proteasome system.28 The levels of SLC38A2 increased consistently after Bafilomycin A1 treatment in all cell lines, suggesting lysosome-mediated degradation of SLC38A2 was also important in these breast cancer cell lines  in normal medium and low Gln medium Fig. . SpheroiThe effect of SLC38A2 knockdown or MeAIB was tested in 2D in the three different cell lines. The effect of the SNAT1/2 inhibitor showed similar pattern to SNAT2kd. The main effect for both treatments was mainly seen in low-Gln conditions except in HCC1806 where the SNAT2 levels are higher Fig. . We then4 Glutamate, derived from Gln, and cysteine are substrates for the first and rate-limiting step in glutathione biosynthesis, a key intracellular anti-oxidant buffer against oxidative stress. We assessed whether SLC38A2 upregulation under Gln deprivation could maintain the Gln intake to modulate ROS production. Gln deprivation elevated ROS levels in all the three cell lines  was shown to mitigate ROS production in cancer cell lines,35 we postulated that exogenous supplementation of NAC might reduce the anti-growth effects of SLC38A2 blockade. We cultured MCF7, MDA-MB-231 and HCC1806 in normal and low Gln medium and we then knocked down SLC38A2 in combination with supplementation in the culture medium of 10 mM NAC. SLC38A2 knockdown reduced cell proliferation in normal medium only in HCC1806, where the SLC38A2 levels are higher, but the inhibitory effect in 2D culture was present in all the three cell lines in low Gln medium . Also, in the TNBC cohort of the Metabric, SLC38A2 clustered with HIF-1α, ATG5 and other autophagic genes like GABARAP and ULK2 , but not with lymph node status  but not at other sites  but not with the “low SLC” (SLC1A5−/SLC7A5−/SLC3A2−) nor the “high SLC1A5” cluster (SLC1A5+/SLC7A5−/SLC3A2−)  Fig. .p = 0.02)  Fig. . There w15 In this study, we showed that the system A transporters (SNAT1 and SLC38A2) are highly expressed in breast cancer cell lines. Interestingly, the cell lines showing high levels of AATs, including SLC38A2, are more sensitive to Gln deprivation and this dependency is also partially mediated by SLC38A2. We demonstrate that the SLC38A2 anti-tumour effect is partially mediated by oxidative stress and that high SLC38A2 protein expression in a large clinical cohort correlates with poor BCSS in patients and particularly those with TNBC.The role of Gln transporters on proliferation has not yet been studied extensively in TNBC.38 but also ER+ cell lines (MCF7) can show Gln sensitivity.39 These data suggest that the early metabolic responses of cancer cells to microenvironment stresses (such as nutrient deprivation) might be shared by different subtypes and going beyond the given genetic background of that cell line.Our results confirmed that Gln sensitivity is overall more prevalent in TNBC subtypes cell lines39 or glutaminase inhibitors.6We found that cell lines with the highest induction of Gln transporters are also more sensitive to Gln deprivation and System A inhibition. This seems to be reflective of an upregulation of the whole Gln metabolic pathway, as these Gln-sensitive breast cancer cell lines are also more sensitive to drugs inhibiting this pathway such as aminotransferase inhibitors40 However, ER+ breast cancer cell lines with high levels of c-MYC are also dependent on Gln for their survival and growth41 and several c-Myc target AATs, including SLC1A5 and SLC7A5,43 correlate with poor survival in ER+ breast cancer, but not TNBC.37 Interestingly, we found that the SLC38A2 protein, which is a target for hypoxia-inducible factor-α (HIF1- α), but not c-Myc12 correlates with poor survival only in TNBC. A possible explanation could be that the activation of HIF gene networks is particularly robust in TNBC patients.44 Moreover, the XBP1 transcription factor, one of the main branches of the unfolded protein response (UPR) is specifically activated in TNBC tumours and transcriptionally cooperates with HIF-1α.45 Both the ATF4 upregulation under ER stress46 and HIF-1α and HIF-2α upregulation under hypoxia can promote the transcription of several AATs including SLC7A5 and SLC38A2.47 Moreover, HIF-1α and c-Myc can modulate adaptive responses to hypoxic environments.48 Thus, TNBC might be more dependent on Gln metabolism, due to the synergy between HIF-1α, XBP1 and c-Myc in modulating Gln metabolism, as well as AAT upregulation.TNBC subtypes have been previously linked to increased Gln sensitivity due to the increased activity of the MYC pathway in this subtype compared to others.SLC38A2 mRNA is highly abundant in breast cancer cell lines, as also seen in Hela and osteosarcoma cell lines.10 However, its abundance is tightly controlled by several mechanisms at the transcriptional49 and post-translational level.50 SLC38A2 protein shows marked heterogeneity in the levels of glycosylation; it undergoes proteasomal degradation under unsaturated fatty acid stress conditions25 and ubiquitination in response to ER-stress.24 The role of post-translational modifications in the SLC38A2 protein might account for the discrepancy between our previous results showing high SLC38A2 mRNA is relevant in ER+ breast cancer tumours,12 while high SLC38A2 protein is relevant in TNBC. Similarly, this might be the cause of the difference in term of the lack of correlation between SLC38A2 and SLC1A5 at mRNA and the positive correlation seen at protein levels between the SLC38A2 and SLC1A5.We showed that 51 However, the presence of SLC38A2 in lysosomes may just reflect its abundance and its turnover in the endocytosis pathway. Due to the similar localisation in LAMP1 vesicles, it would be interesting to investigate whether SLC38A2 might have a potential role also in the lysosomes by supplying the cytosolic AA derived from intracellular (autophagy) stores, as suggested for SLC38A7/SNAT7.52We also confirmed a route for SLC38A2 degradation via autophagy in breast cancer cell lines. The degradation of transporters, such as SLC38A2, through the activation of macroautophagy, might provide a molecular link between metabolic stress and cell death.53 and the autophagy pathway is still functional under glutamine starvation and SLC38A2 knockdown  or intracellular AA pool from this cell line. The KRAS-mutant MDA-MB-231 cells are robustly macropinocytic amongst a wide variety of breast cancer cellsown Fig. .12 In the present study, targeting SLC38A2 also reduces the lysosome energy sensor AMPK and p-AMPK, probably due to the reduced AA pool,54 and increased ROS production in low Gln medium. The increase in ROS was likely due to impaired Gln metabolism and cysteine/glutamate exchange, as has previously been seen for SLC1A5.55 However, in contrast to SLC1A5, we found an increase in ROS production mainly in low Gln medium after SLC38A2 knockdown, confirming recent suggestions that SLC38A2 acts as a rescue transporter providing AA intake under stress conditions.9We have previously shown that SLC38A2 knockdown can modulate mTORC1 and reduce mitochondrial respiration, particularly in low Gln medium.56The role of SLC38A2 in maintaining mitochondrial function and ROS production in low Gln might be beyond the exchange with cysteine. In pancreatic cancer, SLC38A2 represents the major AAT for alanine, which is then deaminated to pyruvate (which can maintain the tricarboxylic acid cycle) in the presence of pyruvate carboxylase.37 The TNBC subtype has still poorer outcome compared to other breast cancer subtypes due to aggressive clinical behaviour and a lack of recognised molecular targets for therapy.57 Blocking cellular Gln transport would potentially impart a greater impact on Gln metabolism in cancer cells than targeting downstream enzyme activity, given the extensive biological plasticity leveraged by cancer cells to maintain intracellular AA pools; new inhibitors with high affinity for AATs have been proposed.58 However, redundancy associated with other AATs after long-term silencing of SLC1A5 has been shown.9Finally, our analysis of clinical data from a large breast cancer cohort suggests that high baseline SLC38A2 protein expression correlates with poor BCSS survival in patients with breast cancer and with TNBC. Moreover, tumours with high SLC38A2 expression were also commonly represented in the poor prognostic high SLC cluster (SLC1A5+/SLC7A5+/SLC3A2+). Interestingly, no significant association between the high SLC clusters and patient outcome was previously reported in TNBC.59 that might inform treatment strategy. In conclusion, this study provides evidence for future investigation of SLC38A2 as a potential target for TNBC breast cancer patients and as a marker for sensitivity to Gln deprivation.Due to its transmembrane localisation and the vulnerability of the TNBC cell lines to Gln depletion and SLC38A2 knockdown, SLC38A2 is potentially a useful target for this cancer subtype. However, apart from the canonical competitive inhibitor of system A transport, MeAIB, no pharmacological agent specifically targeting SLC38A2 has been identified so far. Alternatively, SLC38A2 imaging for clinical selection by AA-based positron emission tomography radiotracers is also a promising approachSupplemental Material


Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions. Since the discovery of the CRISPR/Cas9 genome engineering technology platform , the mosDeletion of selected genetic elements relies on a dual sgRNA-mediated strategy and the repair follows the same principle as above, including excision of the genetic segment by Cas9 and subsequent repair of the cut by NHEJ ,4. For kCRISPR/Cas9 is widely used as a reliable and very precise genome engineering tool. However, accumulating evidence suggests that repair of Cas9-induced DNA double-strand breaks can lead to various degrees of genomic rearrangements. NHEJ is the default repair pathway for cells and is usually highly efficient and accurate to allow maintenance of cell viability , and invIn addition to all of the above-described side products of genome editing, we observe an unexpectedly high level of inverted re-insertion events when using the dual sgRNA approach for genomic deletions. Our study summarizes several independent experiments, and we reveal inverted re-insertions in a median range of 3–20% throughout several different cell lines and at diverse genomic loci. Keeping this hitherto unreported phenomenon in mind, we suggest new measures that are vital for correct genotyping and, moreover, are potentially highly beneficial when establishing cellular model systems in more than diploid cell lines.We present only a brief description of procedures and materials used in this manuscript since the aim is to provide a technical note as opposed to an elaborated protocol. However, we are very happy to share full details of experimental procedures, and any information request can be addressed to the corresponding author at any time.Design of sgRNA reagents. All sgRNAs in this study were designed using the CRISPOR algorithm . Guides Cell lines. Cell lines were obtained from ATCC or SIGMA and cultured, as described . The hiPSC line CTR M3 36S was generated from the reprogramming of keratinocytes from a neurotypical Caucasian male, aged 36 years old, as described . The mou2000  and into HT29-MTX-E12 and E14 by Fugene (Roche). hiPSCs were transfected with pre-assembled RNPs using Alt-R® S.p. HiFi-Cas9 nuclease 3NLS (IDT) and chemically synthesized tracrRNA and crRNA (IDT) using Lipofectamine CRISPRMAX™ reagent , according to a previously outlined protocol [Editing procedure. Two plasmids encoding one single sgRNA and color-coded Cas9  were transfected in either 6 or 12 well format into 293, HCT116, HeLa and 4T1 cells by lipofection with LPFprotocol . All del2O before determination of the DNA concentration .Isolation of gDNA. gDNA was isolated by lysing the cell pellet of a confluent 6 or 12 well plate in 500 µL lysis buffer  by incubation overnight at 56 °C. gDNA was isopropanol-precipitated and  DNA pellets were resuspended overnight at 56 °C in 500 µL ddHScreening and validation procedures. PCR screening procedures in this study involve the following setup: primers for amplification of the genomic fragments were designed as 23mers with the aim to have three 3′ G/C residues and a GC content of minimum 55%. Primary PCR amplification was performed using Qiagen Taq polymerase and standard buffers  in presence of 3% DMSO using an annealing temperature of 62 °C and an elongation time of 60 s for all amplicons below 1 kb using primers denoted FW (forward) and RV (reverse) see . PCR reaAll oligos used in this study are summarized in We routinely use a dual sgRNA-mediated excision approach for both functional gene KO and excision of regulatory genetic elements. Using a dual sgRNA-based strategy has the potential to double the number of potential OFF-target modifications; however, using highly selected sgRNAs, we have successfully generated many model systems and have been unable to detect significant numbers of aberrantly generated models. This can potentially be attributed to the fact that we generally apply careful initial PCR screening procedures that always take into account potential larger-than-expected deletions . The duadeleted only band’ are bona fide KO cell lines  did not express functional protein or detectable amounts of mRNA (data not shown). Investigating this matter by molecular cloning and Sanger sequencing, we found that a substantial number of clonal cell lines displayed inverted re-insertion of the excised fragment . We set We subsequently screened several ongoing projects with this new approach. We observed a median inverted re-insertion rate of 3–20% of all clones screened. We find varying efficiencies in different cell lines and diverse target loci in our set of 12 independent experiments; however, our data strongly suggest a common phenomenon. It is important to note that the median range does exclude sample sizes with very limited numbers of clones available . The consequence is that cells initially screened and identified as WT (by a single PCR) could well turn out to be heterozygous deletions. Importantly, this can be highly beneficial for the generation of gene deficient models in tri- or tetraploid cell lines. Generation of a full KO in those cells is usually difficult and requires screening of tens to hundreds of cell lines to find those with the required three or four simultaneous deletion events. In light of our observations, re-screening of HET clones  could increase the pool of fully deleted clones by identification of inverted re-insertion events without the requirement to screen additional lines. In line with this, we observe that a large proportion of full KO models in tetraploid HeLa and 4T1 cell lines have been generated as combinations of KO and re-inserted inversions, at 30% and 100%, respectively .Interestingly, hiPSCs, which are notoriously hard to modify , did notWe routinely use dual sgRNA approaches to generate deletions in genes or regulatory elements. We consistently find high levels of full deletions in all alleles across a variety of different cell lines, from different species and origins and targeting at several distinct loci .Generally, the regions of interest are short, with critical exons or the targeted regulatory elements usually spanning less than a couple of hundred base pairs. A screen for deletion events can be performed easily using a reliable PCR over the deleted region and this is, importantly, independent of cell ploidy. When analyzing inconsistent gene or protein expression data in genotyped cell clones, we realized that this approach produced unexpected inverted re-insertions at rather high frequencies next to common NHEJ events at either sgRNA target site. We observe a range of 3–20% of events where the target exon and associated splicing signals have been inverted, contributing to the frequencies of heterozygous clones and as well to functional null alleles. Likewise, we observed inversion of regulatory elements targeted using the same dual sgRNA strategy. Our selection of cell lines  is not cOur new data are in line with recent publications where genome engineering resulted in unexpected larger rearrangements ,21,22,23An important lesson we learned is that inverted re-insertions must be considered when selecting appropriate control cell lines. The best control line is always the cells that underwent the same engineering pipeline and that turn out to be “WT” or “HET” in respect to the engineered target. If inverted re-insertions are not properly screened for, such events can be missed. Cells could wrongly be classified “WT” but instead be “HET”, resulting in a “hidden genotype” issue. It is vital to ensure bona fide KO cell lines are compared to the properly characterized control lines. Equally important, inverted re-insertions can be very beneficial when engineering polyploid cell lines. In several cases, we observed that HeLa and 4T1 tetraploid lines with a full KO genotype comprise a combination of deletion and inverted re-insertion alleles to generate the full KO .Albeit we saw a consistent rate of inverted re-insertion throughout several immortalized human and mouse cell lines or murine embryonic stem cells, we failed to detect any event in hiPSCs  last rowThe potential danger of increased OFF-targeting and/or risking potentially higher levels of genomic rearrangements by using two instead of only one sgRNA needs to be carefully considered. In our experience, the benefits prevail, allowing an easy screen and, so far, not experiencing serious OFF-targeting issues. We generally generate three to five independent KO lines and cross-compare their phenotype to ensure that we have generated the model system we intended to create. To be fully aware of any other OFF-targeting events, a comprehensive genome wide analysis is highly recommended, such as targeted locus amplification  or unbiaOur technical note raises awareness of two points: (1) a dual sgRNA-mediated KO strategy is a highly efficient experimental approach to achieve precise deletions that are easy to screen for and, importantly, are irrespective of the ploidy of the cell line concerned; (2) inverted re-insertions occur in a median range of 3 to 20%. It is important to note that potential WT or HET clones required as control cell lines, if not properly screened for, could be wrongly classified and could result in skewed interpretation of scientific data. Additionally, inversions (when guides are properly designed) will equivalently contribute to the pool of functional null alleles and thus can help reduce the number of clones required for screening when generating full KO lines, especially in non-diploid cell systems. This technical note aims to serve as an “eye opener” for screening and to empower new opportunities in model generation.


