Metaplastic squamous cell carcinoma of the breast is a very rare form of breast cancer that consists of both glandular and nonglandular components mixed with epithelial and mesenchymal tissues. Worldwide, the incidence of this tumor is between 0.1 and 2%. Because of the rarity of this tumor and heterogeneous behavior of the tumor cells, it is difficult to establish the standard therapeutic approach. We report 2 cases of metaplastic squamous cell carcinoma of the breast in young patients with different responses to treatment strategies. The first case is a premenopausal female with metaplastic squamous cell carcinoma treated with surgery, chemotherapy, and radiotherapy, and the second case is perimenopausal metaplastic squamous cell carcinoma with sarcomatoid subtype and osteoid matrix production which progressed on chemotherapy and was treated with surgery and radiation. Squamous cell carcinomas (SCC) are common in the skin and respiratory and upper GI tracts lined by squamous cells. Metaplastic breast carcinoma is a rare malignancy , 2. The Broadly, it is categorized under 3 categories: first, metaplastic carcinoma of no specific type that includes low-grade adenosquamous carcinoma, squamous cell carcinoma, spindle cell carcinoma, and fibromatous-like metaplastic carcinoma; second, metaplastic carcinoma with mesenchymal differentiation that includes chondroid differentiation, osseous differentiation, and other types; and third, the mixed type. When the squamous cell component (SCC) predominates by more than 90%, they are pure squamous cell carcinoma and tend to be more aggressive and treatment refractory . For conThere are different hypotheses to explain the histogenesis of squamous cell carcinoma of the breast. It may arise de novo from epithelium lining of the breast or present as a small foci in preexisting adenocarcinoma or deep-seated epidermal cyst , 7.In either case, they express heterogeneous somatic mutations , 9. ThesA 39-year-old African American female presented to her primary care physician with a palpable lump in her left breast. Her past medical history includes hypertension, diabetes mellitus, hyperlipidemia, and seizure. She smoked half a pack of cigarette for the past 16 years. The family history was noncontributory. Ultrasound  and mammA 53-year-old female with a history of abdominal extra-adrenal paraganglioma, status postsurgical removal, endometriosis, hypothyroidism, HTN, and thyroid nodule noticed a left breast lump for a month during her breast self-exam. On the physical exam at the oncology office, her breast was symmetrical. There were no skin or nipple changes. A mass of 2 × 2 cm and 5 cm from the nipple at 5:00 PM was appreciated with no evidence of axillary lymphadenopathy. She promptly underwent a sonogram Figures  and 7 anOn her follow-up clinic visit at 4 weeks post lumpectomy, the mass was felt on the surgical site on the physical exam. MRI bilateral breasts showed an enhancing mass of maximal dimension of 1.7 cm and subtle asymmetric left axillary tail lymph node 1.4 × 0.9 cm . StagingPost mastectomy, the gross specimen was reported as a firm, cystic, hemorrhagic tumor with 4.8 × 4.5 and 4 cm in dimension located 2.5 cm from the closest inked margin. Pathology description was invasive breast cancer with osteoid formation (metaplastic) features, grade 3 with no lymph vascular invasion and negative margin, and DCIS 0/15 lymph node was negative for malignancy. Hormonal receptor and HER2 status were negative. The patient was scheduled to start on dose-dense 4 cycles of AC (Adriamycin and cyclophosphamide) followed by paclitaxel and carboplatin. After 2 cycles of AC, she was admitted with a chest pain. CT chest showed a new mass 3.9 cm complex nodule within the left axilla and 1.8 cm circumscribed nodule within the lateral aspect of the left breast. The biopsy of the lymph node was suggestive of high-grade sarcoma Figures  with a pThe patient underwent further resection of the chest wall mass and axillary lymph node. Pathology disclosed a gross specimen of 1.8 cm chest wall lesion and 3.0 × 3 × 2.5 cm axillary mass with four additional nodules up to 0.5-0.7 cm with histological similarity to the first tissue biopsy and Ki of >90. Next generation sequencing was negative for any mutations. Post resection, she received radiation without any signs of recurrence.It is a well-established fact that metaplastic breast cancer is one of the rare entities.The pathogenesis of metaplastic squamous cell carcinoma of the breast has evolved in the recent few years as our understanding of genetic and nongenetic heterogeneity affecting phenotypical behavior of this type of cancer has broadened. Some authors suggest that it originates from squamous metaplasia –14 whichIn general, DNA array and immunohistochemistry are performed in addition to histopathology to subdivide breast cancer into 3 broad categories: basal, luminal, and HER overexpression. Though metaplastic squamous cell carcinoma is mostly hormone negative , specifiMetaplastic breast cancer harbors different complex mutations. Recently, aberrations in the PIK3/AKT/mTOR pathway are found more frequently in mesenchymal subtype of metaplastic breast cancers. Clinical trials targeting this pathway in combination with chemotherapy have demonstrated very good response –22.Up to 30% of the tumor is found to have lymph node positive even though lymphatic spread is rare. The SCC of the breast present as a larger primary tumor with higher histological grade and lower incidence of axillary node involvement. Radiologically, no typical mammographic appearances are found and they lack microcalcifications in most cases , 24. CysDue to rarity of this tumor, the most appropriate therapeutic regimen for SCC of the breast is not defined . Till daOverexpression of EGFR was found in a metaplastic breast  potentiaAs mentioned earlier, PIK3 inhibitors and mTOR inhibitors in mesenchymal metaplastic breast cancer have shown promising results in early-phase trials.Metaplastic squamous cell breast cancer is a rare malignancy. It appears large and cystic with the absence of microcalcifications on imaging. It usually has a higher histological grade and negative hormonal and HER2 status with lower incidence of axillary node involvement. Though surgery has been a standard treatment for localized disease, we have not been able to achieve the same consensus in regard to chemotherapy. In our reported cases, surgery was the mainstay of treatment while the response to chemotherapy was seen in the 1st case as opposed to the second case which progressed while on chemotherapy. As we wait for clinical trials for MBC, treatment should be individualized in context to histology subtypes for better survival.


Pisum sativum and the release and transport of carbohydrates and amino acids from the source leaves to the pods and seeds. The expression of GFMs for cytokinin biosynthesis (IPT) and destruction (CKX), sucrose transporters (SUT), Sugar Will Eventually be Exported Transporters (SWEET), amino acid permeases (AAP), and cell wall invertases, was determined using RT-qPCR. GFMs were differentially expressed in leaves of different ages. The expression of many gene family members was lower in the expanding sink leaves compared with the senescing leaves, with the exception of two PsAAP GFMs and PsCKX5, which were highly expressed. Expression of specific PsSWEETs, SUTs, and AAPs increased in the mature and/or senescing leaves. Expression of PsIPTs was least in the mature source leaves, but as strong in the senescing leaves as in the young source leaves. PsCKX7 was expressed in source and senescing leaves. We discuss the potential impact of the targeted reduction of specific PsCKX GFMs on source-sink relationships. Gene editing is becoming the plant breeding tool of choice, but prior to targeting a gene for editing, a knowledge of the gene family members (GFMs) controlling yield is required in the specific crop plant. Critical to yield are components from senescing leaves. We targeted genes controlling senescence in  Pisum sativum L. (pea) has been described [For agronomic plants, a critical component that contributes to both yield and nutritional content is the duration of the leaf’s life span and its ultimate senescence . During escribed , little SAGs) [We know from transcriptomic data that the expression of multiple genes changes during the onset and progression of senescence—the so-called senescence associated genes (SAGs) ,6,7. TheSAGs) . The eneSAGs) . Arabidopsis thaliana [AtIPT and of cytokinin response regulators (RRs) and increased expression of an AtCKX. Others have shown decreases in active forms and increases in inactive forms of endogenous cytokinins in senescing leaves of a variety of species [Since the 1960s, cytokinins have been implicated in the enhancement of sink activity in leaves  and in tthaliana  showed d species ,16,17. H species  suggesteSWEET gene family members (GFMs) have been identified in critical roles for plant development and plant–pathogen interactions [SWEETS in source leaves compared with young sink leaves has been shown for cucumber [SWEET10 in sweet potato [SAG29—subsequently renamed as Clade III AtSW15—was shown to increase markedly in senescent Arabidopsis leaves [AtSW15 [OsSW5 [ZmSW13a,b,c were knocked out. PsSWEET gene family members have been shown to be expressed in the germinating seedling [Rhodococcus fascians [Loading of sugars from source and senescing leaves into the phloem requires the co-ordinated activity of the recently identified Sugar Will Eventually be Exported Transporters (SWEETs) and the sucrose transporters (SUTs) ,21. Indiractions . SWEETs ractions ,24,25. Cractions . The actt potato , while Ss leaves . Overexp [AtSW15  and of C5 [OsSW5  resultedseedling  and to bYoung expanding leaves are regarded as sinks for nitrogen, with N transporters effecting uptake of N into the leaves from the xylem and phloem, whereas mature and senescing leaves are sources of N, utilising transporters to export N into the phloem for transport to sinks . N uptakA key breeding target has been identified as the