Drosophila and as part of pathological processes in humans, specifically in tumors and drug-resistant cells. The long-held belief that a physiological gene amplification does not occur in humans was, however, fundamental questioned by findings that showed gene amplification in human stem cells. We hypothesis that the physiological and the pathological, i.e., tumor associated processes of gene amplification share at their beginning the same underlying mechanism. Re-replication was reported both in the context of tumor related genome instability and during restricted time windows in Drosophila development causing the known developmental gene amplification in Drosophila. There is also growing evidence that gene amplification and re-replication were present in human stem cells. It appears likely that stem cells utilize a re-replication mechanism that has been developed early in evolution as a powerful tool to increase gene copy numbers very efficiently. Here, we show that, several decades ago, there was already evidence of gene amplification in non-tumor mammalian cells, but that was not recognized at the time and interpreted accordingly. We give an overview on gene amplifications during normal mammalian development, the possible mechanism that enable gene amplification and hypothesize how tumors adopted this capability for gene amplification.Gene amplifications have been known for several decades as physiological processes in amphibian and flies, e.g., during eggshell development in  Nature by Pelc SR [As early as 1968, studies reported features of gene amplification in normal mammalian cells. These first indications were, however, not followed up upon. Gene amplification, i.e., an increase in the gene copy number as a tool for an increased RNA and protein synthesis in differentiating tissues was first postulated in 3H-dT and purified by CsCl gradient centrifugation. Surprisingly, the buoyant density pattern was different between the DNA from 6-day lactating mammary mice and the DNA from virgin, pregnant or weaned mice. The authors observed an additional labeled band near the heavier region of the gradient for lactating cells and interpreted these results as indication for a high number of actively replicating cells. Since the extra DNA was not found in the involuted mammary glands, the authors hypothesized a periodic of amplification occurrence in parts of the genome during differentiation of mammary gland [The first detailed experimental evidence for gene amplification stems from a study of Banerjee and Wagner, who analyzed mouse mammary gland from virgin, pregnant, lactating and weaned mice . Prior try gland . Notably0t values in spermatozoa and interpreted this observation not only as indication of highly repetitive DNA but also as indication of “periodic” gene amplification in actively differentiating cells [In 1978, Hernández and colleagues determined reassociation kinetics of fragmented DNA from sperm and leucocytes at various time points . They reng cells .Additionally, in 1978 Strom and Dorfman reported evidence for amplification during differentiation in chicken cartilage and postulated that these sequence amplifications were regulatory and “turn on” the synthesis of differentiation-specific genes without replication of the whole genome . In detaAnother study provides evidence for an actin gene amplification during chicken myogenesis. In 1981, Schwartz and Rosenblum speculated that an increased mRNA expression of the chicken actin gene during myogenesis is controlled at the transcriptional level . An addeTen years later, a study reported the enrichment of double minutes after addition of serum to serum-free mouse embryo (SFME) cells. However, the meaning of double minutes as manifestation of gene amplification and a characteristic marker was not recognized in this context ,9. The a2 values between 0.16 and 0.35 in the arrayCGH experiments.Once it has been recognized that serum-free mouse embryo (SFME) cells represent largely neural stem cells, gene amplification were searched for in mouse and human neural stem cells using more sensitive methods. Array comparative genomic hybridization (arrayCGH) detected amplified regions from 250 kb to more than 10 Mb in size with a genome-wide distribution in human and mouse neural stem cells during differentiation including in the above mentioned SFME cells . FurtherCDK4 was amplified during the differentiation into astrocytes, oligodendrocytes, and neurons. MDM2 was amplified in most of the differentiation protocols but not during the first three days of differentiation towards oligodendrocytes. GINS2 was amplified only after one day of differentiation towards oligodendrocytes and neurons but not towards astrocytes [Further and more detailed evidence of gene amplification was found by studies that employed three different induction protocols to differentiate neural stem cells into either astrocytes, oligodendrocytes, or neurons. The pattern of gene amplification during these differentiation processes were exemplarily analyzed for eight genes that were previously found to be amplified in arrayCGH analysis of differentiating neural stem cells. trocytes . These dERBB2 amplifications by dual color silver in situ hybridization (SISH) in human first trimester placental tissue sections [ERBB2 in 29% of HLA-G+ CC trophoblasts [Further example of gene amplification during differentiation and placentation stems from a study by Meinhardt and colleagues who analyzed extravillous trophoblast (EVT) differentiation. They detected sections . Specifihoblasts . Using aDrosophila.Selective amplifications were also detected in parietal trophoblast giant cells of mouse placenta. Whole genome sequencing (WGS) and digital droplet PCR identified five amplified regions including some that mapped on mouse chromosome 13. Interestingly each of the amplified regions on chromosome 13 contain different placenta gene families . AlthougCDK4 and MDM2 that were more generally found not only in hMSCs, but also during adipocyte and osteoblast differentiation, and during neural stem cell differentiation. The analysis of gene amplification in human mesenchymal stem cells also shed light on the challenge of amplification detection. While whole genome sequencing (WGS) detected many amplified chromosome regions, it does not allow to identify all amplifications that were detected by arrayCGH. For example, gene amplification of CABIN1 gene in undifferentiated hMSCs was detected by arrayCGH and on single cell level by FISH but not by WGS [Another example for gene amplification stems from studies on human mesenchymal stem cells (hMSCs) and from mesenchymal stem cells differentiating towards adipocytes and osteoblasts . ArrayCGt by WGS . These eCDK4, MDM2 and NUP133 were investigated for amplification during the primary human myoblast differentiation and ACTA1, CDK4, MYO18B and NUP133 were investigated during the C2C12 myoblast cell differentiation. While, human primary myoblasts showed amplification of CDK4, MDM2 and NUP133, C2C12 myoblast cells showed CDK4 amplification already in undifferentiated cells and the highest level of amplification during differentiation. This analysis also exemplified the challenge of identifying specific gene amplifications. Alpha-actin (ACTA1) gene amplification was not detected during C2C12 differentiation using qPCR with two different ACTA1 TagMan probes [NUP133 gene, which localizes only 10 kb next to ACTA1. Since a genomic probe (BAC-clone) that encompassed ACTA1 and NUP133 for FISH analysis, revealed amplification, it is very likely that the hybridization probe used in former studies on chicken myogenesis included sequences from NUP133 gene and that the hybridization results were misleadingly interpreted as ACTA1 gene amplification. In In addition, during myoblast differentiation gene amplifications were detected. In detail, myoblast differentiation from mononucleate cells towards multinucleate myotubes was investigated on primary human myoblasts and the mouse C2C12 myoblast cell line by qPCR experiments with TagMan probes . Specifin probes . This isn probes . QPCR anAs detailed above, gene amplifications have been known in tumors for several decades and were more recently also reported for both undifferentiated and differentiating stem cells. We summarize similarities of gene amplifications in stem cells and their corresponding malignant tumors.CDK4 and MDM2. QPCR analysis revealed EGFR amplification in NHNP cells and in differentiated NHNP cells [EGFR gene in about one third of the cases [CDK4 gene is amplified and in around 10% the MDM2 gene [ArrayCGH did identify amplification on 93 chromosome regions in normal human neural progenitor (NHNP) cells differentiated for 5 days . The gaiNP cells ,12. GlioNP cells , showed he cases . In abouDM2 gene ,21. In aDM2 gene .ERBB2 amplification was detected [ERBB2 amplification was reported in gestational trophoblastic disease (GTN) that is a rare group of tumors that were mostly benign and origin from placental villous and extravillous trophoblast cells [ERBB2 amplification and expression in combination with hyperploidy leads to a higher proliferation and more aggressive behavior including metastases and carcinoma [As reported above during extravillous trophoblast (EVT) differentiation detected . Likewisst cells ,24,25 ERarcinoma .FISH analysis revealed co-amplification of CDK4 and MDM2 during mesenchymal stem cell differentiation towards adipocytes . Well-diCDK4 and MDM2 occurs during mesenchymal stem cell differentiation towards osteoblasts as shown by FISH and qPCR analysis [CDK4 and MDM2 was found in 67% of parosteal osteosarcoma that is a low-grade differentiated type of osteosarcoma and in 9–12% of classical osteosarcoma [Similar to the aforementioned adipocytic differentiation, co-amplification of analysis . Amplifiosarcoma . Notablyosarcoma .CDK4, MYO18B and NUP133 was detected by FISH and qPCR during the differentiation of mouse C2C12 myoblast cells, and amplification of MDM2, CDK4 and NUP133 during the differentiation of primary human myoblasts. Amplification of CDK4 and MDM2 was also found in leiomyosarcoma and rhabdomyosarcoma which are both myogenic sarcoma [Amplification of  sarcoma ,30.MET gene amplification [Gene amplifications in tumors were often associated with genome instability including double strand breaks. Specifically, the induction of double strand breaks (DSBs) by gamma-irradiation can trigger gene amplifications ,32. In tfication . As showThe appearance of transient strand breaks has been reported during primary chick myoblast differentiation ,36. In dNotably, during adipogenesis of murine and human pre-adipocytes there is an up-regulation of double strand break repair activity due to an up-regulation of DNA-PKcs  expression . During Twenty years after the study of Dawson and Lough, Larsen and colleagues reported another example for a correlation between DSBs and differentiation during myoblast differentiation. They found an increase in double strand breaks after 12 and 24 h of differentiation and a decrease in double strand breaks after 48 to 72 h of differentiation . In addiDNA replication is a tightly regulated process with two main steps, i.e., origin licensing in late mitosis and early G1-phase and origin firing during S-phase. The major mechanism to prevent relicensing during S-phase in mammalian cells is the inactivation of CDT1, which is one of the essential replication initiation proteins ,40. EarlInterestingly in primary cells as BJ and IMR90 cells, CDT1 overexpression leads to limited re-replication at cellular replication origins with none of the replicating cells showing more than 4N DNA content . In addiDrosophila ovarian follicle cells, the gene amplification of eggshell genes is enabled through the over-replication of two clusters of chorion genes [In on genes . Alexandon genes . NotablyAs early as 1984, Stark GR and Wahl GM and Hamlin JL and colleagues postulated a model for gene amplification caused by unscheduled DNA replication . This unDrosophila gene amplification is associated with re-replication and double strand breaks. In human tumors gene amplifications, DSBs and re-replication have been reported but mostly in the context of cell cycle checkpoint failure. As mentioned above we recently reported re-replication in human myoblast cells during differentiation towards myotubes [In myotubes . During Healthy and “normal” stem cells have intact checkpoint and repair mechanisms to protect their genome integrity while using time-restricted re-replication and gene amplification for fast protein expression during differentiation . In cont