Apis mellifera) from fatal levels of virus infection and from the non-target effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for the study of molecular mechanisms of virus–host interaction, for the screening of antiviral agents for potential use within the hive, and for the assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating honey bee cell line has proved challenging. Here, we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) of establishing honey bee cell lines, challenges associated with the presence of latent viruses  in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology. With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees ( Apis mellifera) for pollination, reflecting the importance of the honey bee to agricultural production. However, honey bee colonies in the northern hemisphere have been in decline [Varroa destructor, represents a major threat to honey bee health [Deformed wing virus (DWV) strain [Acute bee paralysis virus (ABPV), DWV, Sacbrood virus (SBV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), Chronic bee paralysis virus (CBPV), and Kashmir bee virus (KBV) [About one third of all agricultural crops are dependent on the honey bee  strain ,21. At l) strain , includius (KBV) ,23. Insect-derived cell lines provide valuable tools for the study of insect viruses under controlled conditions, with their genetic uniformity providing for more consistent results. Cell lines may allow for the study of suborganismal processes that may not be tractable using the host organism. Insect cell lines can also be used for the screening of insecticides or biocontrol compounds against pests, or for the assessment of potential risk to nontarget organisms such as the honey bee . ApproxiApis mellifera-derived AmE-711 cell line, methods for the establishment of virus-free cell lines, and potential applications of these cell lines in insect virology. A honey bee cell line would provide a powerful research platform for the increased understanding of honey bee virology. A honey bee-derived cell line would provide a valuable tool for the study of virus–insect and virus–virus interactions. In this review, we provide a summary of the establishment of primary cultures and continuously replicating hymenopteran cell lines, virus studies using the Pseudomyrmex triplarinus), a parasitic wasp (Mormoniella vitripennis), and the honey bee (A. mellifera)   [26,27,2llifera)  [30,31,3llifera)  [27,35,42O2, and rinsed several times. (2) The tissue was gently homogenized or torn apart in a specific growth medium  with several types of antibiotics , and an antimycotic . Mechanical methods were typically used for the establishment of honey bee primary cell lines, as enzymatic dissociation of tissues resulted in limited numbers of isolated cells and contamination [actin and laminin for confirmation of honey bee cell lines [cytochrome c oxidase subunit I (COI) is also commonly employed for this purpose. Similar procedures were used for the generation of these primary cell cultures, as follows . (1) Beemination . (3) Thell lines ,47. MitoA. mellifera were reported to show attachment and growth in WH2, a medium modified from HH-70 psyllid culture medium, while they grew slowly in two commercial media, Sf-900™III SFM and EX-CELL 405 [The cell culture medium used significantly influenced cell growth rates, suggesting that specific nutrients are required for maintenance of honey bee cells. The media that support the growth of cell lines derived from other insects were mostly insufficient for the maintenance of honey bee-derived cells. The evaluation of different media for cell growth was required, with cells growing extremely slowly in an unsuitable environment. For example, primary cells of CELL 405 . Chan etCELL 405  for the CELL 405 . ClearlyNeodiprion lecontei (Diprionidae) [Trichogramma pretiosum (Trichogrammatidae) [T. confusum, T. exiguum [Hyposoter didymator (Ichneumonidae) [A continuous cell line is often comprised of multiple cell types and can be passaged in culture for many generations or indefinitely . In the ionidae) , Trichogmatidae) , T. conf exiguum  and Hypomonidae)  (Table 2A. mellifera cells derived from honey bee embryos using the human c-myc proto-oncogene for their long-term cultivation [Deformed wing virus (DWV), was also detected in the MYN9 cell line. While MYN9 was a honey bee-derived cell line, whether expression of c-myc in the cells affected endogenous gene expression or not is unknown.The establishment of a continuous cell line from the honey bee has proven difficult, with only two continuous cell lines reported . Bergem tivation . The celApis mellifera cell line from Embryonic tissues, established on 7/2011), was reported by Goblirsch M. et al. [A honey bee cell line derived from embryonic tissues, named AmE-711  Onl. et al. ; (3) The. et al. . The AmE-711 cell line contained bipolar and multipolar fibroblastic cells, elongated in shape with an adherent growth phenotype. Most cells had a diploid karyotype, similar to honey bee cells in nature. Most importantly, the cell line was continuous, as it was maintained long term and passaged at least 18 times, with a minimum of 43 generations ,60. HoweHundreds of insect cell lines have been established since the first ones were produced in the late 1950s and 1960s ,62. SomeDrosophila melanogaster cell lines [In recent years we have been working to establish cell lines from honey bees at BCIRL. The establishment of cell lines derived from honey bees has proven to be very difficult, similar to the situation for a large group of insects from a variety of orders. It is not clear why cell lines are routinely established from some orders of insects, such as Lepidoptera, but not others. Such differences in cell line establishment may relate to fundamental cellular biology. We plan to investigate the point in detail by tracing gene expression patterns during the establishment process, using cell lines from lepidopterans and coleopterans that are routinely established, and from recalcitrant species, similar to work in ll lines . All bee stages were used for culture initiations, including adult workers and queens and specific tissues within the bees. We initiated lines with the midgut, nervous system , aorta, fat body, ovaries, spermatheca, a combination of testes/fat body, muscle, Malpighian tubules, venom sack, and ground pupal heads.g, 5 min, 4 °C), then transferred into either tissue culture plates  or flasks  using cell culture media augmented with selected antibiotics . In some initiations, 0.5 mL of an enzyme mixture  was added to dissociate the tissues. Enzyme-inoculated cultures were incubated at room temperature for 1h with gentle shaking. The dissociated tissues were centrifuged , and transferred to culture containers as described. Cell culture initiations were performed in biosafety hoods with surface sterilized dissecting implements . Before For smaller bee larvae (<4 mm), we minced the whole bodies immediately after sterilization. The eggs were collected into 1.5 mL microfuge tubes containing medium and gently agitated so that they remained in suspension. They were sterilized and washed as above, then either minced with micro-scissors or ground with a pestle. Cell cultures were maintained at 28 or 33 °C and observed daily. Insect cell lines are usually maintained at 28 °C ,66,67,68Over 600 honey bee cell cultures have been initiated using various combinations of tissues, media and media additives . An iterThe influence of the media + FBS on overall cell health was evaluated by visual inspection before testing different combinations of basal media or comparing the effects of supplements  added to the media. For example, we initiated cell lines with CLG#2, a combination of an insect cell culture medium (EX-CELL 420) and a mammalian cell culture medium (L-15), used to establish lepidopteran and hemipteran cell lines ,68). TheAethina tumida) tissues within primary cultures. Adult beetles laid eggs in capped brood cells, as well as throughout the hive, and these eggs could have been mistaken for honey bee eggs [A. mellifera only lay one egg per cell, while A. tumida can lay 10–30 eggs per cell, with the beetle eggs about 2/3 the size of honey bee eggs. A. tumida larvae are smaller than honey bee larvae, but they are more active, especially during their wandering stage . We took care to ensure that only honey bee eggs and larvae were collected when initiating primary tissue cultures.We paid particular attention to potential sources of