manipulation of source/sink relationships during pod and seed development in legumes ,40,41,42We used two popular breeding cultivars of pea, Bolero, a process pea and Bohatyr, a “field” pea as biological replicates to determine which gene family members are consistently expressed despite different breeding histories and growth strategies. Bolero is a “process” pea cultivar with a vining growth habit, with pods harvested immature for both fresh and frozen peas, whereas Bohatyr is a conventional leafed “field” pea, harvested when mature for its protein content as a dried pea. The data are presented as the combined average of the two cultivars, with the data for the individual cultivars shown in The chlorophyll content was similar in the expanding sink leaves and the source leaves supporting pods 5 and 20 days after flowering, and thereafter decreased as the leaves began senescing .SWEETs in leaves of different ages, there was increased expression of several Clade III SWEETs in the mature and senescing leaves. Expression of PsSUTs 1 and 2 increased as the leaves aged. Expression of the PsCWINVs was generally lowest in the mature and/or senescing leaves.Expression data are shown in PsAAP6b and 7b showed strong expression in the expanding sink leaves. The majority of the PsAAP gene family members showed the least expression in the young source leaves, while increased expression of several gene family members was detected in the mature source leaves. Many of the PsAAP gene family members were expressed strongly in the senescing leaves, the exception being Cluster 4B PsAAP8, which showed little expression in leaves of any age (data not shown).PsIPT gene family members expressed in the young source leaves showed decreased expression in the mature source leaves but increased expression again in the senescent leaves. A similar pattern was shown for PsCKX expression, but with PsCKX5 expressed in the sink leaves, and PsCKX7 in the source and senescing leaves.The three SWEET Clade III gene family member  was identified as showing increased expression in senescing leaves of Arabidopsis and accelerated senescence in over-expressed lines [SWEETs in the senescing leaves. A senescing leaf is offloading its carbohydrate, and the increased expression of the SWEETs was matched by increased expression of PsSUT1 and 2 as the leaves aged. A similar coincidence of expression between SWEETs and SUT transporters occurs in various tissues of Brassica napus L. [For translocation through the plant, sucrose must be moved across the plasma membrane to the apoplast and thence into the phloem. In Arabidopsis, Clade III SWEET11 and 12 have been identified as controlling the first step in loading sucrose into the phloem for long-distance transport . Clade Ied lines . In pea,napus L. .PsCWINV family members showed differential expression, as also shown in B. napus leaves [PsCWINVs may have led to increased hexoses in the expanding leaves, providing a strong osmoticum to support expansion. Strong expression of specific PsAAP gene family members during leaf expansion is indicative of the import of amino acids into the developing sink leaves [PsAAPs were expressed in the mature source and senescing pea leaves. However, the expression of PsAAP8 was not detected (data not shown), even though AtAAP8 has been classified as “the long sought after phloem loader” [AAP8 belongs to Cluster 4B, the cluster that PsAAP1 and 6 are allocated to. However, we showed that it is members of Cluster 3A which were most highly expressed in the mature source leaves of pea. Not surprisingly, the PsAAPs were strongly expressed in the senescing leaves, as these AAPs aid in the export of the degraded protein as the leaf senesces [B. napus AAP gene family members [s leaves . Expressk leaves . Several loader”  in Arabi members ,46.PsIPT gene family members in the sink leaves was not unexpected, and has been shown in both brassicas [PsIPT expression in the mature source leaves during pod filling is not surprising either, as the release of nutrients is required, rather than a cytokinin-enhanced sink activity [PsIPT in senescing leaves would not have been expected, although an increase has been reported before in both rapid cycling Brassica rapa [B. napus [LOG7a, was detected in senescing aspen leaves [PsCKX5 occurred in the sink leaves, and increased expression of PsCKX7 occurred in the maturing and senescing leaves. Interestingly, CKX7 was also specifically upregulated in senescing aspen leaves [BnCKX gene family members [Low expression of rassicas ,47 and crassicas . Xylem-srassicas , indicatrassicas . The decactivity . Accordiactivity , an incrica rapa , and forB. napus , and an n leaves . Addition leaves  and Edlun leaves  showed un leaves , as were members .It is possible that the role of cytokinins as scavengers of reactive oxygen species (ROS) is required in senescing leaves. Cytokinin can enhance the activities of anti-oxidant enzymes such as catalase, superoxide dismutase, peroxidase, and ascorbate peroxidase ,54,55,56PsCKX7 was expressed in the young, mature, and senescing leaves. PsCKX7 was also expressed in the meristem of peas, in the pea ovule one day before fertilisation [PsCKX7 in the shoot meristem has been suggested as a target for gene editing, or for Targeted Induced Loci Lesions IN Genomes (TILLING) [PsCKX7 may also lead to increased cytokinin in the shoots, roots, and leaves. Modestly increased endogenous cytokinin has been shown to enhance stress tolerance [PsCKX7. Whether this is beneficial to the yield and protein content of seeds will depend on the resulting strength of the mature and senescing leaves in retaining metabolites [lisation , and durlisation . A deficlisation , and theTILLING) . Downregolerance . Pea yieolerance . Savada olerance  suggest olerance , enhanciabolites .In summary, detailed knowledge of the temporal specificity of gene family members within cultivars of a species is not only useful but required before being able to target specific transporter or cytokinin gene family member(s) for gene editing in order to avoid unintended effects.P. sativum L. cv. Bolero (a process pea) and cv. Bohatyr (a field pea) were sourced from The New Zealand Institute for Plant & Food Research. Both cultivars represent the industry standard and are pure lines. Twenty-two seeds of each line were sown (planted) during summer . Each seed was sown in a pot containing potting mix, mixed with fertilizers. Growth room conditions were 22 °C during the 16 h day and 14 °C during the 8 h night. Plants were watered every two days to keep the plants moist but not wet. Plants were fertilised two or three times during the growing period. Flowers were tagged just as the standard petal opened.Seeds of Plant material for gene expression and chlorophyll analysis was obtained from pooled samples of individual leaves collected from five cv. Bolero and five cv. Bohatyr plants. The leaf samples collected were young expanding sink leaves, source leaves, and senescent leaves. Two sets of source leaves were collected: those supporting pods five days after flowering (DAF) and those supporting pods 20 DAF. Both types of leaves were fully expanded, but those associated with pods 5 DAF are here referred to as “young source leaves” and those collected when pods were 20 DAF are referred to as “mature source leaves”. Leaf samples were collected into 15 mL falcon tubes, immediately frozen in liquid nitrogen, and stored at −80 °C.IPT, CKX, SUT, AAP, CWINV, and SWEET from legume species available in the GenBank database were used as query sequences to BLAST search our P. sativum transcriptome using prfectBLAST 2.0. The putative sequences were verified via BLAST searching the GenBank database and multiple sequence alignment with representative orthologue sequences in closely related species [Sequences of family members from candidate genes of interest were isolated from an RNA-Seq transcriptome as described in Dhandapani et al. . A pool  species .Total RNA was extracted from up to 100 mg of frozen samples using TRIzol Reagent  following the manufacturer’s instructions and immediately stored at −20 °C. The integrity and quality of isolated RNA was assessed by running 1 μL samples on a 1% (w/v) agarose gel. The concentration and purity of the total RNA was assessed using a Nanodrop™ spectrophotometer. Extracted RNA was converted to cDNA through reverse transcription. Approximately 1 µg of total RNA, 50U Expand Reverse Transcriptase , 50 pmol oligo (dT) primers and 100 pmol random hexamer (pdN6) primers were used in a 20 μL reaction. The final reaction mix was incubated at 42 °C for 2 h, and then at 70 °C for 15 min to deactivate the enzyme. The cDNA was diluted 10-fold with nanopure water and stored at −20 °C.® FAST qPCR Kit . Three reference genes, elongation factor (EF), actin (ACT) and GAPDH, were used as internal controls to normalise the data by correcting for differences in the quantity of cDNA used as templates, as described previously [−ΔΔCt method [t of 30 minus the average correction factor derived from the reference genes as the value against which all samples were calculated. Three technical replicates for each of the two biological replicates (Bolero and Bohatyr) were carried out for each sample set.Quantitative RT-PCR was used to measure relative gene expression of the individual family members across the various plant tissues as they developed. Specific PCR primers were designed for each family member of the five genes of interest. A volume of 15 µL was used for all qPCR reactions in a Rotor-Gene Q (Qiagen) real-time PCR instrument, using home-made SYBR Green master mix or a KAPA SYBReviously ,14,62. Tt method  using a total = 7·12A664 + 18·12A647.Chlorophyll from three sub-samples of the pooled leaves for each developmental stage was extracted into DMF and analysed using a Nanodrop spectrophotometer as described in Evans et al. . The tot