Hibiscus sabdariffa L.) is recognized for its phytochemical compounds such as anthocyanins, which possess pharmacological potentials in the treatments of hypertension, diabetes, cancer, hyperlipidaemia and hyperglycaemia. The calyx is the most commercially valuable part of the roselle and usually harvested at maturation. However, genetic study to understand the transcriptome changes in the calyx during maturation has yet to be explored. In this study, we sequenced the transcriptomes of roselle calyces at maturation stages III and IV using Illumina NextSeq 500 platform. These are the two most critical maturation stages in roselle, as these stages are often associated with the quality of the calyx. Over 200 million good quality paired-end reads were generated and de novo assembled into a reference transcriptome consisting of 221,334 transcripts with N50 score of 491bp. Among these transcripts, 92,974 transcripts (42%) were successfully annotated. The total number of significantly differentially expressed genes (DEGs) and the top five most significantly regulated genes in each of the maturation stage were presented. Twenty-one genes implicated in the biosynthesis of anthocyanins and their relative expressions in the calyx tissues at the two maturation stages were reported. Two secondary metabolites biosynthesis pathways that attained a relatively higher number of DEG mappings compared to other pathways were also reported. The findings from this work provide novel insights to better understand the transcriptional changes in roselle during calyx maturation, and the data made available here is intended for continued genetic study on roselle. The work is registered under NCBI Bioproject PRJNA664826. The raw sequencing reads are available in Short Read Archive with the accession numbers SRX9171161, SRX9171162, SRX9171163, SRX9171164, SRX9171165 and SRX9171166.Roselle ( It could also be used as a reference data for researchers working on roselle.•The transcriptome dataset allows for further downstream applications associated with newly constructed genetic resources such as genic SSR marker development and discovery of transcription factors.1. The top five most significantly regulated DEGs in stage III and stage IV are shown is The maturation stage of the calyx is commonly used as a measure to reflect its quality, where it is preferably harvested during stage III over stage IV. To date, no scientific study has been conducted to reveal the transcriptome changes in the calyces of the two stages. Six sequencing libraries prepared from stage III calyx  and stage IV calyx  were sequenced using Illumina NextSeq 500. The datasets contained raw data sequence converted to FastQ format. The raw reads were deposited into NCBI Short Read Archive with te Bioproject number PRJNA664826 . The number of total reads and good quality reads generated in this study is presented in Table 3The two secondary metabolites biosynthesis pathways that attained a relatively higher number of DEG mappings compared to other pathways within the same category were the phenylpropanoid biosynthesis pathway and the isoquinoline and alkaloid biosynthesis pathway.22.1Hibiscus sabdariffa variety UMKL1 (voucher number: CY001) was used in this study. The roselle planting materials were obtained from the Department of Agriculture Terengganu, Malaysia. It is a red variety registered as ‘HS2’ by the Malaya University for national crop list under the Department of Agriculture Malaysia.nd DPA; whereas biological replicates of stage IV calyces  were collected on 59th DPA. Collected samples were immediately flash-frozen in liquid nitrogen and stored in a -80˚C until RNA extraction as a preventive measure against rapid RNA degradation. A separate set of calyx samples were harvested during sampling, observed, and dissected, and the images were captured for the evaluation of phenotypic differences.Calyx tissues were harvested at stages III and IV determined based on the number of days post-anthesis (DPA), in reference to the calyx tissue maturation index guidelines provided by the Federal Agricultural Marketing Authority, Malaysia  [1]. Thr2.2Total RNA was extracted from calyx tissue samples using RNeasy Plant Mini Kit  based on the manufacturer's protocol version 2012. RNA extractions were conducted in triplicate for calyx samples from each maturation stage. RNA quality and quantity were determined using Qubit Fluorometer 2.0  and NanoDrop spectrophotometer  for concentration assessment and Agilent 2100 Bioanalyzer  via a Pico Chip and 1% agarose gel electrophoresis for preliminary assessment of band size and quality. RNA samples having a RIN value above seven and a concentration of more than 1 µM were selected rRNA-depletion.RNA samples were subjected to rRNA-depletion using Terminator™ 5’-Phosphate-Dependant Exonuclease  for the purpose of removing the abundance of ribosomal RNAs (rRNA) from the sample to ensure most of the sequencing output generated accounts for reads derived from messenger RNAs (mRNA) which is important for gene expression analysis. The resulted rRNA depleted RNA samples were assessed using Qubit Fluorometer 2.0  and Agilent 2100 Bioanalyzer . rRNA-depleted samples having a concentration of more than 0.5 ng/µl and a rRNA contamination below 10% were selected to construct the cDNA sequencing libraries.Constructions of the cDNA sequencing libraries were carried out using ScriptSeq™ v2 RNA-Seq Library Preparation Kit  based on the manufacturer's handbook to reverse transcribe the less stable RNA into a more stable molecule, complementary DNA (cDNA) that is used as the template for sequencing. The ScriptSeq cDNA libraries were quantified using Qubit Fluorometer 2.0  and the size distribution was assessed using the Agilent 2100 Bioanalyzer  via a High Sensitivity DNA Chip. cDNA sequencing libraries with more than 60% of the fragment sizes falling within the 200-100 bp range were selected and quantitated using the CFX96 Touch™ Real-Time PCR Detection System . Libraries were sequenced using Illumina NextSeq 500 Sequencing platform , resulted in the generation of 76 bp paired-end sequences.2.32.3.1De novo reconstruction of transcriptomes using the good quality reads was performed using Trinity version 2.2.0 software Raw sequencing reads were cleaned by filtering the PhiX (Illumina co-sequencing positive control) sequences using Bowtie2 version 2.2.3 software 2.3.2de novo assembled transcripts. Transcript abundance for every transcript was quantified in transcripts per million (TPM). Expression profiles of biological replicates were first examined to ensure the well correlation of replicates and to probe relationship among samples. Scatter plots and Pearson correlation coefficient graphs were constructed between biological replicates within samples of each stage to measure their interrelationship. Principal component analysis (PCA) was performed using RStudio IDE (version 4.1.2.) software p-value (FDR) of ≤0.05 were defined as significantly differentially expressed genes (DEGs) and further utilised to form a volcano plot to visualize and highlight the degree of which genes are significantly differentially expressed. A Venn diagram was constructed via Interactivenn software Transcript expression analysis was carried out using the CLC Genomic Workbench (Version 10.1.0). Raw reads were subjected to quality control  and the good quality reads of each sample were mapped to the 2.3.3The KEGG Mapper – Search and Color Pathway software was employed for pathway analysis. Significantly differentially expressed transcripts identified from the differential expression analysis were used to obtain a list of Trinotate-annotated K numbers, which were then used for pathway reconstruction using the Kyoto Encyclopedia of Genes and Genomes web server against the KEGG pathway database. The overall flow chart of the RNA-sequencing pipeline used in this study is summarised in This study does not involve human participants and samples derived from human. It also does not involve animals including live vertebrates and higher invertebrates.Nur Atheeqah Hamzah: Investigation, Methodology, Formal Analysis, Validation, Writing – original draft, Visualization; Christina Seok Yien Yong: Conceptualization, Resources, Writing – review & editing, Visualization, Supervision, Project Administration, Funding acquisition; Meenakshii Nallappan: Supervision, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Mu heavy chain disease is a rare lymphoid neoplasm characterized by vacuolated bone marrow plasma cells and secretion of defective mu immunoglobulin heavy chains. The biological basis of mu heavy chain disease is poorly understood.MYD88 L265P mutation and deletion of 6q, genetic aberrations that are both strongly associated with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Identification of the truncated mu immunoglobulin was facilitated by mass spectrometric analysis of the patient’s serum.We report a case of mu heavy chain disease with MYD88 mutation in mu heavy chain disease, argues that at least a significant subset of cases are more similar to lymphoplasmacytic lymphoma than to chronic lymphocytic leukemia.Mu heavy chain disease has been described as similar to chronic lymphocytic leukemia; however, the frequency of lymphocytosis in mu heavy chain disease has not been previously reported. We reviewed all previously published mu heavy chain disease reports and found that lymphocytosis is uncommon in the entity. This finding, along with the emerging genetic feature of recurrent  MYD88 L265P mutation along with deletion of 6q, supporting a biologic relationship between mu-HCD and lymphoplasmacytic lymphoma (LPL), a lymphoma that most often presents as Waldenström macroglobulinemia (WM).Mu heavy chain disease (mu-HCD) is an exceptionally rare entity described as resembling chronic lymphocytic leukemia (CLL), with vacuolated plasma cells