contamination during cell line establishment. Fungal contamination may occur in bee cell culture initiations, although in most cases, this is controllable through surface sterilization and tissue washing as described. For tissues other than neonates and eggs, a fungicide at low levels  was initially incorporated into culture media to minimize contamination. Another potential source of contamination was the accidental inclusion of small hive beetle  were initiated with TNM-FH and Schneider’s + FBS. The most promising and cleanest cultures were generated from eggs. Promising cultures consist of viable appearing, attached cells, with a clear cytoplasm, no vacuoles or darkened areas, and distinct cell membranes that are actively replicating . CultureOther short-term honey bee cell cultures (<1 month) that exhibit tissue and cell attachment, but no or minimal cell replication, include those initiated from the worker nervous system (in HB-1 or TNM-FH + FBS), larval/worker/pupal midgut (in HB-1 or CLG#2 + FBS + YE), ground pupal whole head (in CLG#2 + FBS), pupal nervous system (in HB-1), queen ovaries , queen midgut (in CLG#2 or TNM-FH or Kimura’s +/- other supplements) and queen/worker Malpighian tubules (in HZ#2 or Kimura’s + FBS). Some ovarian cell cultures exhibited cell networking with contractions. Based on these responses to different media configurations, we propose that each tissue has its own nutrient/medium requirements, which may reflect the in vivo situation. The tissues with the least stringent requirements for generating short-term cultures, aside from egg cell cultures, are those from queen ovarian tissues. This is generally true for cell lines initiated from ovaries and eggs, presumably due to the presence of undifferentiated cells. Clearly, more work is necessary to optimize the medium needed for each tissue isolate.Cricket paralysis virus, which has an unusually wide host range). It follows therefore that honey bee viruses will replicate in honey bee-derived cell lines, and potentially in cell lines derived from other hymenopteran species (Apis cerana) [Insect viruses typically infect cells derived from the host insect or from closely related species, with a few exceptions  . SBV rep cerana)  and was  cerana)  and the  cerana) . Honey b cerana) . InteresCricket paralysis virus, CrPV-1A, was used to induce acute DWV infection and cytopathic effects, confirming the RNAi-mediated suppression of DWV replication in these cells. The AmE-711 cell line was challenging to maintain, likely because environmental stressors  weakened the cells allowing DWV titers to increase, similar to the situation in honey bees [Unfortunately, the AmE-711 cell line was persistently infected with DWV, as confirmed by sequence analysis and observation of DWV virions by TEM . While tney bees ,79. WhilA. mellifera cell lines. One key example is the AmE-711 cell line, established from A. mellifera embryos, which is persistently infected with the DWV [A variety of continuously replicating cell lines, including vertebrate and invertebrate lines, harbor viruses ,81,82,83 the DWV . Two stuTrichoplusia ni, [T. ni cell lines [Spodoptera frugiperda pupal ovaries, along with the subclonal line, Sf9, are well recognized for generating recombinant proteins via the baculovirus expression system [Trichoplusia ni cell line (Tn-368) with similar results [A nodavirus, named “ Tn-nodavirus”, was discovered in the BTI-TN-5B1–4 (Tn5) cell line derived from usia ni,  and subsll lines . The IPLn system . These Sn system ,87. Maghn system . Althougn system . The Sf9 results . +, X-) and uninfected cells. As individual cells failed to survive, a limiting dilution method was used to determine the minimum number of cells required for survival. They transferred 1000 cells/well into one column of a 96-well plate  and made two-fold serial dilutions into subsequent wells. The wells containing the lowest cell numbers that reached more than 40% confluence after 6–8 weeks were transferred into 24-well plates. A total of 115 cell clones were obtained from fifteen 96-well plates and 18 of these tested as negative for Sf-rhabdovirus. Five of the 18 virus-free clones were further cultured for 30 passages, and three of these clones were confirmed to be virus-free [Ma et al.  used limrus-free . RNA-seqhttps://web.expasy.org/cellosaurus), honey bee cell cultures to date have higher doubling times. The AmE-711 cell line, for example, was reported to double every four days [S. frugiperda nervous system cell lines (7:3 conditioned medium to fresh medium). Cells were fed every 7 to 10 days with conditioned medium while in the 96-well plate, and with fresh media after they were transferred into T12.5 flasks. In ongoing research, we will use a similar procedure to isolate individual cell types from cell cultures that may contain both A. tumida and A. mellifera cells at BCIRL. Instead of using conditioned medium from potentially virus-containing parental lines, we will generate conditioned medium from actively growing non-bee cell lines (free of bee viruses) and use it to supplement the fresh medium. While Sf9 and Tn-368 cells are rapidly replicating cell lines with doubling times of ~24 to 27 h  targeting multiple regions of the small positive-strand RNA genome of Turnip mosaic virus . While gRNAs targeting different regions of the virus genome varied in efficiency, gRNAs targeting HC-pro and GFP sequences resulted in a >50% reduction in virus load [An emerging RNA targeting effector Cas13, an RNA-guided single stranded RNA ribonuclease , can be rus load . As CRISrus load ,94, it iThe establishment of virus-free, honey bee cell lines will facilitate a number of avenues of research including (1) screening for antiviral compounds, (2) screening for the potential toxicity of insecticides to honey bees, and (3) elucidation of honey bee-virus molecular interactions. Anopheles and Aedes), but with little or no effect on other insect or human cell lines [Drosophila melanogaster. A honey bee cell line could be employed (1) for the screening of antiviral compounds to reduce viral load within a hive, (2) the screening of current and candidate insecticides for safety to honey bees. The need for such a screening system was highlighted by the impact of neonicotinoid insecticides on honey bee populations [The cell culture system provides a powerful tool for high-throughput preliminary screening of antiviral drugs ,96,97 prll lines . This sculations ,100. D. melanogaster have been used to study the infection cycle, replication of, and RNA interference associated with, small RNA viruses that infect Drosophila [A honey bee cell line would allow for in depth study of virus–host molecular interactions. This will be facilitated in particular by the establishment of infectious clones of honey bee viruses such as those of DWV ,102, thaosophila ,106,107.Dcr2, a key gene in the RNA interference pathway, using the CRISPR/Cas9 technology. In the Dcr2 knockout cell line, Piwi4 retained antiviral activity in the absence of the siRNA pathway [Influenza A virus (IAV) infection, that could serve as targets for novel antiviral compounds [Along with RNAi-, the emerging CRISPR/Cas9 gene editing tool, which has been used in several insect cell lines including Sf9, High Five, BmN , S2 109109,110 a pathway . The CRIompounds . SimilarApis mellifera may support the replication of some viruses, but would be suboptimal for the study of honey bee-specific viruses. Viruses play a significant role in honey bee losses. A honey bee cell line represents a valuable tool to identify solutions to virus infections in apiaries. Previous work with the AmE-711 cell line demonstrated the potential of honey bee cell lines to mirror in vivo virus dynamics. Cell lines derived from hymenopteran species other than Here, we have summarized the establishment of primary and continuous cell lines derived from Hymenoptera. A systematic approach for the establishment of cell lines with the testing of multiple media is warranted for establishment of cell lines from less tractable species such as the honey bee. In addition, methods such as the use of antiviral drugs, sub-cloning and use of CRISPR/Cas13 could be employed for establishment of virus-free, honey bee cell lines. The use of a honey bee cell line in conjunction with virus replicons or infectious clones, and CRIPSR/Cas9-mediated genome editing will facilitate investigation of molecular virus–host interactions. Ultimately, such studies will help mitigate virus-related honey bee losses.