Nicotiana tobacum L.) cultivars, an experiment was carried out in 2014 at the Tobacco Research center of Urmia, Iran under saline and Polyethylene glycol conditions. This experiment was arranged as factorial, based on RCB design with three replications. Treatments were polyethylene glycol  and hydropriming, and salinity levels  in periods 1, 2, 3, 5 and 10 days. Means of Emergence time, emergence rate coefficient, Emergence rate index, Emergence rate, and Emergence percentage decreased with increasing salinity. Emergence time and emergence rate coefficient increased with hydropriming in priming 5 and 10 days. Emergence rate index, Emergence rate, and Emergence percentage increased with 1.5% Polyethylene glycol. Seed priming with Polyethylene glycol was more beneficial in improving Emergence percentage, compared with KNO3 priming.Data on the effect of seed priming on Barli 21 tobacco ( Specifications TableValue of the data•polyethylene glycol) and salinity stress and relationships between seed priming and salinity stress it shows.These data provide the priming of seed and seedling growth of Barli 21 Tobacco under PEG (•polyethylene glycol) treatment may be applied to improving germination of Tobacco seeds as valuable method for increasing hardening at salinity stress conditions.PEG  in periods 1, 2, 3, 5 and 10 days. Seeds of tobacco (Nicotiana tobacum L.) were divided into three subsamples, one of which was kept as control (hydropriming) and two other sub-samples were prepared for priming. A sub-sample was soaked in (PEG 6000) solution and another one was pretreated with KNO3 solution with electrical conductivity of 12.5 dS m−1 at 15 °C for four hours −1) were applied immediately after sowing. Water and saline solutions were added to the Petridis. After emergence, seedling emergence was counted daily with seeds recorded as emerged, when hypocotyls appeared and mean emergence rate was calculated according to Ellis and Roberts (1980) An experiment was conducted to evaluate emergence traits and emergence percentage of tobacco under PEG and saline conditions in different periods. Treatments were polyethylene glycol ) and hydropriming, and salinity levels  was calculated according to the equation of Ellis and Roberts (1980) n is the number of seeds, which were germinated on day D and D is the number of days counted from the beginning of germination.Response to priming was assessed by germination performance . Germination was expressed as the cumulative percentage of germinated seeds. Mean germination time (R) was calculated following modified formula Rate of germination (GU) was calculated following modified formula:t is the time in days, starting from days 0, the day of germination and n is the number of seeds germinate t and MGT.Uniformity (GI) was calculated as described in the Association of Official Seed Analysts (1983) as the following formulae:Germination index (GI = No. of germinated seeds/Days of first count+-------+ No. of germinated seeds/Days of final count2.2p ≤ 0.05. EXCEL software was used to draw figures.Analysis of variance of the data was carried out using MSTATC software. Duncan test was applied to compare means of each trait at


Hevea brasiliensis is a native hyperdiverse tree species in the Amazon basin with great economic importance since it produces the highest quality natural rubber. H. brasiliensis, in its natural habitat, may harbor fungal endophytes that help defend against phytopathogenic fungi. In this work, we investigated the fungal endophytic communities in two pristine areas in Eastern Amazon  at different spatial scales: regional, local, individual (tree), and intra-individual (leaflet). Using a culture-based approach, 210 fungal endophytes were isolated from 240 sampling units and assigned to 46 distinct MOTUs based on sequencing of the nrITS DNA. The community compositions of the endophytomes are different at both regional and local scales, dominated by very few taxa and highly skewed toward rare taxa, with many endophytes infrequently isolated across hosts in sampled space. Colletotrichum sp. 1, a probably latent pathogen, was the most abundant endophytic putative species and was obtained from all individual host trees in both study areas. Although the second most abundant putative species differed between the two collection sites, Clonostachys sp. 1 and Trichoderma sp. 1, they are phylogenetically related  mycoparasites. Thus, they probably exhibit the same ecological function in the foliar endosphere of rubber tree as antagonists of its fungal pathogens. They are internal colonizers of aboveground tissues in all plant species studied to date3. Fungal endophytes of woody plants are mainly horizontally transmitted, as evidenced by their scarcity in tree seeds5. Horizontal transmission occurs preferentially by spores and/or hyphal fragmentation from senescent plant tissues6, and these propagules may be disseminated by herbivores or by abiotic agents such as wind or rain7.Fungal endophytes are defined functionally by their occurrence within asymptomatic tissues of plants without causing any apparent symptom of disease on the host plants8. The mechanisms underlying species coexistence within fungal community assembly are a dichotomy of deterministic and stochastic processes. They are used to support the niche and neutral theories separately as well as their synthesis9. The niche theory suggests that differences in species ecological traits determine which species will establish in a niche space10. On the other hand, the neutral theory posits that species composition is related to stochastic events or is shaped by geographic distance, considering the neutrality in traits11.Assembly of local communities occurs by the sequential and repeated immigration of species from the regional species pool13. Geographic distance reflects environmental differences, and these differences may serve as ecological “filters” which selects for taxa that are better adapted to local conditions14. Ecological “filters” mediate the community assembly through processes of habitat filtering and species interactions16, and include both abiotic variables  and biotic variables related to plant traits 15.Community composition of fungal endophytes is usually dependent on the geographic scale analyzed17. Conversely to previous classification systems7, this theoretical framework claims to fully encompass the range of plant—fungal interactions and their unique characteristics in nature17.Recently, a novel framework for examining fungal endophyte biology was proposed. The so-called dual-axis framework is based in two core axes: (i) mode of host-to-host transmission and (ii) degree of specificity to a particular host species or cladeHevea brasiliensis (Willd.) Muell.-Arg., is the primary commercial source for natural rubber production18. Although H. brasiliensis is a native neotropical tree species of the Amazon biome and commercial rubber plantations in the Americas have largely failed due to South American Leaf Blight (SALB), a disease caused by the phytopathogenic fungus Pseudocercospora ulei (Henn.) Hora Júnior & Mizubuti19. Unlike plantation grown trees, incidence of disease among H. brasiliensis trees in their natural, undisturbed, habitat is low19. One of the hypotheses for this low incidence of SALB in native rubber trees is that they may harbor a protective endophytic mycobiota, which may be relatively abundant in plant tissue and directly acquired from their natural habitat21.The rubber tree, Hevea brasiliensis in two Eastern Amazonian pristine conservation units.Given that fungal endophytes are mainly horizontally transmitted from the surrounding environment and that native habitat harbor protective fungal endophytes, we posit the following hypotheses: i) fungal endophytic communities significantly differ between study areas, ii) some of the most abundant fungal endophytes potentially represent mutualistic species that can be used as biological control agents of fungal diseases of rubber tree. In order to test these hypotheses, our study aimed to characterize the fungal endophytic communities of the foliar endosphere of native At the regional level, a total of 210 fungal endophytes isolates were obtained from the 240 leaflet fragments while, at the local level, 110 and 100 fungal endophytes isolates were obtained from ANP and CNF, respectively. A total of 46 putative species (OTUs) were retrieved, and these putative species are from two phyla, four classes, ten orders, 14 families and 21 (or 22) distinct genera. Two of the putative species (OTU 45 and 46) were not resolved to genus level and may be indeed a new genus (or genera). The majority of isolates were Ascomycota (95.71%) and only 4.28% were Basidiomycota (Table Most of the Ascomycota isolates were Sordariomycetes (85.23%), and only 8.57% comprised Dothideomycetes, with an even lower value for Eurotiomycetes 1.90%). The most abundant order belonging to Sordariomycetes were Glomerellales (55.90%), followed by Hypocreales (27.93%), Xylariales (11.12%), Diaporthales (13.04%), and Calosphaeriales and Amphisphaeriales representing only 0.56% each. The other two classes of Ascomycota, Dothidiomycetes and Eurotiomycetes, were both represented by only one order (and each order by only one family): Botryospheriales (Botryospheriaceae) and Eurotiales (Trichocomaceae). The Basidiomycota putative species were all of one single class, Agaricomycetes, distributed in two orders, Russulales (88.89%) and Polyporales (11.11%), which is both represented by a single family, Peniophoraceae and Polyporaceae, respectively (Table .90%. TheColletotrichum (47.62%), followed by Clonostachys (14.29%), Trichoderma (8.57%), Lasiodiplodia (4.76%), Peniophora (3.81%), which together comprised 69.16% of all the isolates  are either exclusively of ANP (32.60%) or, even more, of CNF (52.18%). Colletotrichum sp. 1 was the only putative species obtained from all host trees in both study areas and was recovered with a relative abundance of 48.0% in ANP and 44.5% in CNF  and among individual trees  did not significantly differ, which corroborates the previous results showing that two putative species dominate the communities, regardless considering or not the singletons. The only exception was for the individual trees when the singletons were