and secretion of a defective mu heavy chain . We repo9/L. A PET/CT scan did not demonstrate any lytic bone lesions or enlarged lymph nodes; the spleen size was reported as at the upper limit of normal. A bone marrow biopsy and aspirate were obtained. The bone marrow was markedly hypercellular due to a diffuse proliferation of small lymphocytes and plasma cells with prominent cytoplasmic vacuolization . The patient had an absolute lymphocyte count (ALC) of 2.2 × 10MYD88 L265P mutation and deletion of 6q, both common abnormalities in LPL [MYD88 L265P mutations, although vacuolated plasma cells were described in only one case and none were illustrated [MYD88 mutations are recurrent in mu-HCD, suggesting a relationship with LPL. The generalizability of this finding is difficult to assess as mu-HCD is extraordinarily rare, with only 30–40 cases previously reported; however, additional indirect evidence suggests that a relationship with LPL is plausible in many mu-HCD cases.This case of mu-HCD with classic morphologic, phenotypic, and serologic features demonstrates genetic similarity to LPL with a s in LPL , 5. Verg9/L, and the patient was clinically diagnosed with CLL [sine qua non of CLL, but the frequency of lymphocytosis in mu-HCD is not established, as it has not been summarized in previous reviews of the entity. We attempted to review all prior reports of mu-HCD, collecting peripheral blood white blood cell and differential count information from 29 previously reported patients with mu-HCD [9/L), and only 5 reported patients have had ALC greater than 10 × 109/L  was negative for CD5 [Nearly all cases of CLL are positive for CD5, but the immunophenotypic characteristics of mu-HCD are not well established, as many of the reports of mu-HCD predate comprehensive immunophenotyping of lymphoid neoplasia and the recognition of the CD5 antigen on CLL cells . However for CD5 , as was  for CD5 . CD5 may for CD5 , and so  for CD5 .These features suggest that most previously reported cases of mu-HCD are unlike CLL as currently understood. Our case and those of Vergneault and colleagues  establisMYD88 mutation and establish a rationale for potential LPL-directed management of this rare entity.This report additionally underscores the value of novel techniques such as MASS-FIX in the detection of abnormal serum proteins. mu-HCD may be underrecognized, and patients with increased free serum light chains or Bence-Jones proteinuria without a corresponding serum heavy chain should be considered for further assessment using methods such as MASS-FIX to better evaluate for abnormal proteins that could be missed using conventional serologic techniques. Discrimination between a B-cell neoplasm with plasmacytic differentiation and a plasma cell neoplasm is highly clinically relevant, and accurate M-protein characterization is a critical component of the diagnosis. Our findings suggest that mu-HCD cases should be screened for


Aortic stenosis is the most common cardiac valve disorder requiring clinical management. However, there is little evidence on the societal cost of progressive aortic stenosis. We sought to quantify the societal burden of premature mortality associated with progressively worse aortic stenosis.In this observational clinical cohort study, we examined echocardiograms on native aortic valves of 98 565 men and 99 357 women aged 65 years or older across 23 sites in Australia, from Jan 1, 2003, to Dec 31, 2017. Individuals were grouped according to their peak aortic valve velocity in 0·50 m/s increments up to 4·00 m/s or more (severe aortic stenosis), using 1·00–1·99 m/s (no aortic stenosis) as the reference group. Sex-specific premature mortality and years of life lost during a 5-year follow-up were calculated, along with willingness-to-pay to regain quality-adjusted life years (QALYs).Overall, 20 701 (21·0%) men and 18 576 (18·7%) women had evidence of mild-to-severe aortic stenosis. The actual 5-year mortality in men with normal aortic valves was 32·1% and in women was 26·1%, increasing to 40·9% (mild aortic stenosis) and 52·2% (severe aortic stenosis) in men and to 35·9% (mild aortic stenosis) and 55·3% (severe aortic stenosis) in women. Overall, the estimated societal cost of premature mortality associated with aortic stenosis was AU$629 million in men and $735 million in women. Per 1000 men and women investigated, aortic stenosis was associated with eight more premature deaths in men resulting in 32·5 more QALYs lost  and 12 more premature deaths in women resulting in 57·5 more QALYs lost  when compared with those without aortic stenosis.Any degree of aortic stenosis in older individuals is associated with premature mortality and QALYs. In this context, there is a crucial need for cost-effective strategies to promptly detect and optimally manage this common condition within our ageing populations.Edwards LifeSciences, National Health and Medical Research Council of Australia, and the National Heart, Lung, and Blood Institute. Paradoxically, having avoided early and often fatal forms of heart disease, these individuals might still be at risk of dying prematurely.6 The development of less invasive, transcatheter techniques for aortic valve replacement has altered the benefit-to-risk ratio of aortic stenosis management and assessment of treatment safety and efficacy is ongoing in patients with less severe symptoms.7 Regardless of treatment modality, the increasingly prevalent condition of aortic stenosis poses an enormous challenge to already limited health-care resources.8 Crucially, there is increasing evidence showing that less severe forms of aortic stenosis are associated with premature mortality, whether by causation or correlation.9 However, there remains minimal evidence of the societal cost of progressive aortic valve disease.10Traditionally, surgical aortic valve replacement is reserved for patients with severe symptoms.Therefore, in this study, we firstly aimed to identify the sex-specific distribution of progressive aortic stenosis among older individuals being routinely investigated for heart disease. We then determined the association of progressive aortic stenosis with quality-adjusted life years (QALYs) lost due to premature mortality, compared with individuals presenting with normal aortic valve function.11 using the Reporting of Studies Conducted Using Observational Routinely Collected Health Data guidelines.12 NEDA has obtained ethical approvals across Australia from all relevant institutional, university and government Human Research Ethics Committees (HREC). A patient consent waiver has been authorised by each HREC for retrospective data, and prospective consent is obtained by verbal script at each centre using an opt-out process. The NEDA cohort comprises individuals aged 65 years or older with native aortic valves who received echocardiograms investigating aortic stenosis severity  across 23 centres in Australia from Jan 1, 2003, to Dec 31, 2017 (11 NEDA independently extracts (thereby minimising bias) the age, sex, anthropometric profile, and a standard list of left and right heart parameters of patients being routinely assessed by echocardiography. All identified patients were individually linked from the time of their last echocardiogram to the well validated National Death Index13 to determine all-cause mortality up to and including Dec 31, 2018.We conducted an observational clinical cohort study based on the previously described National Echo Database of Australia (NEDA),31, 2017  p 2. We 15 The proportion of men and women categorised within each group was then calculated.The primary outcomes of this study were years of life lost (YLL) and the associated societal costs attributable to premature mortality during a fixed 5-year follow-up from last echocardiogram according to an individual’s baseline aortic valve function . For all outcome analyses, Vmax (which increases linearly with aortic stenosis severity) recorded on the last echocardiogram was aggregated into six prospectively selected groups. The group with the lowest Vmax values Vmax (indicating no aortic stenosis) was defined as the reference group for all subsequent analyses. The prospectively selected groups were: Vmax 1·00–1·99 m/s (no aortic stenosis or reference), 2·00–2·49 m/s (mild aortic stenosis), 2·50–2·99 m/s (mild-to-moderate aortic stenosis), 3·00–3·49 m/s (moderate aortic stenosis), 3·50–3·99 m/s (moderate-to-severe aortic stenosis), and 4·00 m/s or higher (severe aortic stenosis).16 For each Vmax group, the number of YLL during complete 5-year follow-up was calculated. To examine the societal burden of premature mortality according to progressive aortic stenosis (relative to the no aortic stenosis group) we calculated: (1) the total QALYs lost in each group during 5-year follow-up, and (2) willingness-to-pay to regain each QALY if there were the capacity to restore equivalent levels of premature mortality and associated YLL to those observed in the no aortic stenosis group. For these estimates we used data from Lee and colleagues,17 who estimated that the quality of life (using the EQ-5D-3L questionnaire) in people with severe aortic stenosis who were receiving transcatheter aortic valve implantation to be 0·71 at baseline (mean age of 86 years). With no equivalent estimates for those living with mild-to-moderate forms of the condition , for our main analyses we applied the same quality of life levels to the remaining, non-severe aortic stenosis groups.Age at death was then used to identify premature mortality based on sex-specific life-expectancy thresholds in Australia in 2020 (80·7 years for men and 84·9 years for women).18 estimated that one QALY was worth AU$42 000 over a 2-year rolling window using Australian population-based data. Cost analysis was done from a societal perspective and results are reported in 2020 values after adjusting for inflation using the consumer price index from the Australian Bureau of Statistics and 2018 price level as the base. An inflation-adjusted willingness-to-pay of $43 038 was used for our baseline cost analysis. Currently, NEDA does not capture detailed clinical data  and therefore the reported pattern of mortality within each discrete study group has not been adjusted for these potential confounders.Given the substantive size of the patient cohort being studied and our intention not to formally compare groups and outcomes, no formal power calculations of the sample size needed were conducted. The sex-specific distribution of progressive aortic stenosis according to Vmax and the associated pattern of actual 5-year mortality (with no imputation of data) was firstly described for the entire NEDA cohort. For each group, the number of all-cause deaths, premature deaths and associated YLL and QALYs were calculated based on actual person-years accumulated during a fixed 5-year follow-up. QALY was calculated as a product of YLL and quality of life value. For comparative purposes, these data were then converted into the distribution of Vmax groups and proportional outcomes per 1000 men and 1000 women undergoing echocardiographic investigation. All subsequent comparisons are described  as the excess number of events and costs  relative to the reference group. Huang and colleagues19 