The effect of Klotho on lung structure, PH and cardiac function was assessed. As compared to controls, preterm infants with BPD or BPD–PH had decreased cord Klotho levels. Early Klotho supplementation in neonatal hyperoxia-exposed rodents preserved lung alveolar and vascular structure, attenuated PH, reduced pulmonary vascular remodeling and improved cardiac function. Together, these findings have important implications as they suggest that perinatal Klotho deficiency contributes to BPD–PH risk and strategies that preserve Klotho levels, may improve long-term cardiopulmonary outcomes in preterm infants.Preterm infants with bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) have accelerated lung aging and poor long-term outcomes. Klotho is an antiaging protein that modulates oxidative stress, angiogenesis and fibrosis. Here we test the hypothesis that decreased cord Klotho levels in preterm infants predict increased BPD–PH risk and early Klotho supplementation prevents BPD-like phenotype and PH in rodents exposed to neonatal hyperoxia. In experiment 1, Klotho levels were measured in cord blood of preterm infants who were enrolled in a longitudinal cohort study. In experiment 2, using an experimental BPD–PH model, rat pups exposed to room air or hyperoxia (85% O It is characterized by alterations in lung development, vascular remodeling and lung dysfunction that range the spectrum of mild to severe and even fatal disease. Preterm infants with BPD often grow poorly2 and in the most severe cases develop pulmonary hypertension (PH)3, and myocardial dysfunction5. According to data from the National Institute of Child Health and Human Development, during the years 2003 to 2007, the incidence of BPD in infants less than 29 weeks gestational age was 42%6 with an increasing trend over the past 10 years7. Not only does BPD increase infant mortality but survivors have cardiopulmonary dysfunction extending long into adulthood8.Bronchopulmonary dysplasia (BPD) is a multi-factorial disease which affects premature infants9 while hyperoxia10, infection11 and poor nutrition12 are among the many postnatal insults which increase lung oxidative stress and create a pro-inflammatory milieu. This suboptimal intrauterine and neonatal environment, during a pivotal period of organogenesis, alters key developmental signaling pathways, disrupts intercellular communication, induces cellular apoptosis and promotes early lung aging.Various prenatal and postnatal factors contribute to the development of BPD and its sequelae. Infants with placental vascular malperfusion are at an increased risk for BPD and PH13. Homozygous Klotho deficient mice have premature aging, spontaneous fibrosis, pulmonary emphysema and vascular dysfunction, while mice that over-express Klotho have extended life span and relative protection from cardiopulmonary injury13. Klotho is primarily expressed in the kidneys and choroid plexus13, but it is also present in the placenta14, lungs, parathyroid glands and heart13. Klotho exists in two main forms: full-length transmembrane Klotho and soluble Klotho, the latter being formed by proteolytic cleavage of membrane Klotho.Klotho is an anti-aging gene first identified by Kuro-o et al. in 199715. Other reports demonstrate that soluble Klotho may act as an on demand co-receptor for fibroblast growth factor 23 (FGF-23)16 or it may act independently of FGF23 and interact with monosialogangliosides in lipid rafts of the plasma membrane, altering their lipid organization and modulating biological pathways that regulate oxidative stress18, apoptosis19, stem cell renewal20, fibrogenesis21 and angiogenesis22. Soluble Klotho reduces oxidative stress by promoting forkhead transcription factor activation and nuclear translocation, leading to upregulation of manganese superoxide dismutase (MnSoD)23. It also suppresses fibrosis by binding to type II transforming growth factor-beta 1 (TGF-β) receptor, preventing TGF-β binding and inhibiting SMAD 2/3 signaling21.The role of soluble Klotho in disease and repair is not fully understood. Emerging evidence suggest that soluble Klotho exhibits humoral activity as Klotho deficient mice have functional deficits in organs which do not express Klotho25, little is known about the role of Klotho in neonatal diseases. Intra-amniotic infection and smoking is associated with decreased maternal plasma Klotho concentrations26 and pregnancies complicated by pre-eclampsia and small for gestational age infants exhibit lower soluble Klotho levels28. Interestingly, while the latter perinatal insults contribute to BPD and poor long-term outcomes, to the best of our knowledge, there are no published data evaluating the relationship between cord Klotho levels and BPD or PH. Moreover, whether early administration of Klotho can reduce BPD, PH and long-term myocardial dysfunction has not been previously explored.Although multiple age-related morbidities exhibit reduced Klotho levelsIn the present study, we tested the hypothesis that decreased cord Klotho levels in preterm infants is associated with increased BPD–PH risk and that early Klotho supplementation decreases oxidative stress, suppresses fibrosis, improves lung vascular development and prevents BPD-like phenotype and PH in rodents exposed to neonatal hyperoxia. We demonstrate in a cohort of prospectively followed preterm infants that decreased cord blood Klotho concentration is associated with BPD and BPD–PH. In addition, using an in vivo preclinical model of severe BPD–PH, we show that early administration of soluble Klotho improves lung vascular development, attenuates PH, and reduces myocardial dysfunction. In vitro, we demonstrate that Klotho treatment of hyperoxia-exposed human pulmonary artery endothelial cells (HPAECs) increases cell survival, proliferation and capillary-tube formation. Together, these findings have important implications as they suggest that perinatal Klotho deficiency may increase the susceptibility to BPD and PH in preterm infants. Moreover, we speculate that strategies which increase soluble Klotho levels, may improve cardiopulmonary outcomes in preterm neonates.9. This study was approved by the Institutional Review Board of Northwestern University and all methods were carried out in accordance with approved guidelines. Informed consent was obtained from all mothers prior to participation.We performed a nested case–control study of infants < 29 weeks GA from an ongoing prospective longitudinal cohort study at Prentice Women’s Hospital in Chicago, IL, USA from 2008-present. Eligible patients of the parent study are live births with 23–41 completed weeks of gestation with available cord blood. For this study, all infants less than 29 weeks gestation from 2017–2018 with available cord blood were included. Comprehensive maternal and infant data were collected. Infants were classified as BPD using the NIH consensus definitions. BPD–PH was defined using echocardiography parameters at 36 weeks corrected gestational age as previously published in this cohortVenous cord blood was collected at birth by trained delivery staff. Samples were centrifuged for 10 min at − 4 °C at 3,000 rpm. Plasma was removed by pipetting and aliquots were archived at − 80 °C. Samples were retrieved from the Prentice Birth Cohort Repository and thawed on ice. Only samples without hemolysis were included. Soluble Klotho levels were measured via enzyme-linked immunoassay  using the protocol written by the manufacturer using 1:4 dilutions in duplicate.Pregnant Sprague Dawley rats were purchased from Charles River Laboratories . Rats were treated according to National Institute of Health guidelines for the use and care of laboratory animals following approval of the study protocol by the University of Miami Animal Care and Use Committee.2) or hyperoxia (85% O2), were randomly assigned to receive intraperitoneal (IP) injections of recombinant Klotho 30 mcg/kg , or phosphate buffered saline as placebo (PL) every other day from postnatal day 1 to 21. This dose was based on pilot studies in our laboratory. Oxygen exposure was achieved in a Plexiglas chamber by a flow-through system and the oxygen level inside the chamber was monitored daily with a Maxtec Oxygen Analyzer . Dams were rotated every 48 h between hyperoxia and normoxia chambers to prevent oxygen-induced damage to their lungs. Litter size was adjusted to 10–12 pups to control for the effect of litter size on nutrition and growth. The animals were recovered in normoxia for three additional weeks prior to sacrifice. Transthoracic echocardiography, lung morphometric and molecular studies were performed at postnatal day 42.Sprague Dawley pups assigned to normoxia . This solution was delivered at an air driven pressure of 40 cmH2O for 5 min and then the left atrium was punctured after distension. After completion of the vascular perfusion, a vascular fixative containing 4% paraformaldehyde was delivered at the same pressure and duration. The airways were perfused through the trachea at a transpulmonary pressure of 15 cmH2O for 5 min with 4% paraformaldehyde. The lungs were fixed overnight in 4% paraformaldehyde and then transferred to 70% ethanol and then subsequently embedded in paraffin. Serial Sects. (5 μm) thick were taken from the upper and lower lobes and stained