removed. The distance decay analysis also corroborated this finding . PERMANOVA analyses showed that the community composition (presence and absence) of the study areas  and of the individual trees  were significantly different, regardless considering or not the singletons.The first two components in the PCA explained 80% of the variation, and there was a clear separation among the samples from the two study areas Fig. . PERMDISP = 0.14), and the mean number of isolates was not statistically different among the individual trees in both study areas and between them  in ANP, when one of the taxa occurred (regardless if one or more than one isolate was retrieved), the other necessarily did not occur (P = 0.01). This very high negative correlation reached the maximum in CNF: in 100% of the sampling units (leaflet fragments) when one of the taxa occurred , the other necessarily did not occur (P = 0.01).Colonization Rate (CR) was similar in CNF and ANP Table   in the Eastern Amazon. A high number of leaflet fragments showed at least one fungal endophyte isolate (CR- ANP: 61.7 and CR-CNF: 85.8), and similar values were obtained for Hevea brasiliensis  in southern China22, from Theobroma cacao 23 and from Heisteria concinna (Olacaceae) and Ouratea lucens (Ochnaceae) in Panama4.We performed a multiscale study of culturable fungal communities of the foliar endosphere of a native hyperdiverse tree (20. Most of the putative species were single occurrences (singletons), and similar values was previously found by Gazis & Chaverri20, who studied the same host tree species in Western Amazon, as well as in many tropical trees in Barro Colorado Island in Panamá2. The singletons may represent rare species24, and probably, the increase in the sampling effort would capture mainly these species22. After removing the singletons, the accumulation curves approximated to an asymptote  was used with a nutritional supplement (2% dextrose). These differences in the culturable methodologies could explain the discrepancies in the diversity indices values obtained in the present work when compared to other similar studies.Methods based on culture are influenced by the composition of the culture media, the physiological adaptations of the fungi, and the sampling proceduresHevea brasiliensis was explored at the regional scale, over distances of approximately 1,000 Km. The non-systemic fungal endophytes are horizontally transmitted by hyphal fragmentation and/or by spores from plant to plant26. Thus, the mycobiota surrounding the host trees in each study area are probably responsible for the fungal endophytes that reach and colonize the tree hosts, regardless if the hosts are from the same tree species27. Although the fungal endophytes are capable of dispersing and reaching host trees, they are subjected to environmental variables, which act as filters and select those better adapted to local conditions14. In our work, the environmental variables were quite similar between the two study areas and, thus, the main factor contributing to the community composition differences (PERMANOVA) were, most probably, the fungal source at local scale since the tree species diversity is distinct between the two regions29.The beta diversity of fungal endophyte communities associated to Colletotrichum sp. 1 was the most abundant species in both study areas. The genus Colletotrichum is a very speciose genus with 805 putative species30  and comprises hemibiotrophic phytopathogens of major importance, causing diseases of a wide variety of woody and herbaceous plants, primarily with tropical and subtropical distribution31. Colletotrichum species are primarily described as causing anthracnose diseases, mainly necrotic lesions on leaves, besides on flowers and fruits32.Colletotrichum sp. 1 in both collection sites correspond to the UNITE Species Hypothesis SH103151.07FU33. This SH group comprises 1,086 records of Colletotrichum gloeosporioides species complex from all over the world, including 53 records from Hevea spp. (38 from Hevea brasiliensis) with genetic distances equal or less than 1% compared to ANP and CNF Colletotrichum sp. 1 isolates. The gloeosporioides species complex is a collective of C. gloeosporioides and 37 closely related species that mainly encompass plant pathogens, with some species also isolated as endophytes34. Furthermore, Colletotrichum gloeosporioides species complex has already been recorded associated to more than 1,000 distinct plant species35 . One of the five major leaf diseases that can cause damage to Hevea brasiliensis in different countries is the Colletotrichum leaf disease, which is caused precisely by a Colletotrichum gloeosporioides36, which appears in older literature as Colletotrichum heveae37 or Colletotrichum gloeosporioides f. heveae38.All the isolates identified as Colletotrichum appears as the most abundant genus or one of the most abundant genera in different fungal endophyte culture-based studies globally, especially in tropical tree hosts39. It was the most abundant genus obtained from plantations of H. brasiliensis (Euphorbiaceae)20 and Musa acuminata (Musaceae) in China40 as well as from native Manilkara bidentata (Sapotaceae) in Guyana31, Myrceugenia ovata (Myrtaceae) in Brazil12, and Plumeria rubra (Apocynaceae) in India41. Colletotrichum was also the third most abundant fungal endophyte from native H. brasiliensis in Peru37 and Tectona grandis (Verbenaceae) in India42. According to the dual-axis framework for examining fungal endophyte diversity17, fungal endophyte genera that contain many pathogenic members often show very broad geographic distribution and low host specificity: this is exactly the case of Colletotrichum, suggesting that our putative species Colletotrichum sp. 1, our most abundant endophyte in both study areas, may be a latent pathogen43.Colletotrichum sp. 1 distribution, indicating that there is a higher odds ratio of finding one isolate of this species when the host tree species are near each other, regardless if these fungal propagules are from the same leaflet or individual host tree. Moreover, Colletotrichum sp. 1 did not exhibit host preference by Hevea brasiliensis trees evaluated in this study, which corroborates the idea that this species is widespread and ubiquitous at regional scale43. On the other hand, we found statistically significant evidence for host colonization preference by two different hypocrealean endophytes. The second most abundant genera obtained from the host trees in ANP and CNF was Clonostachys sp. 1  and Trichoderma sp. 1 , respectively.The distance between host tree species inside each study area was statistically significant Table  to explaClonostachys sp. 1 was the second most abundant species in ANP host trees and showed an association at the leaflet and individual host tree levels . Besides other life-styles, species of Clonostachys include destructive mycoparasites, some of which are used as biocontrol agents of fungal plant pathogens44. All the isolates identified as Clonostachys sp. 1 correspond to the UNITE Species Hypothesis SH182678.07FU33. This SH group comprises 294 records of Clonostachys rosea from distinct countries. Clonostachys rosea is a necrotrophic mycoparasitic fungus, used as a biological control agent of many phytopathogenic fungi, such as Alternaria spp., Bipolaris sorokiniana, Botrytis cinerea, Fusarium culmorum, Fusarium graminearum and Sclerotinia sclerotiorum45 and Moniliophthora perniciosa46.ls Table . TherefoTrichoderma sp. 1 was the second most abundant genus obtained from the host trees in CNF. Trichoderma is also a common fungal endophyte genus and was one of the most abundant from native Hevea spp. in Western Amazonia47. Trichoderma is the anamorph of the genus Hypocrea and comprises 367 putative species30 . Trichoderma spp. are among the most frequently isolated soil fungi and present in plant root ecosystems48. These fungi are avirulent plant symbionts, and parasites of many phytopathogenic fungi, thus protecting plants from diseases. Trichoderma species are among the most studied fungal Biological Control Agents (BCAs) and commercially marketed as biopesticides49. All the isolates identified as Trichoderma sp. 1 correspond to the UNITE Species Hypothesis SH190868.07FU33. This SH group comprises 2,161 records of Trichoderma harzianum from distinct countries all over the world. The Trichoderma harzianum species complex is a collective of 14 closely related species that mainly encompass mycoparasites or fungicoles with a long history in agricultural applications, especially those related BCA of phytopathogenic fungi47. Therefore, Clonostachys and Trichoderma have been long considered effective biocontrol agents against diverse fungal phytopathogens in nature and agroecosystems50.16. In our work, we suggested that the fungal endophyte Colletotrichum sp. 1 is widespread in the environment and reaches and colonizes the foliar tissue of all plant hosts.The generalist fungal endophytes exhibit fast growth, the hyphal extension increases their competition by resources, and their foliar colonized area is larger, which make them more easily isolated in culture using the standard media15. Clonostachys sp. 1 and Trichoderma sp. 1 are from the same order , exhibited a lower abundance compared to the most abundant taxon, and, most importantly, are mycoparasitic or fungicoles, suggesting that these two different taxa share the same ecological function in the foliar tissues of the host. Furthermore, previous works reported that slower growth fungal endophytes are able to produce secondary metabolites that inhibit possible pathogens46. Although we had not evaluated the antibiosis potential of Clonostachys sp. 1 and Trichoderma sp. 1 against Colletotrichum sp. 1 in in vitro antagonistic bioassays, they would probably be potential candidates as biological control agents of H. brasiliensis fungal pathogens by secondary metabolite production.The traits related to the environment where the organisms establish in communities are evolutionary conserved, as a result, related species may function ecologically similar23: They are dominated by very few core taxa and highly skewed toward infrequently isolated rare taxa. These core taxa comprised the most abundant putative species, Colletotrichum sp. 1, a probable latent pathogen, and the phylogenetically related hypocrealean putative species, Clonostachys sp. 1 and Trichoderma sp. 1, which are mycoparasites that potentially represent mutualistic species performing the same ecological function in ANP and CNF, respectively. The next steps in our research program will be to test both in vitro and in planta the antagonism of Clonostachys sp. 1 and Trichoderma sp. 1 against Colletotrichum sp. 1 and Hevea brasiliensis phytopathogenic strains, Colletotrichum gloeosporioides and Pseudocercospora ulei, causal agents of Colletotrichum leaf disease and SALB, respectively.Our study showed that the fungal endophyte community composition of the foliar endosphere of native rubber trees in Eastern Amazon significantly differed between the study areas. Nevertheless, the endophytomes exhibited two general features in common, which are in complete accordance with the dual-axis framework for decoding fungal endophyte diversityHevea brasiliensis, whereas ANP is in the center of origin of the genus Hevea51. Fieldwork permits were obtained from the Brazilian Ministry of Environment (MMA) under the access code SISBIO 42316-2.Fieldwork was conducted in two protected areas in Eastern Amazonia, distant each other approximately 1000 Km: (i) Caxiuanã National Forest (CNF) and (ii) Anavilhanas National Park (ANP). CNF is the eastern limit of the natural geographic distribution of 29.CNF is situated in the state of Pará, Brazil , with an altitude ranging from 0–80 m and a total area of 330,000 ha. The majority of the landscape is composed of non-flooded forests (85%) and seasonally or permanently flooded forests (15%). The regional climate is classified as Am in the Köppen climate classification system, with a mean annual temperature of 25.9 °C, a mean annual rainfall of 2011 mm, mean annual air relative humidity of 83%, and a short dry season29.ANP is situated in the state of Amazonas, Brazil , with an altitude ranging from 40–75 m and a total area of 350,018 ha. The majority of the landscape is composed of non-flooded forests (70%) and seasonally or permanently flooded forests (30%). The regional climate is classified as Af in the Köppen climate classification system, with a mean annual temperature of 26 °C, a mean annual rainfall of 2286 mm, mean annual air relative humidity of 83%, without a dry seasonHevea brasiliensis were randomly selected from each study area. Five visually healthy compound leaves (three leaflets per leaf) with homogeneous green coloration, and without any wilting or necrotic lesions were sampled from each one of the four adult individual trees. All leaves were at D developmental stage (mature leaves)19 and were collected at 2.5–3.5 m height. Geographical coordinates were recorded for each sampled tree using a handheld GPS unit . All the compound leaves were maintained in individualized sterile plastic bags and the fungal isolation was performed in the maximum period of time around 3–4 hours, the samples were not maintained cool The median leaflet from each compound leaf was detached and rinsed under running tap water to remove dirt and debris. After which, the leaflet was surface-sterilized via sequential dipping in 70% ethanol (1 min), 2% sodium hypochlorite (3 min), and sterile distilled water (2 min). Six fragments (approximately 5 mm2) were excised from each leaflet in specific positions: one from the base near petiole, two from the middle vein, one from the left margin, one from the right margin and from the tip26  and Rose Bengal  (30 mg/L−1) to inhibit the bacterial contamination and rapidly growing fungi52. The plates were incubated at room temperature, in the dark, for up to 60 days. The effectiveness of the surface sterilization was tested by plating 100 µl of the water used in the last step and incubated under the same conditions as the media plates. Emerging fungal colonies were purified on MEA without bactericidal or bacteriostatic compounds. The isolated were preserved in sterile distilled water at room temperature53. Vouchers (testimony specimens) were preserved in sterile distilled water and stored in CCMB (Culture Collection of Microorganisms of Bahia) (UEFS - Brazil) under the accessions CCMB660 to CCMB870.The leaflet fragments were plated onto malt extract agar (MEA) , supplemented with chloramphenicol  . The internal transcribed spacer (ITS) regions of rRNA gene were amplified using ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′)55. PCR was performed according to Vaz et al.56 with modifications: it was added 10 uL of Betaine 1 M, 1 μL of Dimethyl sulfoxide (DMSO) 50 and 1.5 μL of Bovine Serum Albumin (BSA) 0,031 μg/μL in PCR reaction. Successfully amplified PCR products were purified using an ethanol/ethylenediaminetetraacetic acid 125 mM precipitation protocol and the sequencing reactions were performed at Myleus Biotechnology  on an ABI 3130 automated sequencer  Sequences were edited using Geneious (version 9.1.6)57.Mycelial mats were ground using liquid nitrogen. Ground up mycelia (~150 mg) was then placed into a 1.5 mL tube containing 600 µL of extraction buffer  and Proteinase K 50 µg/ml. Samples were incubated at 60 °C for 60 minutes, after which 800 µL of chloroform-isoamyl alcohol (24:1) was added to each tube. Samples were then incubated on ice for 30 minutes. After incubation period, DNA was precipitated according to De Hoog 58 in Geneious (version 9.1.6)57. Default parameters were selected, along with the adjust direction option. Subsequently, sequences were clustered into operational taxonomic units (OTUs) using the furthest neighbor method in MOTHUR v. 1.36.159 with a 99% similarity criterion60. One representative sequence from each putative species was chosen for OTU taxonomic classification. BLASTn61 was used to compare sequences against the GenBank nucleotide database excluding uncultured/environmental sample sequences. GenBank sequences of the matched taxa for all OTUs were collected. When possible, well-curated published sequences with voucher/culture collection numbers were selected. Representative sequences from our study were then aligned with GenBank sequences and clustered into OTUs as previously described. The percent similarity at which all the GenBank representative sequences clustered together exclusively, with no other representative sequences from other genera were clustered with them, was considered the genus limit for the OTU. The final edited sequences were deposited in NCBI Genbank under accessions MG490657-MG490860; MG800849-MG800854.Full-length ITS sequences (450–650 bp) of fungal isolates were aligned with MAFFT 7.30562. The diversity was estimated using the Shannon (H’) (H = −Σ ni/n ln (ni/n) and Simpson indices (D = 1−Σ (ni/n)2), where ni is the number of individuals of the taxon i, and n is the total number of individuals. To find the effective number of species these both indices were modified: Shannon normalized (exp (H’)) Simpson normalized (1/(1 − D))63. The homogeneity of abundances (evenness) was measured using the Pielou formula (H’/H’max)64. The Chao2 (Schao2 = Sobs + (m − 1/m) (q1(q1 − 1)/2(q2 + 1)), where Sobs is the total observed number of species, m is the number of samples, q1 is the number of uniques (species that occur in one sample), q2 is the number of duplicates (species that occur in two samples)65. For statistical analysis, each individual fragment was considered a sample unit and a total of 240 sample units were evaluated. Rarefaction curves was performed to indicate if the number of sampling units was sufficient to wholly capture the diversity and to extrapolate the species richness using iNEXT package67.The species diversity was measured through species richness, abundance and diversity index, which combine both richness and abundance68. The rate of distance decay of the fungal endophyte communities was calculated according to Nekola & White69, with the assumption that community similarities decrease with increasing geographical distance. A randomization procedure with 1,000 iterations was implemented to test whether the slope of the distance decay curve was significantly different from zero.The data was evaluated at four different spatial scales: regional, local, individual (tree), and intra-individual (leaflet). The geographic distance among was measured for regional  and local (0–100 km) scales. There was no possible to determine the geographic distance to the other because individual scale correspond to the samples collected from the same individual host tree and the intra-individual the samples took from different parts from the same leaf. Principal Component Analysis (PCA) was conducted to visualize the trend and grouping to the fungal endophytes at regional and local scales using the vegan packageIn order to test the heterogeneity of the community between the study areas  and individuals host trees , the permutational test of multivariate dispersion (PERMDISP) was used. Leaflet fragments that did not exhibit fungal endophyte growth were removed from the datasets prior to PERMANOVA and PERMIDISP analyses. Therefore, the size of the Anavilhanas dataset (67 leaflet fragments) differed from the Caxiuanã dataset (93 leaflet fragments).70:Colonization rate was calculated using the following equation71. In order to evaluate if there was any statistically significant association between the most abundant taxa, a correlation analysis was performed using the non-parametric Spearmann’s D (rank-order correlation coefficient) as the test criterion and 5% significance level using Hmisc package72.The Friedman test, followed by the Tukey post hoc test, was used to determine whether the CR differences, either among host tree individuals  or among leaflets of an individual host tree , were statistically significant67 . The ALR models were analyzed at all fungal taxonomic levels for all genera obtained. The mean and structure was modeled with the marginal odds ratio73. A pairwise odds ratio significantly different from the null suggests the presence of fungal endophytes within a nesting level. The distance reflects the odds ratio of finding another fungal endophyte of the same taxonomic level previously found when comparing two individual host trees spaced one meter apart. Fungal endophyte singletons were not included in the modeling analysis. The ALR analysis was performed using the ordgee function from geepack package74. All analysis was carried out using the vegan package75 and the R script.In order to test the probability of re-encountering a particular fungal taxon within a leaflet fragment, leaflet, host tree, or study area, an Alternating Logistic Regression (ALR) was used