Second, we applied a quality-of-life utility score of 0·55 derived from a US cohort based on Kansas City Cardiomyopathy Questionnaire scores for patients with intermediate-risk aortic stenosis receiving the next-generation SAPIEN 3 transcatheter heart valve intervention.20 Together, these values provide upper and lower bound estimates for our baseline QALY estimates. Thirdly, we applied a higher cost per QALY ($68 675) based on a 5-year rolling window using Australian population-based data to assess long-term willingness-to-pay.18We performed three sensitivity analyses. First, we relaxed the assumption that patients with mild-to-moderate aortic stenosis have the same quality of life (utility score of 0·71) as patients with severe aortic stenosis, replacing this value with a recent estimate for chronically ill patients in Australia (utility score of 0·79).Confidence intervals were calculated using the standard formula:Data were prepared and analysed using SPSS version 26.0 and STATA version 13.0.The funders of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Of the 217 599 participants aged 65 years or older with a native aortic valve  p 2, we Actual 5-year mortality increased with progressively higher Vmax levels in both men  per death in men and 7·6 YLL (5·2) per death in women, the societal cost of premature mortality (25 833 deaths) over the 5-year follow-up within the no aortic stenosis (reference) group was $2·70 billion in men and $2·89 billion in women. The total equivalent cost of premature mortality and associated YLL among individuals with any form of aortic stenosis, despite their advanced age, was also substantive ($629 million from 20 593 premature deaths in men and $735 million from 24 058 premature deaths in women). Due to a combination of more premature deaths overall and greater YLL per death, societal costs were higher among men and women with mild-to-moderate forms of aortic stenosis than among those with more severe forms of aortic stenosis . Among mSensitivity analyses  p 4 reve15 would qualify for an aortic valve replacement if also symptomatic .21 However, we also found that among those individuals presenting with mild-to-moderate aortic stenosis (affecting 18·7% of men and 16·8% of women), levels of premature mortality and associated QALYs lost within 5 years were substantially high. Despite a similar distribution of aortic stenosis, overall, women had a greater number of YLL, QALYs lost, and societal costs than men due to their higher expected longevity. Similarly, the relative high impact of mild-to-moderate forms of aortic stenosis on both premature mortality and QALYs reflected the dynamics of three key factors: (1) the larger numbers of women and men with mild-to-moderate forms (compared with those with severe aortic stenosis), (2) their younger age profile, meaning more potential for premature mortality  and, (3) increasingly higher 5-year mortality in individuals with any form of aortic stenosis (compared with those with no aortic stenosis). Within the mild-to-moderate aortic stenosis cohort, who typically remain untreated based on current guidelines,15 the societal cost of premature mortality was estimated to be $1·40 million per 1000 men and $2·48 million per 1000 women, over and above that observed in the no aortic stenosis group. Even when considering the potentially lower estimates derived from sensitivity analyses, these findings suggest that patients with non-severe forms of aortic stenosis still have a high societal burden, either by causation or correlation.To our knowledge, this represents the first study to examine the sex-specific pattern of premature mortality and subsequent YLL associated with progressive aortic stenosis in a large, real-world cohort of individuals being routinely investigated for heart disease. Due to a combination of factors, including the age profile of those presenting with less severe forms of aortic stenosis, we identified that the greatest societal burden attributable to premature mortality is not confined to those presenting with severe aortic stenosis. As expected, patients with severe aortic stenosis (comprising 1·9% of women and 2·3% of men) had typically high 5-year mortality and according to current guidelines15 For example, despite their proven efficacy for other forms of heart disease, statins do not slow progression of aortic stenosis;22 which can be rapid in some individuals.24 The same applies to other cardiac prevention strategies. As a direct therapeutic measure, both surgical6 and transcatheter7 forms of aortic valve replacement are proven strategies to prolong survival in severe cases of aortic stenosis, particularly when normalised Vmax levels are reached.25 However, as highlighted by a 2022 analysis of the caseload of severe cases in the UK (an estimated 1·45% of the population aged 55 years or older),5 there is limited scope for the National Health Service to scale-up surgical services.26Unfortunately, there is a scarcity of evidence to support the prevention of this common condition.27 The inexorable ageing of many high-income populations will inevitably impart an increasingly challenging and unsustainable societal burden and health-care expenditure if there is no development and application of pragmatic preventive strategies for key contributors of future costs such as aortic stenosis.Even in the most well resourced health systems, aortic valve replacement rates remain lower than expected.28 The positive results from clinical trials are clinically relevant and important given that in a parallel cohort of US patients matched with the same Vmax levels of those in the NEDA cohort, we found the proportion of individuals concurrently presenting and being treated for heart failure progressively rose from 38·6% in men and 36·8% in women, among individuals with no aortic stenosis, to 59·5% in men and 65·7% in women, among those presenting with severe aortic stenosis.29 The SGLT2 inhibitors appear to convey benefits across the entire spectrum of heart failure. As recently shown within the NEDA cohort, any decline in ejection fraction is of prognostic importance in many individuals.30 Given the high cost of health-care expenditure for patients with heart failure and the proven cost-effectiveness of readily available medical therapy,28 there is a compelling argument for alerting an individual’s health-care team of the need for more proactive surveillance and management (including evidence-based heart failure treatments) once mild-to-moderate aortic stenosis has been detected.Even if aortic stenosis cannot be effectively managed directly without resorting to resource-intensive and costly surgical options, the evidence-based treatment of concurrent heart failure has been transformed in recent years with positive results from clinical trials of angiotensin receptor-neprilysin inhibitors and SGLT2 inhibitors to supplement the proven benefits of other neurohormonal antagonists.31 The calculated proportion of premature deaths and associated YLL would be lower for countries such as the UK where life expectancy is reportedly 2–4 years lower.5 This same heterogeneity would apply to each society’s willingness-to-pay for each QALY lost, but our findings reinforce a greater focus on quantifying the quality of life of those affected by mild-to-moderate forms of aortic stenosis. Given the close association between progressively worse aortic valve disease and advancing age , there is a potential argument that this condition is an inevitable consequence of ageing populations that now survive previously fatal events at a younger age.1 Although this argument might apply to a good proportion of individuals who exceeded average life expectancy in Australia (around two-thirds with severe aortic stenosis within our cohort), there was still a large component of individuals  who did not. By any measure, the current societal cost of progressive aortic stenosis is substantive, and this will undoubtedly translate into substantive health-care costs. Compared with other OECD countries, Australia typically spends more on health care and provides core services to the majority of the population.31 However, Australia currently lags with regards to the provision of primary care services and preventive health services, and these might be the most important investments to tackle an evolving burden of heart disease.31The dynamics of sex-specific, premature mortality vary from country to country as do the costs of managing common forms of chronic heart disease. According to the Organisation for Economic Co-operation and Development (OECD), Australia is one of the top five countries for longevity for both men and women.11 Although we report on actual mortality and standardised premature mortality or YLL in a very large clinical cohort, by necessity, we did have to apply several assumptions. Firstly, we cannot exclude the possibility that some individuals did undergo surgical replacement of their aortic valve following their last echocardiogram. Secondly, we assumed that mortality rates across the Vmax groups remained the same in participants of the NEDA cohort with later echocardiograms . Although previous analyses have shown improving survival rates across historical epochs within the NEDA cohort,30 there is no evidence to suggest that these improved survival rates favoured those with aortic stenosis. Despite the greater number of individuals with non-severe aortic stenosis, compared with those with severe forms, there remains a paucity of data on the quality of life of individuals with non-severe forms. We therefore assumed indices of QALY and subsequent willingness-to-pay to regain QALY lost were largely static across the aortic stenosis groups. Our sensitivity analyses revealed consistent and substantial costs of untreated mild-to-moderate aortic stenosis. Our cost estimates are very useful as baseline costs for evaluating the cost-effectiveness of health-care interventions that seek to improve the longterm survivorship of patients with mild-to-moderate aortic stenosis. Because NEDA has yet to capture detailed clinical data, we report on the association between varying levels of aortic stenosis and mortality, rather than establishing causality. We also did not undertake any adjusted analyses to determine if progressive aortic stenosis independently contributes to the observed pattern of increasing mortality. However, we recently completed two collaborative studies with equivalent Northern American29 and European32 cohorts with detailed clinical phenotyping. In both analyses, direct comparisons between these cohorts and the NEDA cohort showed similar patterns of increasing mortality with the same unit increases in Vmax levels.32 Moreover, in both these studies, increasing Vmax levels remained a strong and independent prognostic marker for increasing mortality after extensive adjustment for potential clinical confounders.32 NEDA also does not capture data on the race and ethnicity of individuals, which have been shown to be important predictors of premature mortality in other health systems .33 Beyond replicating our broad findings in other large clinical cohorts, future research is needed to better delineate the clinical trajectory and cause of death of individuals affected by mild-to-moderate forms of the disease.24We purposefully focused on individuals aged 65 years or older within the NEDA cohort who had a Vmax level measured across their native aortic valve given that this is the age bracket in which aortic stenosis is most common. We also excluded individuals with severely compromised cardiac function who typically have high mortality rates owing to their advanced heart disease.5 and countries in continental Europe and North America.4 The individual and societal imperative to more readily detect and clinically manage less severe forms of this common clinical condition to reduce the societal cost of future premature mortality, as well as a probable high burden of related health expenditure, is likely to apply to any country with substantial population ageing.Although we report on the pattern of premature mortality and YLL associated with progressively worse aortic stenosis within an Australian clinical cohort, our findings are broadly generalisable to other countries with a large burden of disease imposed by aortic stenosis, including the UK1