with hematoxylin and eosin. Images from five randomly selected, non-overlapping parenchymal fields were acquired from lung sections of each animal using an Olympus Qcolor 3 color camera interfaced with a light microscope (Model Leica DMI 4000B) at 20X magnification. Alveolarization was determined by calculating mean linear intercept (MLI) and septal thickness. Mean linear intercept (MLI) was calculated by determining average distance between intersects of alveolar septal tissue and a superimposed counting grid30. Septal thickness was assessed on hematoxylin and eosin stained lung sections by averaging 100 measurements per 10 representative fields at 40X magnification.Lung alveolarization was evaluated as previously described30. Briefly, sections were de-paraffinized, rehydrated, and stained with polyclonal rabbit anti-human Von Willebrand Factor . The number of vessels (20–50 μm diameter) per high power field (HPF) was quantified in five randomly selected, non-overlapping, parenchymal fields from lung sections of each animal.Lung vascular density was evaluated as previously described30. Briefly, paraffin embedded sections were stained with polyclonal rabbit anti-human vWF and monoclonal mouse anti-α-smooth muscle actin . Medial wall thickness (MWT) of partially and fully muscular arteries (20–50 μm) was determined by using the formula: 2(MT) × 100/ED, where MT is the distance between the internal and external boundaries of the α-SMA layer and ED is the external diameter. The percentage of peripheral pulmonary vessels (< 50 µm in diameter) stained with α-SMA > 50% of the circumference was determined from ten random images on each lung section and all analyses were performed by a blinded observer.Pulmonary vascular remodeling was evaluated as previously describedRats underwent echocardiography using a Vevo2100 Imaging System . Echocardiography was performed under anesthesia via isoflurane inhalation (2% to 3%), controlled heart rates (≥ 400 bpm), and core body temperatures (37 ± 1 ℃). Left ventricular (LV) ejection fraction was assessed using M-mode and tricuspid annular plane systolic excursion (TAPSE) was measured using a modified 4-chamber view. Pulmonary artery Doppler was used to assess pulmonary artery acceleration time (PAAT) and pulmonary artery ejection time (PET). The average of 3–5 representative cardiac cycles was used to calculate values, using Vevo LAB Software (version 3.2.0).Paraffin embedded sections of lungs and right ventricles were de-paraffinized, rehydrated and stained with mouse monoclonal antibody to 3-Nitrotyrosine . 3-Nitrotyrosine expression (brown) was qualitatively determined by examining slides under a light microscope (Model Leica DMI 4000B) at 40X magnification.Paraffin embedded sections of lungs and right ventricles were de-paraffinized, rehydrated and stained with Masson’s trichrome as per manufacturer’s instructions . Fibrosis was qualitatively determined by examining slides under a light microscope (Model Leica DMI 4000B) at 40X magnification.29. The polyclonal antibodies for α-Klotho , MnSOD  and TGF-β  were obtained from Abcam . CC3 polyclonal antibody  was obtained from Cell Signaling Technology . Total protein was extracted from frozen lung, RV and LV tissues with a RIPA buffer according to the manufacturer’s protocol . Protein concentration was measured by BCA protein assay using a commercial kit from Pierce Biotechnology Inc. . Lung, RV and LV lysate (50 μg/sample) were fractionated by SDS-PAGE on 4–20% Mini-Protean Tris–Glycine Extended Precast Protein Gel  and transferred to nitrocellulose membranes . Immunodetection was performed by incubating the membranes with the primary antibodies diluted in blocking buffer overnight at 4ºC and then for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. Antibody bound protein was detected using enhanced chemiluminescence methodology (Amersham). Band intensity was quantified with Quantity One Software , with β-Actin acting as the normalization protein  for lung samples and GAPDH as the normalization protein for heart samples .The protein expression of α-Klotho, MnSOD, TGF-β, and CC3 were determined by Western blot analysis as previously describedLung Klotho gene expression was determined by real time RT-PCR. RNA from lung tissue was extracted  and reverse transcribed . Real time RT-PCR using TaqMan Fast Advanced Master Mix  was performed on an ABI Fast 7500 system (Applied Biosystems). Primers for Klotho (Cat# Rn0058012) and GAPDH (Cat# Rn99999916) were pre-developed by Applied Biosystems. The relative mRNA expression of Klotho was normalized to GAPDH expression.Rat Klotho serum concentration was determined by ELISA kit as per manufacturer instructions .2, 5% CO2) or hyperoxic  conditions for 24 h.HPAECs  were cultured to passage 3 to 6, plated in 100 mm dishes and serum starved for 48 h. Serum-starved HPAECs were treated with varying doses of recombinant human Klotho  and cultured in normoxic . Bright field images were collected at 12 h. All experiments were done in triplicate and tube formation was assessed using Image J Angiogenesis Analyzer.The effect of Klotho on capillary tube formation was determined by matrigel assay as previously described4 cells/well) cultured overnight in 96-well plates and treated with Klotho (0.01–0.5 µg/ml) were exposed to normoxia  or hyperoxia  for 24 h. Twenty microliters of MTT labeling solution was added 4 h before the end of the incubation. The plate was then incubated at 37 ºC for 5 min following which it was transferred to a plate reader  and absorbance was measured at a wavelength of 550 nm. The experiment was performed in triplicate.The effect of Klotho on HPAECs viability was determined by MTT assay . HPAECs  staining of HPAECs was performed using a TUNEL kit  as per manufacturer instructions. Five randomly selected fields were photographed, and the number of apoptotic nuclei as well as the total number of nuclei was counted per HPF. The apoptotic index was obtained by means of the formula: number of apoptotic cells per field / total number of cells per field as previously describedP values less than 0.05 were considered statistically significant.Human clinical data were analyzed using Kruskal–Wallis non-parametric testing when comparing median biomarker concentrations as distribution of Klotho was not normal by Shapiro–Wilk testing. Cord blood Klotho values are displayed as median [interquartile range]. Preclinical results reported as mean ± standard error of mean (SEM), were analyzed by two-way ANOVA with post-hoc Holm-Sidak test using Sigma Stat software. The clinical data of 40 infants including  are summarized in Table P < 0.05  for 3, 5, 14 and 21 days and lung Klotho gene and protein expression along with circulating soluble Klotho levels were assessed. The mRNA Klotho gene expression in the lung tissue of neonatal rat pups exposed to hyperoxia for 3 days was comparable to that of the group exposed to normoxia  from postnatal day (P) 1 to 21 were randomly assigned to receive every other day injections of recombinant Klotho or placebo (PL). The effect of Klotho on lung vascular development was assessed on P42. Lung sections were immunostained with vWF and the number of vessels (20–50 µm) per HPF quantified. PL-treated hyperoxia exposed rats had reduced lung vascular density, , but this significantly improved following early Klotho supplementation, , treatment with Klotho increased HPAECs viability, number of capillary tube like structures and HPAECs survival   Fig. a–d.FigurP < 0.05; N = 5/group) and this was significantly improved following early Klotho administration , and increased Tei index , Klotho administration significantly improved these measurements of left ventricular function  is an echocardiographic measure of RV function. PL-treated hyperoxia exposed rats had evidence of RV dysfunction as demonstrated by shortening of TAPSE but early administration of Klotho did not significantly alter TAPSE in hyperoxia-exposed animalsP < 0.05; N = 6–10/group) and septal thickness in the hyperoxia PL-treated animals. Early Klotho supplementation significantly improved these measures of alveolarization  and there was no significant difference with Klotho treatment, . In contrast, whereas the survival of normoxia treated rats was similar in both groups, , there was a trend for improved survival in the Klotho supplemented hyperoxia-exposed rats, which did not reach statistical significance .Since infants with BPD complicated by PH grow poorly1. Here, we provide the first published evidence demonstrating that soluble Klotho, an anti-aging protein with potent antioxidant, pro-angiogenic and anti-fibrotic properties, is reduced in the cord blood of preterm infants who subsequently develop BPD–PH. We also show, in a rodent model of severe BPD complicated by PH, that early Klotho supplementation improves lung vascular development, reduces PH, attenuates vascular remodeling and prevents long-term cardiac dysfunction, through antioxidant, pro-angiogenic and anti-fibrotic effects. Together, our present findings provide novel insight into the potential contribution of Klotho deficiency in BPD–PH pathogenesis and suggest a new target to improve long-term cardiopulmonary outcomes in preterm infants. This is particularly important, as there are currently