The use of transparent polymers as an alternative to glass has become widespread. However, the direct exposure of these materials to climatic conditions of sunlight and heat decrease the lifetime cost of these products. The aim of this study was to minimize the harm caused by ultraviolet (UV) radiation exposure to transparent poly(methylmethacrylate) (PMMA), which usually leads to changes in the physical and chemical properties of these materials and reduced performance. This was achieved using environmentally friendly cellulose acetate butyrate (CAB). The optical, morphological, and thermal properties of CAB blended with transparent PMMA was studied using UV-VIS spectrophotometry, scanning electron microscopy, X-ray diffraction, dynamic mechanical analysis, and thermal gravimetric analysis. The results show that CAB was able to reduce the effects of UV radiation by making PMMA more transparent to UV light, thereby preventing the negative effects of trapped radiation within the compositional structure, while maintaining the amorphous structure of the blend. The results also show that CAB blended with PMMA led to some properties commensurate with the requirements of research in terms of a slight increase in the value of the modulus and the glass transition temperature for the PMMA/CAB blend. Polymer blends are able to provide materials with extensive functional properties beyond the range that can be gained from single polymer equivalents. Research has confirmed that di-block copolymers are more efficient in decreasing the phase size and tri-block copolymers are more effective in developing the mechanical properties . They arPolymers are vulnerable to ultraviolet radiation degradation because many types of bonds in organic polymers are able to absorb ultraviolet light . PolymetMn ~ 12,000) and white powder poly (methylmethacrylate)  were supplied by Sigma-Aldrich .White powder cellulose acetate butyrate  and a hot press from Hsin-Chi Machinery Co. Ltd. . The materials were dried in a vacuum oven at 50 °C for 4 h before mixing. To prepare PMMA/CAB samples, a fixed weight of PMMA was melt-kneaded in the extruder at a rotation rate of 50 rpm at 130 °C for 10 min. Then, variable percentage weights of CAB  were added to the molten PMMA. Mixing continued until a constant torque was reached, which took about 10–15 min. The samples were transparent and homogeneous. After that, each blended sample was pressed in a hot press at 110 KPa and 130 °C to form a sheet 70 mm × 90 mm, 1 mm thick.The CAB/PMMA blend was prepared using a twin screw Thermo-Haake Poly Drive  (The nature of the transparency and absorbance of the samples was ascertained using ultraviolet-visible (UV-VIS) spectroscopy  according to ASTM D1003. Surface topography images were obtained by scanning electron microscopy (SEM) studies on a Hitachi S-3400N  microscope. Sample morphology and crystallinity were determined by XRD analysis using a X-ray diffractometer . The diffractometer data were obtained from 2Ɵ = 20° to 80° at a scanning speed of 5°/min. Thermogravimetric analysis (TGA) was undertaken on a TGA/DSC1 STAR System  at a heating rate of 20 °C/min from room temperature to 1000 °C in a continuous highly-pure nitrogen atmosphere. Dynamic mechanical analysis (DMA) was performed on a PerkinElmer Pyris Diamond  apparatus in tension mode at a frequency of 1 Hz and a heating rate of 5 °C/min in a liquid nitrogen atmosphere.Full scans over the ultraviolet and visible spectra were made from 220 to 800 nm for highly-transparent pure PMMA samples using a Shimadzu UV-3600 spectrophotometer. The results show that the absorbance peak in the ultraviolet region for pure PMMA was at 226 nm, which represents the UV damage threshold for the polymer ,19,20, wThe absorbance and transmittance curves, as well as the spectra and the visual appearance of the PMMA/CAB blends, are shown in vs. concentration for these five ascending concentrations showed that the lowest value of absorbency was at 10% CAB, i.e. about 2.184 (knowing that the absorption of pure PMMA is 3.321), with an increase in transparence up to 92%. The next step was to prepare new samples  to make a full survey of the low-absorbance area (10% CAB). Three samples were tested from each concentration and then measurements were taken to calculate the values of absorbance and transmittance. All absorbance and transmittance results were identical to those previously reported in Initially, the CAB concentrations in PMMA were 5%, 10%, 15%, 20%, and 25%. The curve of absorbance In the PMMA/CAB blends, it was noted that the samples were transparent up to 20% CAB; after that, the samples started to become hazy with increasing CAB concentrations in PMMA. Samples with concentrations greater than 20% were discarded because they did not meet the required purpose d.The SEM images taken for the internal morphological study of pure PMMA and PMMA/10%CAB blend are shown in . The SEMThe single phase of the PMMA/10%CAB blend surface with inconsistent dispersal of CAB in PMMA domains appear as white areas, without clear phase separation, which indicated average miscibility of CAB with PMMA on a microscopic scale. Macro phase separation in the PMMA/CAB blends did not occur at the mixing and injection molding temperature (130 °C) . Figure The X-ray diffraction pattern of pure PMMA and PMMA/10%CAB blend are shown in . The patThe dynamic mechanical analysis involves studying the dynamic mechanical reaction of viscoelastic materials to sinusoidal changeable strain and is useful in improving materials . The dynvs. temperature for pure PMMA, and PMMA/10%CAB blends are shown in , loss modulus E" (energy dissipation in the form of heat upon deformation), and phase angle tan δ  shown in .The storage modulus curve for PMMA and PMMA/10%CAB a shows tTg area. The peak at the lower temperature for PMMA/10%CAB was a signature of a secondary relaxation or β-relaxation process [Tg) for pure and blended (atactic) PMMA [There was a slight increase in modulus after CAB addition to PMMA, which led to an increase in the material’s ability to store potential energy (stiffness). Moreover, it should be noted that in going from pure PMMA to PMMA/10%CAB, the hydroxyl groups’ mole fraction increases. Similarly, due to the hydrogen bonding between the hydroxyl groups, the steric limitation to polymer chain motion is probable, which may slow the relaxation frequency . The two process . The tanic) PMMA .The thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) diagrams for pure PMMA and the PMMA/10%CAB blend are shown in a,b. All Multi-stage processes, such as those observed in pure PMMA, may cause differences in apparent activation energy values over the transformation range; however, unequivocal interpretation of such changes is not easy. Thus, 10%CAB inhibits activation energy values of the blend . The brew/w CAB in the PMMA/CAB blend, demonstrating high transparency and low ultraviolet light absorption. The results also showed clear improvement in some properties, such as an increase in modulus, loss factor, stiffness, thermal stability, and glass transition temperature for PMMA/10%CAB blend.Environmentally friendly transparent PMMA/CAB blends were successfully prepared by melt-blending in a twin screw extruder. The polymer blends were found to be immiscible (PMMA/CAB) with different levels of mixing. The properties of the blends were not only a function of the blend composition but also clearly depended on the degree of dispersion of CAB in PMMA, as well as the phase interaction between the components of the blend. The incorporation of CAB into PMMA reduced the absorption of ultraviolet rays at the damage threshold for both polymers, while the amorphous state for PMMA remained the same after adding CAB. The best blend sample contained 10%


PER1/2/3, CLOCK, CRY1/2, and ARNTL) were low, but that the expression levels of core clock genes were downregulated by the higher levels of DNA methylation in most tumors. The circadian clock index (CCI) was established through a principal component analysis, and this measure well represents the overall expression of the core clock genes. In fact, the CCI was significantly lower in hepatocellular carcinoma with HBV infection than in other cancers. Furthermore, pathways such as the MAPK, JAK‐STAT, and immune‐related signaling pathways were enriched in tumors with high CCI values. Interestingly, the CCI was generally positively related to the immunophenoscores and immunophenotypes of tumors. Additionally, the expression levels of core clock genes and the CCI were also generally positively related to survival across cancers. Taken together, the results of this study provide a comprehensive analysis of circadian clock aberrances in cancer, and the results should aid further investigations of the molecular mechanisms of cancer and the development of therapeutic strategies.It has been proposed that the circadian rhythm generally plays important roles in tumor suppression, but there is also evidence that disruption of the canonical circadian pathway has anticancer effects. In this study, we systematically analyzed the aberrances of circadian clock genes across cancers based on data from The Cancer Genome Atlas (TCGA). These data showed that the frequencies of mutations and copy number alterations in core clock genes ( Hall, Michael Rosbash and Michael W. Young for their