Local cancer therapy by combining real-time surgical exploration and resection with delivery of a single dose of high-energy electron irradiation entails a very precise and effective local therapeutic approach. Integrating the benefits from minimally invasive surgical techniques with the very precise delivery of intraoperative electron irradiation results in an efficient combined modality therapy.Patients with locally advanced disease, who are candidates for laparoscopic and/or thoracoscopic surgery, received an integrated multimodal management. Preoperative treatment included induction chemotherapy and/or chemoradiation, followed by laparoscopic surgery and intraoperative electron radiation therapy.In a period of 5 consecutive years, 125 rectal cancer patients were treated, of which 35% underwent a laparoscopic approach. We found no differences in cancer outcomes and tolerance between the open and laparoscopic groups. Two esophageal cancer patients were treated with IOeRT during thoracoscopic resection, with the resection specimens showing intense downstaging effects. Two oligo-recurrent prostatic cancer patients  had a robotic-assisted surgical resection and post-lymphadenectomy electron boost on the vascular and lateral pelvic wall.Minimally invasive and robotic-assisted surgery is feasible to combine with intraoperative electron radiation therapy and offers a new model explored with electron-FLASH beams. Laparoscopic resection is a surgical standard: IOeRT is feasible to be integrated to boost post-resection pelvic areas at high risk.In experienced groups, oncologic surgery  is performed using laparoscopy . In patiThe positive experience with laparoscopic surgery to early-stage rectal cancer led us to incorporate this approach in the multimodal management of locally advanced rectal cancer (LARC) patients. Preoperative treatment including induction chemotherapy and chemoradiation was followed by laparoscopic surgery and IOeRT delivered to the posterior pelvic wall (post-resection and pre-reconstruction).Methodology and patient selection: single-institution IOeRT expert experience.2) combined with leucovorin (20 mg/m2) on days 1–5 every 21 days was recommended following the institutional protocol. Radical surgery was scheduled for 6 weeks after completion of CRT. Compliance with the principles of TME was mandatory. After surgery and before pelvic reconstruction, 10–15 Gy (median 12.5 Gy) was delivered in a single fraction  to the posterior presacral space, using a median energy of 12 MeV .Table Patients in the laparoscopy group lost less blood and had a shorter hospital stay than those in the open surgery group. Postoperative complications (at least 1 postoperative complication) were reported in 14 of 44 patients (32%) in the laparoscopy group and in 36 of 81 patients (44%) in the open surgery group. The proportion of patients who underwent a second intervention within 28 days after surgery was similar between the two groups. Intraoperative, acute, and late toxicity was similar among groups.p = 0.25). The median distance to the circumferential resection margin was 7 cm in the laparoscopic group and 8 mm in the open surgery group (p = 0.42). The median number of lymph nodes harvested after surgery was not significantly different between the groups. Tumor and nodal downstaging after preoperative treatment did not differ significantly between the two groups. Median follow-up time for the entire cohort of patients was 59.5 months (range 7.8–90). Seven patients had a loco-regional recurrence (5.6%), and 22 out of the original 125 patients (18%) developed distant metastases. Two out of the seven patients with loco-regional recurrence were rescued with a second surgical procedure and achieved long-term survival (24 and 55 months).The distribution of the different procedures was similar in both groups. An abdominoperineal procedure was performed in 34% of the patients in the laparoscopy group and in 26% in the open surgery group. Laparoscopic procedures were converted to open surgery in 5 of 44 patients. Macroscopically incomplete resected specimens were obtained in 1 of 44 patients (2%) after laparoscopic surgery and 5 of 81 (6%) after open surgery. The median distal resection margin was 2.84 cm in the laparoscopic group and 3.17 cm in the open surgery group . However, laparoscopy prolonged surgical times . In the laparoscopy group, bowel function was regained sooner  and hospital stay was shorter . Macroscopically, the completeness of resection was not different between the groups . A positive CRM (< 2 mm) was noted in 10% of patients in both the laparoscopy and the open surgery groups (p = 0.850). Postoperative morbidity  and mortality  within 28 days after surgery were similar.The results of several prospective randomized trials on laparoscopic resection for mid- to low rectal cancer have been reported , 2, 8–10A recent meta-analysis  concluded that laparoscopic rectal cancer resection does not compromise long-term oncologic outcomes compared with open surgery with potential survival benefits  for a minimal access approach in patients with stage II and III rectal cancer .An important remark is that most of the laparoscopic data were generated excluding patients with T3 rectal cancer within 2 mm of the endopelvic fascia or T4 cancers. Therefore, their findings require validation in LARC patients after contemporary multidisciplinary treatment including exposure to intensive preoperative systemic and radiation treatments. The demonstration of the equivalence and possibly benefits in some of the parameters analyzing clinical tolerance and the induction of similar downstaging effects, without increased toxicity in tissues exposed to extensive combined modality therapy and different surgical approaches, will form solid evidence to promote the use of minimally invasive surgery and IOeRT also in patients with locally advanced disease at diagnosis.Most of the challenges in the treatment of rectal cancer concern the lowest located and the locally advanced tumors. Several reports have described the availability and effectiveness of IOeRT combined with preoperative chemoradiation for controlling LARC  and promLocally advanced rectal cancer has broadly been defined as T3, T4, or lymph node-positive disease. Preoperative chemoradiation is the standard of care, based on acceptable toxicity and reduced local recurrence rates, as well as higher rates of sphincter preservation compared with postoperative chemoradiation. Both short-course radiation and long-course chemoradiation followed by TME and adjuvant chemotherapy are currently accepted methods, with recent trials showing equivalence in outcomes, with longer follow-up ongoing. MRI is increasingly being used to determine pCR following preoperative therapy to either predict optimal surgical candidates or as a mechanism for monitoring patients without immediate surgical intervention. Currently, TME remains the cornerstone of treatment for locally advanced rectal cancer, nonoperative management is an emerging alternative treatment paradigm for achieving comparable oncologic control with encouraging early results .Outcomes including intraoperative electron irradiation in the multimodal approach have been reported in the treatment of locally advanced gastric cancer –22, gastP = 0.50). The results supported findings observed in a previous meta-analysis showing a statistically significant loco-regional control benefit with the addition of IOeRT in patients with resectable gastric cancer and the indication that IOeRT may provide promising results on overall survival for the subgroup of patients with stage III disease [A meta-analysis including 11 studies, 9 for gastric cancer and 2 for esophageal cancer reported  on 1581  disease .n = 26; 44%) or esophago-gastric carcinoma , with disease confined to loco-regional area, were evaluated. Thirty-seven patients also received a perianastomotic reconstruction IOeRT boost over the tumor bed in the mediastinum and upper abdominal lymph node area. Loco-regional recurrence rate was 15% (n = 8). Five-year overall survival and disease-free survival was 48 and 36%, respectively. Univariate and multivariate log-rank analyses showed that receiving IOeRT was associated with lower risk of local recurrence  .Since the first laparoscopic procedure, there has been a steady increase in advanced minimally invasive surgery. These procedures include oncological colorectal, hepatobiliary, and upper gastrointestinal surgery. Implementation of these procedures requires different and new skills for the surgeons who wish to perform these procedures. To accomplish this surgical teaching program, a mentorship seems the most ideal method to teach the apprentice surgeon these specific skills. A pioneering teaching program for a minimally invasive esophagectomy for esophageal cancer was started in 2009 . As partThe theoretical advantages of robotic surgery are the enhanced three-dimensional and magnified views of closed spaces that can be obtained as well as increased dexterity with fine precision surgical instruments, which make it ideal for operating in confined anatomical spaces such as the pelvis. Considering the great complexity of pelvic anatomy, robotic surgery has been reported to be feasible and related to beneficial outcomes in urological malignancy including prostatectomy  and nodaFLASH irradiation with electron beams has proven to be protective in normal tissue damage in animal models, while maintaining similar tumor control effect . ElectroIOeRT can be delivered as a boost component after preoperative chemoradiation in gastroesophageal cancer as a dose-escalation strategy to improve local tumor control in the nodal regions of the upper abdomen  and in the lower mediastinal space covering the circumferential margin tumor extension (thoracoscopic approach). The normal tissues exposed to electron boost are relatively dose resistant  promises to further improve the tolerance of normal tissues. In this scenario, escalating the intraoperative dose delivered during minimally invasive surgery might significantly improve local tumor control without jeopardizing normal tissue tolerance.