few effective strategies to treat BPD and PH. Indeed, strategies targeting inflammation39, oxidative stress40 and angiogenesis41 have shown promise in preclinical models, but owing the multifactorial etiology of BPD and PH, few have been used clinically.Emerging evidence suggest that perturbation of key molecular aging pathways play a key role in BPD–PH pathogenesis42. We speculate that variations in Klotho circulating in cord blood at birth are heavily influenced by placental function (or dysfunction). This has been described in our recent publication27. Specifically, maternal vascular malperfusion (MVM), which is a histologic marker of upstream placental dysfunction arising during early placentation, is associated with decreased cord blood Klotho levels. Placental MVM is also prominent in extremely preterm births in which BPD-associated PH developed 43.In our current study, we first show that decreased cord Klotho levels are associated with an increased risk for severe BPD and PH. Klotho is known to be secreted by the placenta and whereas cord Klotho levels are typically higher than adult levels, plasma Klotho concentration is greater in full term than preterm infants at 14 and 28 days of life44 and they also importantly suggest that not only does placental insults lead to Klotho deficiency, but postnatal factors further reduce lung and circulating Klotho levels.Neonatal hyperoxia exposure is another key factor in BPD and PH pathogenesis. In our present study, employing an established experimental model of BPD and PH, we also demonstrate a marked reduction in lung Klotho expression and circulating Klotho levels following neonatal hyperoxia exposure. Our findings extend those of other investigators who demonstrate reduced Klotho expression in hyperoxia-exposed alveolar epithelial cells 45 and there is substantial evidence linking altered pro-angiogenic signaling pathways to BPD and PH pathogenesis46. In our study, we not only demonstrate that Klotho improves viability, proliferation and capillary tube formation of hyperoxia-exposed HPAECs but early supplementation of Klotho increased the number of peripheral pulmonary vessels in neonatal hyperoxia-exposed rodents. Our findings are consistent with other studies, which demonstrated improved tube formation, and survival of hydrogen peroxide injured Klotho-treated human umbilical venous cells19. Moreover, although the mechanisms by which Klotho regulates endothelial function and angiogenesis are still under investigation, emerging evidence suggest that the vasculoprotective effects of soluble Klotho may be secondary to its regulation of VEGFR2/transient receptor potential canonical-1 mediated Ca2+ influx22, modulation of endothelial nitric oxide production48 and its anti-oxidant properties. We speculate that Klotho’s direct pulmonary vascular protective properties in our study are secondary to its antioxidant effects and decreased TGF-β levels. This latter finding is supported by prior studies showing that TGF-β via its activation of the Smad 2/3 signaling pathway negatively regulates angiogenesis49.In order to ascertain whether early Klotho supplementation could reduce BPD and PH, we utilized a neonatal hyperoxia exposure rodent model of severe BPD–PH. Infants with BPD and PH exhibit decreased density of peripheral pulmonary arteries50. Additionally, Klotho inhibits renal fibrosis by antagonizing Wnt/β-catenin signaling51. While these mechanisms may contribute to the effects of Klotho evidenced in our study, we postulate that our current findings are secondary to decreased TGF-β pro-fibrotic signaling.Another important finding in our study is the significant reduction in vascular remodeling evidenced in neonatal hyperoxia-exposed rodents who received early Klotho supplementation. Other studies have demonstrated that Klotho-overexpressing mesenchymal stem cells reduce vascular remodeling in rodents with monocrotaline-induced PH by reducing inflammation, and increasing endothelial nitric oxide synthase levels52. Along with RV dysfunction, infants also develop LV dysfunction often secondary to septal bowing, myocardial ischemia and toxic effects of hyperoxia on the developing myocardium. Surprisingly, in our current study, while early Klotho supplementation significantly improved LV function, there were no significant changes in TAPSE, a measure of RV function. We speculate that with improvement in PH, the ventricular septal position shifts, allowing for volumetric changes in the left ventricle and improvement in the ejection fraction. RV function also impacts LV function through ventricular interdependence such that changes in RV geometry/function may also impact LV function. Importantly, TAPSE is only one measure of RV function, representing the RV longitudinal function, and it does not account for other aspects of RV function including circumferential shortening. Echocardiographic measures of RV function have always been limited owing to the complex geometry of the right ventricle, thus it is possible that there may have been improvement in the RV function, which was not accounted for by TAPSE. Our current finding of improved LV function is nonetheless in keeping with other investigators who demonstrated improved cardiac function in mice with doxorubicin-induced cardiac injury following Klotho administration53.Cardiac dysfunction is a significant cause of mortality in preterm patients with BPD and PH55. While it is plausible that the improvement in alveolarization in treated animals may be related to increased lung angiogenesis, we speculate that our finding of decreased apoptosis in the treated animals may have contributed to this preservation of alveolar structure. This reduced apoptosis in the treated animals may be due to decreased oxidative stress or maybe related to decreased TGF-β related pro-apoptotic pathways.In our present study, lungs from all experimental groups were also uniformly inflated in order to assess the effects of Klotho on lung alveolarization. Klotho supplemented hyperoxia-exposed rats had a significant improvement in alveolar structure. Lung vascular development is closely linked with alveolarization and impaired pulmonary function is seen in preterm survivors of BPD–PH even into adulthoodThe present study has several limitations. While cord blood Klotho was decreased with MVM in the current study (data not shown), the much smaller sample size included here did not allow us to comprehensively analyze the interactions between Klotho, MVM and BPD–PH outcomes collectively. The role of the placenta in mediating BPD–PH via Klotho cannot be determined by the clinical observations alone in this study, but combined with the animal findings presented here, these potential interactions are supported. Further studies incorporating larger infant cohorts and animal models of placental dysfunction are warranted. Also, while there are currently no clear genetic links to low Klotho levels in cord blood, investigation of genetic factors that influence Klotho is also warranted. The BPD–PH rodent model used was also severe and may not reflect the disease evidenced in many preterm survivors. In addition, while exposure to high oxygen levels is important in BPD–PH pathogenesis, other factors including intermittent hypoxia, pre-and postnatal exposure to infection, mechanical ventilation and poor nutrition, contribute to this disease. It is therefore possible that the additive effects of these insults on the preterm cardiopulmonary system will potentially alter the efficacy of Klotho. We also exposed the rat pups to a high oxygen concentration from birth to postnatal day 21, which corresponds to the saccular-alveolar stage of lung development. Most preterm infants at highest risk for BPD are born in the cannalicular and saccular stages of lung development, with repair and recovery occurring in the alveolar phase. Further dose–response studies will also need to be performed, as early Klotho supplementation did not return lung structure or cardiac function to those observed under normoxic conditions. It will also be crucial to perform studies evaluating the effect of later administration of Klotho in established neonatal PH models. Moreover, since Klotho affects calcium and phosphorus metabolism, it will be important to follow calcium and phosphorus levels longitudinally.In conclusion, to the best of our knowledge, this is the first study which evaluates cord blood Klotho levels in preterm infants who develop BPD and PH. We show that Klotho deficiency predicts BPD and PH risk in preterm infants. Moreover, through anti-oxidant, anti-fibrotic, anti-apoptotic and pro-angiogenic mechanisms, early Klotho supplementation improves lung structure, attenuates vascular remodeling and reduces cardiac hypertrophy and dysfunction in rats with experimental severe BPD complicated by PH. Together, our findings suggest that Klotho deficiency is a potential mediator and biomarker for BPD and PH risk and early interventions, which augment Klotho levels, may be an effective strategy to improve cardiopulmonary outcomes in preterm infants. Further studies will be needed evaluating the effects of Klotho on other developing organ systems as this will be crucial to translate the findings of our studies to the bedside.Supplementary information.