leading discoveries of the molecular mechanisms controlling the circadian rhythm.CLOCK and ARNTL , and the repressor genes, including PER1, PER2, CRY1, and CRY2.bHLH)‐PER‐ARNT‐SIM (PAS) transcription factors, CLOCK and its heterodimeric partner ARNTL form a complex that binds to the regulatory elements of core clock genes, including repressors  and the cryptochrome (CRY1 and CRY2),NR1D1 (REV‐ERBα) and NR1D2 (REV‐ERBβ), which are regulated by CLOCK and ARNTL, could be driven by RORα and RORβ to repress the transcription of ARNTL and NFIL3, whereas NFIL3 could, in turn, repress D‐box‐binding protein (DBP) to regulate ROR nuclear receptors.et al recently constructed a regulatory network for the mammalian circadian clock.CLOCK, ARNTL, PER1/2/3, and CRY1/2, form the core components of the mammalian circadian clock (CCMCCs), and these establish complicated molecular circuits that orchestrate the different phases of the circadian rhythm.The circadian rhythm in humans is orchestrated by the autoregulatory transcription and translation feedback loops of core clock genes, which comprise the activator genes, including et al have found that the downregulation of PER1 is correlated with high‐grade colon tumors,et al have revealed that the circadian rhythm is disrupted in colorectal liver metastases.et al have demonstrated that SNPs in core clock genes are significantly associated with susceptibility to prostate cancer.PER3 is hypermethylated,PER1 due to inhibition of miR‐34a decreases the growth of cholangiocarcinomas.ARNTL has been detected in hematologic malignancies,ARNTL could increase oxaliplatin sensitivity in colorectal cancer.et al have found that RAS can deregulate the mammalian circadian clocket al have performed comprehensive analyses that indicated widespread dysregulation of the circadian clock in cancer,et al have observed that most circadian clock genes are related to survival, oncogenic pathways, and anticancer drug sensitivity.Krugluger In this study, we systematically analyzed the molecular dysregulation, functional implications, and clinical relevance of the circadian clock across 20 cancers based on data from The Cancer Genome Atlas (TCGA). The results revealed that the circadian clock genes were most often dysregulated through DNA hypermethylation‐based disruption of expression rather than mutations or copy number alterations. Based on the principal component analysis, we established the circadian clock index to represent the overall expression of core circadian clock genes and discovered that it is closely related to viral infections, various signaling pathways, such as the MAPK, JAK‐STAT, immune‐related, protein export, nucleotide excision repair, mismatch repair and cell cycle pathways, immunophenotypes, and survival. Our study provided a comprehensive analysis of the role of the circadian clock in cancer, and the results should be helpful for further investigations of circadian clock‐related molecular mechanisms and the development of therapies for cancer.22.1CLOCK, ARNTL, PER1, PER2, PER3, CRY1, and CRY2,BTRC, CSNK1D, CSNK1E, CUL1, DBP, FBXL21, FBXL3, NFIL3, NR1D1, NR1D2, PRKAA1, PRKAA2, RORA, RORB, and SKP1.http://gdac.broadinstitute.org, 2016 January). The analyses were performed using genomic and transcriptome data from both tumor and normal tissues for the following 20 types of cancer: bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), cervical and endocervical cancers (CESCs), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC). The somatic copy‐number alterations (SCNAs) of the core clock genes were obtained from cBioPortal,https://tcia.at/) with substantial assistance from the authors.The circadian rhythm‐related genes were curated from a previous systematic review.2.2https://genepattern.broadinstitute.org)P < 0.05. The correlation between mutations of 375 driver genesP values across 375 genes were corrected for multiple hypothesis testing using method of Benjamini & Hochberg, and only genes under 10% false discovery rate (FDR) were considered as significantly mutated. Genes and promoter regions were annotated with the Bioconductor (Release 3.7) R package IlluminaHumanMethylation450kanno.ilmn12.hg19 (version 0.6.0) for DNA methylation 450 K data. Only the methylation sites within the promotor regions of the core clock genes were considered in the differential methylation analysis, which was performed with Wilcoxon rank sum tests.Mutation data were obtained from the downloaded MAF files, and only nonsilent mutations were reserved for the analysis. The frequency ratio was calculated as the mutation frequency divided by the mutation burden to eliminate the diversity of mutation burden among cancers. A frequently mutated gene (FMG) in one type of cancer was identified based on a mutation frequency greater than 5% in that type of cancer, whereas the enrichment of FMGs in the set of rhythmic genes was performed based on hypergeometric statistics. Driver gene analysis for each cancer type was conducted using MutSigCV v1.2 with the GenePattern online server  was conducted with R packages.https://pypi.python.org/pypi/gseapy), and the other visualizations were generated with ggplot2Normalized RNA‐Seq v2 data were downloaded for the gene expression analysis and are summarized in Table 33.1PER2 in CHOL, STAD and UCEC, PER1 in CHOL, and PER3 in UCEC. Thus, since the mutation rates of core clock genes were found to be low, the circadian rhythm system might not be disrupted by mutation in cancers. In general, PER1/2/3 exhibited the highest mutation frequencies among the core clock genes. In fact, the mutation‐based driver gene analysis showed that only PER3 might be a potential driver gene in CESC and ESCA. Furthermore, the mutation rates of other CCMCCs in cancer were also low , ESCA (P = 0.0339), KIRC (P = 0.0060), and UCEC (P = 0.0074), which indicated that rhythmic genes have relatively high mutation rates in these cancers. Furthermore, the results obtained using MutSigCV software showed that a number of rhythmic genes were driver genes in these cancers, although rhythmic genes were not significantly enriched with driver genes. For example, more than 10% of driver genes were also circadian genes in KIRP and THCA. Thus, rhythmic genes might be increasingly involved in rhythmic dynamics and could be preferentially mutated in several cancers.To investigate aberrances in rhythmic genes across cancers, the mutations in the 1350 experimentally identified rhythmic genes curated from the CGDB were systematically analyzed in various cancers. The genes with mutation frequencies greater than 5% were defined as FMGs, and enrichment analyses were then performed across cancers based on hypergeometric test. The results revealed that FMGs were enriched in rhythmic genes in cancers, including COAD (3.2P < 0.05) are shown in Figure PER1/2 in KIRC, ARNTL in KRIC and HNSC, THCA and CRY2 in KICH, and CLOCK in CESC and LUAD. Thus, the expression levels of core clock genes are generally downregulated in tumors. Since there is no timepoint information for the samples in TCGA, the observed differences in expression should be considered as the differences between tumor and normal tissues. Furthermore, we performed a GSEA with the core clock genes among cancers, and the results shown in Figure The circadian rhythm has been generally proposed as a tumor‐suppressive mechanism that is disrupted in cancers,PER1/2/3 and ARNTL, and the analysis showed that the promoter regions of PER1/2/3 and ARNTL were generally highly methylated , CHOL (P = 0.0021), PAAD (P = 0.0024), and CESC (P = 0.0142), and thus indicated that disruption of the circadian rhythm might strongly influence gene expression in these cancers. Although the regulation of gene expression is complicated, the disruption of oscillatory expression in cancer is likely to be related to the dysregulation of gene expression in tumors.Furthermore, the expression and methylation of CCMCCs were analyzed, and the results presented in Figure 3.3Since circadian rhythm is critical for normal physiological functioning and is disrupted in tumors, we analyzed the signaling pathways correlated with a disrupted circadian rhythm for the results might be helpful for future studies aiming to understand underlying molecular mechanisms and functions of the circadian rhythm in cancer. GSEA of signaling pathways was performed between high‐ and low‐CCI tumors Figure . As showThe circadian rhythm recently emerged as an important regulator of the immune system.Furthermore, we attempted to systematically investigate the relationship between the CCI and immunophenotypes based on previous immunogenomic analyses.Furthermore, based on estimations of the abundances of immune cell types by CIBERSORT, the correlations between the CCI and immune cell types were analyzed, and the results are shown in Figure 3.4CLOCK was negatively correlated with survival in most cancers. Thus, it is obvious that high expression levels of the core clock genes generally predict better survival among cancers. The exceptions might be related to the multifunctional roles of core clock genes and need further investigation. Furthermore, the prognostic ability of the CCI was also studied. The survival analysis with the best cutoff value showed that a high CCI predicted better survival in BRCA, CESC, KIRP, and LIHC  and LUSC , while BMI was negatively correlated with CCI in READ . Furthermore, positive correlation was observed between age and THCA . However, no significant association between the CCI and alcohol was observed across cancers. Furthermore, we analyzed the correlation among tumor stages and circadian rhythm through GSEA of the core clock genes between high‐ and low‐stage tumors among cancers and found that the downregulated core clock genes were significantly enriched in high‐stage tumors in KICH and KIRC (Figure A previous study found that the circadian rhythm is associated with survival in colorectal cancer patients.C Figure B‐E. BesiC Figure .FBXL3 predicted better survival, while RORB, CSNK1D, and CSNK1E significantly predicted poor survival (Figure FBXL3 was related to poor