Habitability of exoplanet’s deepest oceans could be limited by the presence of high-pressure ices at their base. New work demonstrates that efficient chemical transport within deep planetary ice mantles is possible through significant salt incorporation within the high-pressure ice. The largest amount of liquid water in our universe by volume might be found in the gigantic oceans of water-rich exoplanets and icy moons. These vast ocean worlds might host some of the most promising environments for extra-terrestrial life to emerge and prosper. Nonetheless, the incredible pressures found within the water-rich layers will freeze the oceans from the bottom into dense high-pressure (HP) ices, separating the liquid layer from the nutrient-rich rocky interior  when compared to these worlds that may contain up to several 10 s wt% of H2O. This would result in up to thousands of kilometers thick water-rich envelopes, called “hydrospheres”, covering a rocky-metal core  and mini-Neptunes (1.8–4·REarth radius). Mini-Neptune’s water-rich fluid envelopes are expected to be at several thousands of Kelvin, making them less relevant for astrobiology. Ocean exoplanets, such as the Trappist 1 e, f, g and h planets9, are prime targets for habitability within their temperate oceans, possibly in equilibrium with their atmosphere, allowing for potential characterization and biosignature detection by upcoming ground and space based observatories10. The icy worlds of the outer solar systems , scaled-down versions of ocean exoplanets, also host large liquid water oceans below their frozen surfaces. Interest in the geophysical and astrobiological attributes of these icy moons has motivated the most ambitious planetary exploration missions of the upcoming decade with NASA’s Europa Clipper11 and Dragonfly12, and ESA’s JUICE13. Furthermore, the search for biosignatures and habitable environments on exoplanets is one of the four main science goals of the newly launched James Webb Space Telescope6.The growing catalog of exoplanets has revealed the widespread existence of a type of planet unknown in our solar system: ocean worlds. These planets, first suggested by ref. y dry <0. wt% H2O 6 , the pressure within the hydrosphere of such planets can form dense HP water ices , stable below the liquid ocean6 Fig. . The pre16. This remains to be studied for thicker exoplanet hydrospheres, but efficient liquid formation at the base of the HP mantle seems to be the rule rather than the exception17. Secondly, chemical physics advocates for efficient chemical transport through HP ices mantle.This assumption is actively challenged by experimental and theoretical studies of icy mantle dynamics and HP ices chemical physics. Both suggest efficient transport of solutes from the rocky core to the upper ocean may be possible. First, dynamically, it has been demonstrated that efficient convection can happen within HP ice mantles in large icy moons like Titan and Ganymede, involving transport of liquid pockets from the hydrosphere-rock boundary to the ocean20. These new type of crystalline solids in salt-water systems are also called “solid solutions” as the amount of salt is not stoichiometrically fixed, like with common types of salt hydrates such as hydrohalite (NaCl•2H2O).To understand the importance of this new study by Hernandez et al., one needs to re-examine a pillar of undergraduate physical chemistry teachings: “ice grows chemically pure from a saline solution by expelling all the salt ions into the liquid”. This is generally true for low pressure common hexagonal ice (ice Ih). However, one remarkable discovery of ice physics of the last decade is that HP water ice with cubic crystalline structure above 20,000 atmospheres (phase VII and X) can incorporate significant amounts of simple ionic salts  up to several weight %, while maintaining the same a crystalline structure21. Furthermore the presence of ions in the lattice extend to higher pressure the stability of ice VII (molecular ice with H2O molecule linked by H-bonding) at the expense of ice X (higher-pressure ionic ice with hydrogens in an symmetric position between oxygens)23. Controversy remained regarding the maximum amount of salt soluble24, as well as the mechanism of salt ions inclusion being either interstitial (between H2O molecules) or substitutional (replacing H2O molecules). Furthermore, there is still uncertainty about solute incorporation effects on the lattice volume and density of salty ice. Experimental work by ref. 18 reported a reduction of the lattice volume, however, other recent experimental and theoretical work reported an increase22. How much salt can be incorporated and how it could either reduce or increase the volume of HP ice (and affect its density), has crucial implications for our ability to describe the interior structure and dynamics of ocean exoplanets. Too high a density increase due to high salt solubility, or possible immiscibility within the HP mantle would inhibit convection and solute vertical transport20. Finally, limited data exists above 300 K on incorporation into HP ices, conditions found in exoplanets. To determine if solute transport is possible through planetary HP ice mantle, one needs to know, over the relevant range of pressure and temperatures, the solute solubility and its effect on density. Until now, we were lacking such a unified physical model and thermodynamic representation.Several groups have reported intriguing properties of these salt-bearing ices. Salt incorporation distorts the HP ice crystalline lattice by breaking some of the hydrogen bound network8 provide a comprehensive study of NaCl incorporation in HP ice using state-of-the-art molecular dynamics simulations (first principle DFT-MD) over a wide range of conditions. By simulating crystallization of salty solutions, this extensive study binds together within a self-consistent physical model, several observations from previous studies more limited in scope and at lower temperatures. They report up to 2.5 wt% NaCl incorporation on substitutional sites that results in an increased lattice volume of salty HP ice. The study also provides a thermodynamic representation (equation of state) for salty HP ices that allows the authors to show that convective chemical transport through the dense salty HP ices is possible. Hernandez et al.8 demonstrate that the density increase due to NaCl incorporation does not hinder vertical transport through solid-state convection. Along with previous studies on convection of HP ice and melt vertical transport in mantles in smaller icy moons16, this substantiates the notion that HP ice mantles do not act as chemical barriers between the rocky core and the liquid water ocean. The authors also suggest solute recycling mechanism with diffuse return flow of salty ice across the icy mantle-ocean boundary due to crystallization of salty ice , or phosphates (PO43−). Secondly, it has been shown that salt solubility can decrease by an order of magnitude in lower HP ice, like ice VI20 expected in thick hydrospheres6, or at high temperatures24. Due to these transitions, solute-rich ice could release its solutes within the HP ice mantle, forming a deep reservoir  will directly test some of these conclusions by looking at the geophysical and surface signatures of the ocean composition and estimate the permeability of HP ice mantles (Fig. The study by Hernandez et al.les Fig. . These m


GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase. While dietary restriction of galactose is lifesaving in the neonatal period, the development of complications including primary ovarian insufficiency is not mitigated. Additionally, the pattern(s) of follicle loss have not been completely characterized. The chronic accumulation of aberrant metabolites such as galactose-1-phosphate and galactitol are suspected culprits in the development of the sequelae, yet the mechanisms remain elusive.Primary ovarian insufficiency is characterized by accelerated loss of primordial follicles, which results in ovarian failure and concomitant menopause before age 40. About 1–3% of females in the general population are diagnosed with POI; however, greater than 80% of females with the inherited disease Classic Galactosemia will develop POI. Classic Galactosemia is caused by mutations in the GalT gene-trapped mouse model to study the pathophysiology of primary ovarian insufficiency in Classic Galactosemia. We recently showed that differences in the Integrated Stress Response pathway occur in mutant ovaries that likely contribute to their primary ovarian insufficiency phenotype. Using immunofluorescent staining of histological sections of ovaries at progressive ages, we saw evidence of altered Integrated Stress Response activity in granulosa cells and primordial oocytes consistent with accelerated primordial follicle growth activation, aberrant DNA damage and/or repair, and increased cellular stress/death. Overall, our findings indicate that abnormal Integrated Stress Response in the Classic Galactosemia model ovary results in accelerated primordial follicle growth activation, sometimes referred to as “burnout.” These aberrant early events help further clarify when/how the primary ovarian insufficiency phenotype arises under galactosemic conditions.Our group uses a  GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase in the galactose metabolic pathway [Classic Galactosemia (CG) is an inherited disease caused by mutations in the  pathway , 3. Give pathway –6. Data  pathway –12.As mentioned, POI affects most females with CG . POI is Human females are born with dormant primordial follicles consisting of immature eggs (oocytes) surrounded by somatic cells (granulosa cells) that number in the hundreds of thousands . The remAs previously mentioned, the pattern(s) of follicle loss due to severe POI associated with CG have not been completely characterized. In human females with CG, there is evidence of normal appearing ovaries/follicles at birth and in early childhood. However, by adolescence, evidence of rapid primordial follicle depletion and lower numbers of developing follicles support postnatal insult , 23. NonGalT gene-trapped  mouse model to study the pathophysiology of GALT deficiency in a physiologically relevant mammal [GalTKO whole ovary specimens [Our group uses a t mammal , 25–29. t mammal . The ISRt mammal  Fig. 1)GalT genet mammal , 33. In t mammal . The phot mammal , 36 and t mammal  are thoupecimens  suggestipecimens .Fig. 1LeThe goal of this study was to determine the earliest timing of onset of altered ISR signaling in oocytes and granulosa cells of immature follicles in the context of galactosemia, and to expand our knowledge of the specific ovarian cell types involved. Here we report our findings of the early onset of molecular changes in primordial oocytes and granulosa cells of various stages of follicle development in juvenile mice.GalTKO mice used in this study were established as previously described; all animals were maintained on normal chow, and the genetic background of all mice was confirmed by genotyping using previously published protocols [All animal studies were conducted in full compliance by the guidelines outlined for the care and use of laboratory animals and were approved by the University of Utah Institutional Animal Care and Use Committee (IACUC). rotocols . Table 1GalTKO mice as described previously [Ovaries were extracted from 