Artemia), roundworms (nematodes), and tardigrades (water bears). Discovery of molecular protectants that allow each of these three animals to survive drying diversifies our understanding of desiccation tolerance, and convergent themes suggest mechanisms that may offer a general model for engineering desiccation tolerance in other contexts.Water is critical for the survival of most cells and organisms. Remarkably, a small number of multicellular animals are able to survive nearly complete drying. The phenomenon of anhydrobiosis, or life without water, has been of interest to researchers for over 300 years. In this review we discuss advances in our understanding of protectants and mechanisms of desiccation tolerance that have emerged from research in three anhydrobiotic invertebrates: brine shrimp ( The metabolism of life occurs in an aqueous environment, and the biological constituents of cells – DNA, RNA, protein, and membranes – are all unstable during desiccation. However, some animals defy this reasoning, surviving in the near-absence of water by entering a quiescent state and later resuming activity upon rehydration. Antonie van Leeuwenhoek first described reviving dehydrated “animalcules” from the sediment in his gutter in 1702 . Other iTo affirm that a being, whose animation has been suspended for an immoderate length of time, even for years, can, in virtue of certain conditions, be revived, has so singular and paradoxical an appearance, that reason finds it repugnant to admit the fact. But to produce an animal which has been stiff and motionless, withered, disfigured, and contracted, utterly incapable of any corporeal function… and, by a particular treatment, to make it renovate every action that it could perform before; …not only will it bear perfect conviction to the mind that it has come from a state which, if it was not death, certainly cannot be called life, but that it again lives as completely as before its animation was suspended or destroyed. Some animals in the world enjoy this wonderful prerogative , roundworms (with a focus on Caenorhabditis elegans), and tardigrades. Artemia, roundworms, and tardigrades all undergo substantial physical and physiological changes. Artemia are able to survive dehydration as diapausing embryos  that are exposed to desiccation conditions are able to survive as larvae or adults, whereas diapause . Tardigrdiapause . Thus, aArtemia, nematodes, and tardigrades have been somewhat complementary and revealed different aspects of the physiology and molecular details of desiccation resistance. In this review we explore the molecules and mechanisms of desiccation tolerance, highlight common themes, and suggest open questions whose answers will be instrumental to the advancement of our understanding of desiccation tolerance.The approaches researchers have employed in studying Artemia). Johannes Albertus Schlosser is credited with the discovery and initial description of Artemia ; however, when environmental conditions are poor, females will instead release gastrula-stage embryos (oviparous development) that diapause until conditions amenable to hatching and development are present  are a family of ATP-dependent chaperones essential for protein folding. Knockdown of one family member, Hsp70, in mpbellii . Knockdompbellii . J-domaimpbellii . Two J-drHsp40-2 . KnockdorHsp40-2 . DeveloprHsp40-2 . This tiArtemia  is a common mechanism contributing to anhydrobiosis in a variety of organisms. One of the most prominent examples of an IDP is the family of late embryogenesis abudant (LEA) proteins. LEA proteins were originally identified in plants, but subsequently have been found in multiple animal species, including Artemia . There aArtemia . Artemia animals . These L animals .Artemia embryos suggests a possible function in desiccation resistance. A common experimental strategy to test for LEA function has been to assess chaperone activity or protective capacity in heterologous or in vitro contexts. LEA proteins from Artemia are capable of protecting liposomes, stabilizing membranes, attenuating protein aggregation, and conferring resistance to osmotic and desiccation stresses  regulates expression of Artemin and the small heat shock proteins p26, Hsp21, and Hsp22  expression profiling to identify genes whose mRNA abundances change during desiccation and (2) disruption of candidate genes’ functions to identify genes that are required for desiccation tolerance. These approaches have identified molecules that suggest a striking diversity of physiological adaptations to desiccation, some prominent examples of which we discuss in the sections below.C. elegans, the upregulation of trehalose synthesis during gradual drying is essential for worms to survive desiccation  and the subsequent dephosphorylation of trehalose 6-phosphate by a protein encoded by gob-1    . Each ofgulated) . The iniscussed) . Despitean cells . LEA-1 ficcation .Aphelenchus avenae as a model, C. elegans LEA-1 , several glutathione peroxidases  and catalase (ctl-1) are required for desiccation survival, suggesting that upregulation of these genes  is an adaptive physiological response to gradual desiccation , Δ6 desaturase (fat-3), and Δ5 desaturase (fat-4). Of note, the ω3 desaturase, fat-1, does not impact desiccation survival, suggesting that arachidonic acid may play a particular role in desiccation resistance  must have mechanisms to recognize gradual loss of water and to induce the responses described above. Even animals like fruit flies that cannot survive total desiccation can sense and respond to humidity  in thermosensory neurons regulates hygrotaxis  channel gene, survival . TRP chagrotaxis . In C. egrotaxis . Additiogrotaxis . It is shsf-1 regulates the heat shock response and many chaperones, skn-1/Nrf2 is a master regulator of the oxidative stress response, and daf-16/FOXO transcriptionally regulates many genes involved in the glyoxylate shunt and trehalose synthesis during starvation , a patched related protein (DAF-6), and mitogen-activated protein kinase (MAPK) signaling . Specifiarvation . HoweverArtemia and tardigrades. More genes than discussed above play roles in desiccation tolerance. These genes   . HoweverAdorybiotus coronifer and those of the family Macrobiotidae have been shown to produce trehalose in response to desiccation  is considerably lower than the level of trehalose accumulation reported in Artemia (15%) or nematodes (∼13%)  . The appProtectants that function during desiccation must retain their protective properties even during conditions in which most proteins are prone to aggregation and dysfunction. Thus, protective proteins are likely to be resistant to aggregation and retain solubility even during extreme conditions if they are to interact with and preserve other cellular constituents. If protectants that function during desiccation are themselves resistant to severe dehydration stress, this may predispose them to withstand other stresses as well. To identify possible protectant proteins, one approach has been to determine the portion of the proteome that is highly stable and soluble even during stress. The most straightforward method for this has been to search for heat soluble proteins – those that can retain solubility even after transient exposure to near-boiling temperatures. Analysis of the tardigrade proteome has revealed such heat soluble proteins .Ramazzotius varieornatus, and homologs for some of the heat soluble proteins are also present in other tardigrade species including H. exemplaris and Milnesium tardigradum , mitochondrial abundant heat soluble (MAHS), and secretory abundant heat soluble (SAHS) proteins . The SAHdigradum . Furtherichtersi . Some CAichtersi . RNAi knichtersi . These eThe potentially broad function of TDPs piques interest in understanding the mechanisms by which they exert their protective effects. As with LEA proteins, the disordered nature of CAHS proteins and other TDPs goes against traditional intuition of structure-function analysis. However, CAHS proteins take on a glass form during desiccation, suggesting that disordered proteins in general may contribute to cellular vitrification – a mechanism we will discuss in more detail below .Artemia researchers, was used along with immunolocalization in tardigrades to determine the subcellular localization of a group 3 LEA protein of Ramazzottius varieornatus, RvLEAM. This LEA is mitochondrially localized, similar to AfrLEA3m  are also upregulated during dehydration  that would occur in an aqueous environment . A partiA glass is an amorphous, non-crystalline solid. Through the process of vitrification, biological glasses form and protect cells from damage during dehydration . During Artemia and tardigrades, suggest that glass formation is a widespread mechanism contributing to desiccation tolerance (Evidence for glassy solids in plants and animals, including olerance . While sThe term “molecular shield” was first proposed to describe the function of LEA proteins that may provide a protective shell that shields proteins during desiccation and prevent aggregation . The proWhile the water replacement hypothesis, vitrification, and molecular shielding are sometimes discussed as individual mechanisms for desiccation survival, they likely overlap and synergistically promote desiccation tolerance. This can be appreciated when individual molecular protectants contribute to more than one of these models. For example, LEA proteins contribute to each of the three models for protection, and trehalose plays a prominent role in both the water replacement hypothesis and vitrification . Thus, fArtemia, nematodes, and tardigrades has advanced our understanding of the protectants and mechanisms that contribute to desiccation survival. Comparisons of the types of protectants involved in desiccation tolerance in each animal converges on a few key molecules (see Artemia-specific protein, Artemin. Each organism has brought with it unique experimental advantages as well – Artemia have a unique life cycle and intergenerational diapause, C. elegans has provided a genetic model to greatly facilitate gene identification and functional studies, and tardigrades are emerging as another model for exceptional stress tolerance. A comparative organismal approach to the study of desiccation tolerance, using these organisms and others such as rotifers and midges, has enriched our understanding of mechanisms that multicellular organisms employ to survive desiccation, and no doubt will continue to do so.Our knowledge of the mechanisms of desiccation tolerance has been largely informed by research on a small number of anhydrobiotic organisms. Work on ules see . HoweverDesiccation tolerance is sometimes conceived as primarily a cellular survival strategy. While individual cells must synthesize the necessary protectants to survive, the coordination of the desiccation response in multicellular organisms presents an expanded set of challenges – to sense and respond appropriately to environmental cues that signal dehydration, and coordinate a response across tissues to allow the animal to survive. This necessitates exquisite spatial and temporal regulation. While some mechanisms by which this sensing and coordination are accomplished have been discussed, the basis of the intermediate regulatory steps between survival of an organism and survival of a cell remain largely unknown.in vivo experiments and drawing conclusions about protectants in their native context will help to distinguish between possible protectants and those that are essential. Still, part of the promise in studying mechanisms of desiccation tolerance is the application of discoveries to better preserve biomaterials. Therefore, discovery of new, uncharacterized protectants continues to be of interest. In over 300 years since the initial observation of desiccation survival, our understanding has grown considerably. Yet, in many ways the molecular basis of anhydrobiosis and mechanisms by which multicellular animals survive drying are only beginning to emerge. Continued research in Artemia, nematodes, and tardigrades in coming years will likely contribute significantly to our growing understanding of desiccation tolerance.In many cases, heterologous expression of proteins demonstrates the possible activity of protectants for desiccation survival. However, the endogenous functions of proteins are often poorly characterized. Conducting more JH drafted the original manuscript. All authors edited and revised the manuscript, approved the final version, and contributed to the conception of this review and discussion of its contents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.