5D, 14D, and 30D wildtype (WT) and eviously  and placGalTKO samples for each antibody.Briefly, after deparaffinization and rehydration, antigen retrieval was performed by incubating sections in either 10 mM citrate buffer, pH 6.0 or 1X EDTA buffer, pH 8.0 at 120 °C for 20 minutes. Blocking was performed with 1% (wt/vol) bovine serum albumin plus 10% normal goat serum at room temperature for 60 min. After blocking, sections were incubated overnight with the selected primary antibodies at 4 °C. After washes with Tris-buffered saline containing 0.05% Tween 20, sections were incubated for 60 minutes at room temperature with Alexa Fluor 647  secondary antibodies. Nuclei were counterstained with 300 μM DAPI  for 4 min. Slides were mounted with EMS mounting medium , the covers applied and held in place with clear nail polish. Secondary antibody only sections were subjected to the same procedures except blocking buffer only was added in place of the primary antibody for the overnight incubation. Imaging of slides was performed with a Nikon Widefield microscope with 20X lenses. All ovary sections were imaged using the same camera settings across WT and Background autofluorescence from secondary antibody-only controls were subtracted from primary antibody-stained samples using NIS-Elements Analysis software . Follicle and cell types were sorted in the DAPI channel by their morphology, with the oocytes and granulosa cells analyzed separately for each follicle type. Oocytes were differentiated from granulosa cells by separate regions of interest in follicles where visible nuclei were present. Staining intensities were quantified in oocytes and granulosa cells of different classes of follicles by measuring average signal intensity in the region of interest; for ɣ-H2AX and CCASP3 in secondary follicle granulosa cells, individual stained cells were counted, and the ratio of positive stained cells compared to the total area of the ovary sample measured, instead of intensities averaged. For average intensities calculated, the signal was quantified and divided by the area for each region of interest.One ovary from separate animals at either 14D or 30D was harvested and fixed in Dietrich’s fixative for 12 hours, rehydrated and embedded as described above. Pole-to-pole serial sections of 5 μm were cut and placed on slides by AML Laboratories  or the Research Histology Core Facility of the ARUP Laboratories . Sections were then stained with Weigert’s Iron Hematoxylin  and Picric Acid -Methyl blue  with cover slides mounted using Permount . Each sample was assigned a unique code for blinding purposes.Blinded histomorphometric evaluation of ovarian follicles was performed . The metp value of less than 0.05 was considered statistically significant.Statistical significance was determined as indicated, by using t test or Mann-Whitney U test (MW-U), and Kolmogorov-Smirnov (KS) test for non-parametric data and distributions, respectively. The statistical program R was used to analyze and graph signal intensity distributions with KS test. GraphPad Prism version 9.0 for Windows was used for all other statistical tests and graphing. A Granulosa cells of primary and secondary follicles in ovaries isolated at 5D, 14D, and 30D  or the number of positive cells  in secondary follicle granulosa cells.Finally, we extended our study to 30D to account for the mid-juvenile period, but no significant differences were detected between Primordial follicle oocytes were identified and assessed separately from granulosa cells at the noted ages. For primordial oocytes, staining intensity was the measurement for all target markers.GalTKO primordial oocytes from 5D mouse ovaries revealed dramatically lower staining intensities of ISR effector P-eIF2ɑ . In addition, mitotic figures in ten small secondary and larger preantral follicles per ovary, per genotype, were quantified at 14D to assess the proportion of growing follicles, as we found increased evidence of growth and subsequent death in the molecular markers in 14D Fig. A and no 14D Fig. B, GalTKOGalTKO mouse model, there are marked decreases in primordial follicles at 6 months of life, longer times to pregnancy, and fewer litters and number of pups born per litter [GalTKO mouse model [GalTKO mice.POI is a devastating complication in human CG patients, with most females affected and no preventative treatment. The molecular mechanisms contributing to follicle loss and ovarian insufficiency in CG specifically are only now being elucidated. In our r litter , making se model , 30. HerGalTKO ovarian follicles compared to WT controls. This is summarized as follows: Immature follicles within GalTKO ovaries exhibit altered ISR activity  leads to a cascade of cellular signaling that promotes a state of cytoprotection and cell cycle arrest until the damage can be resolved. This arises due to the ISR signaling convergence upon eIF2ɑ phosphorylation, which slows global protein translation, but favors the translation of reparative proteins. In the normal ovary, it appears that baseline activation of the ISR is integral in follicle dormancy and arrest . For exaty Figs. A-C, 5A&Bty Figs. D-G, 5A&Bty Figs. H&I, 6A-Dty Figs. E&F, 5C.GalTKO ovaries. Consistent with elevated levels of BiP in primary follicle granulosa cells at 5D, a GALT-knockout granulosa cell line (GALTKO OV3121) developed by Rushing and colleagues also exhibits increased BiP at baseline in those cells compared to the parent cell line . Decreased BiP at 30D in primordial oocytes and primary follicle granulosa cells may indicate mutant ovaries at this age have a dysfunctional stress and growth response [Specifically, we saw evidence of increased ER stress response marker BiP in primary follicle granulosa cells at 5D and then decreased BiP in primary follicle granulosa cells and primordial oocytes at 30D in response , but furGalTKO ovaries; this is matched by significantly more primary follicles and a significantly lower ratio of primordial to primary follicles at 30D in GalTKO ovaries. Lower staining intensity for P-eIF2ɑ in primary and secondary follicle granulosa cells at 5D and 14D  and elevated DNA damage (below) in GalTKO ovarian follicles remains unclear.The concomitant increase of growth marker Ki67 at 5D and 14D in primary follicle granulosa cells reveals what is likely to be accelerated follicle growth in GalTKO ovarian follicles also show an increase in the DNA damage/repair marker ɣ-H2AX and the apoptosis onset marker CCASP3. This was found in the granulosa cells of secondary follicles at 14D at the same time as the detected decrease in P-eIF2ɑ. Increased staining for these markers, along with decreased mitotic figures as follicles reach the secondary stage, are consistent with observations in humans with CG that secondary and preantral follicles appear fewer and arrested as early as childhood [Cells of hildhood . Secondahildhood .GalTKO ovaries. Decreased staining for P-eIF2ɑ in mutant primordial oocytes suggests GALT-deficiency leads to loss of constitutive ISR activation in the primordial oocyte. We also saw significantly decreased DNA damage and repair marker ɣ-H2AX in mutant primordial oocytes at 5D and 14D days but increased at 30D. This is an unexpected finding, as ɣ-H2AX foci are primarily considered markers of stress/DNA damage and subsequent cell death. However, it could indicate inappropriate DNA damage repair mechanisms, or perhaps resolution of prophase 1 of meiosis [GalTKO primordial oocytes, we will need to perform additional experiments by measuring other downstream targets of the ISR and related DNA repair pathways. At 30D, GalTKO primordial oocytes had increased ɣ-H2AX, as might be seen in oocytes exposed to DNA-damaging stimuli [GalTKO ovaries.In the primordial oocytes, we detected significant differences as early as 5D in  meiosis –46. Mara meiosis . To dete stimuli . This diGalTKO POI phenotype. Based on the data above, we propose the following model: Impaired ISR action due to GALT deficiency results in an accelerated transition of primordial to primary follicles. Increased follicle growth is matched by increased atresia in secondary follicles at young ages in GalTKO ovaries. This may be occurring in a way that depends upon levels of potentially toxic metabolites that accumulate, and this is a key focus of ongoing studies. Accelerated loss of the primordial follicle pool is a situation reminiscent of follicle “burnout” [Galactosemic conditions result in quite dynamic alterations in follicular damage and repair markers in immature ovarian follicles early in life and these plausibly contribute to the burnout”  that hasburnout” , which p


Primary percutaneous coronary intervention (PCI) is considered the most preferred strategy in ST-segment elevation myocardial infarction (STEMI). However, the prognostic role of spontaneous re-canalization in STEMI patients is still not clear. The purpose of this study is to evaluate the impact of pre-procedural TIMI flow grade in the culprit coronary artery on the short and long term prognosis in Egyptian patients presented with STEMI and treated with primary PCI.P value = 0.033). At 1 year follow up; 5 mortalities were recorded in group A with no mortalities at all in group B . There was a trend towards an increase in acute heart failure incidence in group A yet no statistically significant value was achieved . Target lesion revascularization was reported in 8 patients in group A and in only 3 patients in group B .A dual center, prospective observational study that was conducted in the period from January 2019 till June 2020 and enrolled 150 STEMI patients presented within 24 h from onset of chest pain. Initial angiography was done with analysis of TIMI flow grade in the infarct related artery. Of the 150 enrolled patients; 93 patients (62%) were found to have initial TIMI flow grade 0 (group A) and 57 patients (38%) had initial TIMI flow grade I–III (group B). There was a strong association between cardiac mortality and pre-procedural TIMI flow grade. 12 mortalities  were recorded during our study period; in-hospital mortality was reported in 7 patients in group A, yet no mortalities were recorded in-hospital in group B (Despite the evolution in primary PCI strategies and the continuous advancement in anti-thrombotic treatment; pre-interventional infarct related artery TIMI flow grade I–III is associated with better in hospital and 1 year outcome, specifically significantly lower cardiac mortality compared to patients who had TIMI flow grade 0 at initial angiography. Primary percutaneous coronary intervention (PCI) has been shown to improve the outcome of patients with ST-segment elevation myocardial infarction (STEMI) in comparison to thrombolysis, mainly due to restoration of angiographic thrombolysis in myocardial infarction (TIMI) III flow grade in the vast majority of patients with further improvement of adjunctive pharmacological agents and mechanical devices. However, the prognostic role of early spontaneous re-canalization before undergoing angioplasty in these patients has not been thoroughly investigated. Although there is an excellent outcome conferred by primary PCI in patients with STEMI, the prognostic role of spontaneous re-canalization in these patients is still not clear .Previous clinical studies have shown that the initial TIMI flow grade in the culprit coronary artery was an independent predictor of better one year survival in STEMI patients and that the TIMI flow grade 0 before primary PCI is associated with a worse clinical outcome. However, it is unclear whether the same is true with modern guideline-adherent therapy and reperfusion strategies –4.The purpose of this study is to evaluate the impact of pre-procedural TIMI flow grade in the culprit coronary artery on the short and long term prognosis in Egyptian patients presented with STEMI and treated with primary PCI.Our study was a cohort, prospective and dual-center observational study. We started recruiting patients in January 2019 till June 2020. The study was conducted on 150 patients presenting with STEMI from both genders within 6 h from symptoms onset or between 6 and 24 h if they have continuous signs and symptoms of ongoing ischemia. Coronary angiography was done and coronary blood flow was assessed using TIMI flow grade. Patients with past ischemic history or patients who received thrombolytic therapy were excluded from the study.Approval of university ethical committee was taken , and informed consent was obtained from all enrolled patients. All patients were subjected to the following: (a) detailed medical history with special emphasis on chest pain analysis, (b) general examination and local cardiac examination with assessment of Killip class, (c) 12 leads ECG within 10 min from first medical contact, (d) blood samples included , (e) Trans-thoracic echocardiography: a rapid bed-side echo to assess left ventricular ejection fraction, resting wall motion abnormalities, and screen for mechanical complications, (f) pre-loading with 300 mg Aspirin orally and a P2Y12 inhibitor . Coronary angiography was done via right radial or right femoral approaches according to the operator experience and tools availability with commenting on initial TIMI flow grade in the infarct related artery, non-culprit lesions, methods of antegrade flow restoration and glycoprotein IIb/IIIa inhibitor usage if done. Drug eluting stents were implanted in all indicated patients with sizing according to operator judgment. Final reporting on post PCI TIMI flow grade was mentioned.After initial angiography; patients were divided into 2 groups: group (A) with initial TIMI flow grade 0 (107 patients ~ 62% of study population) and Group (B) with initial TIMI flow grade I-III (43 patients ~ 38% of study population). Our primary end point was occurrence of major adverse cardiac events (MACE) in the form of: non-fatal stroke, acute heart failure, target lesion revascularization and cardiac death either in-hospital or during the one year follow up period.Chi-square test and/or Fisher exact test when the expected count was found less than 5%.The comparison between two groups with quantitative data and parametric distribution were done by using Independent t test. While the comparison between two groups with quantitative data and non-parametric distribution was done by using Mann–Whitney test. The comparison between two paired groups with quantitative data and parametric distribution were done by using Paired t test. While the comparison between two paired groups with quantitative data and non-parametric distribution was done by using Willcoxon test. The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the P value was considered significant if P < 0.05.Data were collected, revised, coded and entered to the Statistical Package for Social Science (IBM SPSS) version 23. The quantitative data were presented as mean, standard deviations and ranges when parametric and median, inter-quartile range (IQR) when data found non-parametric. Also, qualitative variables were presented as number and percentages. The comparison between groups with qualitative data was done by using P value = 0.016. Details of the procedural data are shown in Table Of the 150 studied patients, 126 patients 84%) were males and 24 patients (16%) were females with age ranged from 23 to 76 years with mean ± SD of 53.87 ± 9.64. Demographic and clinical data of our study population are shown in Table % were maAs shown in Table P value of 0.033.There was a strong association between cardiovascular mortality and pre-interventional TIMI flow grade. In-hospital mortality was reported in 7 patients in group A, yet no mortalities were recorded in-hospital in group B yielding a statistically significant P value 0.005. There was a trend towards an increase in acute heart failure incidence in group A yet no statistically significant value was achieved. Target lesion revascularization was reported in 8 patients in group A and in only 3 patients in group B .After one year of follow up; mortality was much higher in group A  than in group B which recorded no mortalities at all in the follow up period with statistically significant The univariate and multivariate analysis are shown in Table In the present study; pre-interventional TIMI flow grade was found to be a strong independent factor for survival in STEMI patients treated by primary PCI. The importance of early spontaneous recanalization is justified by the larger extension of myocardial loss observed with poor pre-interventional TIMI flow grade. Evidence of patent infarct related artery both prior to and after primary PCI is supposedly to have better clinical outcomes. Compared with early spontaneous recanalization, delayed recanalization is associated with an older, organized intra-coronary thrombus which will result in more impairment in myocardial perfusion limiting myocardial salvage .The TIMI flow grade was initially established to ensure a uniform method of documenting epicardial perfusion in coronary angiography. We investigated the prognostic role of early re-canalization as assessed by TIMI flow grade in the pre-interventional coronary angiography in these patients.The diagnosis of STEMI is the clinical representation of an acute occlusion of an epicardial coronary artery and thus it’s not surprising that the majority of patients with STEMI have a TIMI flow grade 0 at time of initial angiography. In some patients with STEMI, spontaneous recanalization occurs before angiography and PCI. Stone et al. analyzed the four Primary Angioplasty in Myocardial Infarction (PAMI) trials with 2507 enrolled patients and showed that normal pre-interventional TIMI flow grade is an independent determinant of survival .This finding was confirmed by De Luca et al., in whose study initial TIMI flow grade was significantly correlated with one-year mortality in high-risk TIMI risk score patients .These results were further supported by the databases from the CADILLAC and HORIZONS-AMI trials in which an initial TIMI I-III flow grade was independently associated with a 65% decrease in 1-year all-cause mortality. They also showed that initial TIMI flow grade I–III was associated with a significant improvement of final TIMI flow grade which in terms shows a better perfusion at the end of PCI and was correlated with better outcomes and lower 1 year mortality . Our datTotal occlusion of infarct related artery was noted in almost 70% of patients in most of clinical trials investigating the clinical impact of pre-interventional TIMI flow grade –4. In ouWe found that group A patients are still having poorer early clinical outcomes compared to those with initial TIMI flow grade I-III and this was still valid till the later period in the one year follow up. 12 mortalities  were recorded during our study period; 7 of them (4.7%) were recorded in-hospital and 5 mortalities (3.3%) were recorded over the one year follow up. In-hospital mortalities occurred in those patients who had Killip class II–III at time of presentation with longer chest pain onset compared to the other studied patients.In comparison to mortalities recorded in a study done by Rakowzki et al., our study showed higher percentage of cardiac mortalities either in-hospital or during the follow up period. This may be attributed to longer onset of chest pain as mentioned in our data. However, both studies were similar in that the mortality was higher in patients with TIMI 0 flow grade than that in patients with TIMI I-III flow grade . This waOur data was also consistent with what Stone et al. concluded regarding the pre-interventional TIMI flow grade 0 and both in-hospital and one year follow up mortality rates .On contrary, Bauer et al. proved no significant correlation between pre-interventional TIMI flow grade and adverse cardiac events in the ATLANTIC study which enrolled 1862 STEMI patients. However; their study design enrolled only low risk patients and excluded any patient presented with cardiogenic shock or severe hemodynamic instability although the benefit of early recanalization is more pronounced in higher risk patients [Brener et al. showed paradoxical results in contrary to all clinical trials investigating the pre-interventional TIMI flow grade impact in STEMI patients. They mentioned that patients with TIMI III flow at initial angiography had higher clinical risk features than that with TIMI flow grade 0 with diabetes mellitus, smoking and longer period to primary PCI as independent predictors to pre-interventional TIMI III flow .In our study, we didn’t have any clinical or demographic factor with that could significantly affect pre-interventional TIMI flow grade except for chest pain onset which has some contributing factors especially in Egypt that can lengthen the time to first medical contact and proper management strategy including the primary PCI decision. This delay is concerning and included both patients’ related factors and system related factors. This elaborates the importance of public health awareness of recognizing alarming symptoms of acute myocardial infarction as well as importance of immediate medical care.Based on the importance of time delay to primary PCI and the beneficial effect of early recanalization, the concept of pharmacologically induced recanalization was established. Theoretical advantages of early infarct related artery patency included easier guidewire crossing, better stent sizing selection and lower thrombus burden with lower risk of distal embolization.This strategy has been tested in many clinical trials using thrombolytics or GP IIb/IIIa inhibitors or combination of both, but hasn’t shown clinical benefit over primary PCI mainly because of increased bleeding events .Our data warrants intensified risk factors control with pharmacological and non-pharmacological management together with stricter follow up and surveillance in STEMI patients who had initial TIMI grade 0 as they showed to have a higher incidence of adverse effects and more importantly higher mortality rates.Despite the evolution in primary PCI strategies and the continuous advancement in anti-thrombotic treatment; pre-interventional infarct related artery TIMI flow grade I-III is still associated with better clinical outcomes at 1 year follow up, specifically lower cardiac mortality compared to patients who had TIMI flow grade 0 at initial angiography.The present study has number of limitations. Our study recruited 150 patients; we recommend increasing the number in future studies. We followed up our patients for one year; a longer follow up period maybe needed to insure the clinical outcomes.Patients with past ischemic history were excluded from our study so our results couldn’t be applied to this category of patients.Detection of myocardial infarction size and distribution could have been done using cardiac magnetic resonance imaging.