survival in STAD (Figure To further reveal the prognostic ability of the circadian clock genes, the survival analysis was performed among the CCMCCs. As shown in Figure l Figure . HoweverD Figure . Thus, t4Although it has long been known that the circadian rhythm plays crucial roles in physiology, this system had not attracted much attention until recently.et. al. reported that in hematologic malignancies ARNTL was transcriptionally silenced by the hypermethylation of its promoter CpG island.ARNTL was significantly lower in most solid tumors than in nontumor tissues; in addition, hypermethylation of ARNTL was also observed in PRAD (Figure ARNTL in other cancers was not available. Furthermore, hypermethylation of PER1/PER2/PER3 was observed in most cancers, although the hypermethylation of PER3 was only statistically significant in HNSC, BRCA, and THCA. Thus, the main cause of the lower expression levels of circadian clock genes might be hypermethylation of ARNTL and PER1/PER2/PER3, while the mechanisms for CLOCK and CRY1/CRY2 require further investigation.Previously, Taniguchi D Figure A and STACRY1 in GBM, PER1/PER2 in KIRC, and CRY2 in KICH had significantly higher expression levels in tumors than in nontumor tissue, and the CCI was also higher in tumors (Figure PER1/PER2, and CRY2 are negative arms of the circadian feedback loop, while the role of CRY1 is unclear.et al might be helpful for further analyses of aberrances of the circadian clock in these three cancers. Furthermore, this study clarified that the CCIs of the core clock genes were closely correlated with immunophenotypes and cancer patient survival. However, the relationships were not consistently positive or negative among cancer types. Thus, further detailed investigations should be performed to determine the mechanisms and functions of the dysregulation of the circadian clock in different cancer types.In general, the proportions of PC1 were sufficiently high to represent the expression of the core clock genes in PCA Figure A. Howeve5The circadian rhythm and its regulatory circadian clock genes play crucial roles in cancer, and further investigation could advance the understanding of the functional roles of the circadian clock and the circadian rhythm in cancer and aid the development of potential therapies.The authors declare that they have no competing interests. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


To estimate the effect of prompt admission to critical care on mortality for deteriorating ward patients.We performed a prospective cohort study of consecutive ward patients assessed for critical care. Prompt admissions (within 4 h of assessment) were compared to a ‘watchful waiting’ cohort. We used critical care strain (bed occupancy) as a natural randomisation event that would predict prompt transfer to critical care. Strain was classified as low, medium or high . This instrumental variable (IV) analysis was repeated for the subgroup of referrals with a recommendation for critical care once assessed. Risk-adjusted 90-day survival models were also constructed.p < 0.0001) as strain increased from low (22%), to medium (15%) to high (9%); the median delay to admission was 3, 4 and 5 h respectively. In the IV analysis, prompt admission reduced 90-day mortality by 7.4%  overall, and 16.2%  for those recommended for critical care. In the risk-adjust survival model, 90-day mortality was similar.A total of 12,380 patients from 48 hospitals were available for analysis. There were 2411 (19%) prompt admissions  and 9969 (81%) controls; 1990 (20%) controls were admitted later . Prompt admissions were less frequent  contains supplementary material, which is available to authorized users. Recent policy stresses the importance of identifying and responding to the deteriorating ward patient . CurrentNon-randomised evaluations are primarily confounded by treatment allocation bias . PatientAn alternative to experimental randomisation is to seek an instrument that naturally randomises patients to prompt admission or not. This natural randomisation is known as instrumental variable (IV) analysis, and has been similarly used to remove unmeasured confounding in the assessment of influenza vaccine efficacy and cardiac catheterisation , 5.Here, we used critical care unit strain, measuring bed occupancy rates at the specific time of the patient’s assessment, for this purpose. This approach circumvents selection bias found in previous observational studies comparing prompt to delayed admission –13. DelaA fair comparison requires prospective follow-up of all watchful-waiting controls. Our prospective study does this and also exploits the learning opportunity created by a constrained supply of critical care beds. Critical care bed provision in the UK is lower than in the majority of European  and North American health care systems , 15. In We first describe the effect of critical care strain on decision-making, and the delivery of critical care for all deteriorating ward patients referred to critical care. We explore whether delays to admission engendered by high strain allow us to estimate the effect of effect of delay on patient outcome. Finally, we focus on the subgroup recommended for critical care by the bedside clinician.The full study protocol is available on the Intensive National Audit and Research Centre’s (ICNARC) website. In brief, the (SPOT)light study was a prospective cohort study of the deteriorating ward patient referred for assessment by critical care. The assessment had to be conducted on an inpatient ward by either a member of the critical care medical staff or the critical care outreach team (CCOT). Repeat visits and re-admissions were excluded as were patients where intensive care units (ICU) admission was either a priori refused (treatment limitation orders) or inevitable . Admissions following surgery, where delay was due to the process of care, were also excluded.The study was registered on the National Institute of Health Research (NIHR) research portfolio (No. 9139). Hospitals were eligible for inclusion if they participated in the national clinical audit for critical care—the ICNARC case mix programme (CMP). Research teams at each hospital attended a data set familiarisation course and were given a manual of data definitions. The ICNARC clinical trials unit provided support for the study.Reporting was via a secure online web portal that performed real-time field and record level validation. Hospitals were asked to report all consecutive ward referrals to critical care. Contemporaneous data collection was recommended, but missed referrals were sought and accepted retrospectively. We used the proportion of unplanned ward admissions to critical care in the CMP that were successfully linked to the (SPOT)light database to monitor data capture each month. Data from specific months in which data linkage rates fell below 80% were excluded from the primary analysis . Further online validation reports were completed by all hospitals before the database was locked in September 2012. Fact and date of death were then requested from the NHS Information Service. CCOT provision was reported by participating hospitals. CMP and hospital episode statistics (HES) data were used to define critical care provision and hospital characteristics.Physiology measurements at the time of the ward assessment were abstracted. From these, the ICNARC physiology score, the NHS National Early Warning Score (NEWS) and the Sequential Organ Failure Assessment (SOFA) score were calculated with missing values given zero weights as recommended –18. The The indicator of critical care unit strain was the difference between the maximum number of beds reported to ICNARC and the number of actively treated patients  occupying those beds at the time the ward patient was assessed. Units were defined as being under low, medium or high strain corresponding to having two or more, one, or zero or fewer empty beds (the last of these where strain exceeded reported capacity) respectively.The aim of the primary analysis was to estimate the effect of prompt critical care admission versus watchful waiting on 90-day mortality. We repeated the analysis in the subgroup recommended for critical care at the bedside assessment.We first built orthodox proportional hazards models with risk adjustment to handle the anticipated treatment allocation bias according to those risk factors that were observed. We then built IV models in two stages. In the first stage, a selection model was constructed to predict prompt admission including the effect of strain. In the second stage, an outcome model was built replacing strain with the fitted prediction of prompt admission from the selection model. All models were adjusted for patient-level confounders including age, sex, reported referral delay, sepsis diagnosis, peri-arrest status, existing CCMDS level of care, and severity of illness using the ICNARC, SOFA and NEWS scores.F test, and used Huber–White (robust) standard errors to allow for hospital-level clustering and potential heteroscedasticity   most often in a single physical location . Each unit admitted a median 20 unplanned admissions (IQR 14–26) from the ward per month, representing 36% of all ICU admissions (IQR 31–43%). Critical care outreach was available 24/7 in 14 hospitals, daily but not overnight in 19 hospitals, weekday daytime only in 13 hospitals and was not offered in two hospitals.Table A total of 4560 (37%) patients were recommended for critical care at the bedside assessment. These patients were younger  and more acutely unwell  than those not recommended. Patients older than 80 years were less likely to be recommended for critical care  even after risk adjustment .Overall, 36% (4401 patients) were admitted to critical care in the following week rising to 72% (3296) of those recommended on assessment. The median time from assessment to admission was 3 h (IQR 1–9) overall and 2 h (IQR 1–5) for those recommended. Admissions were prompt for 19% (2411 patients) rising to 49% (2250 patients) for those with a bedside recommendation.A total of 1636 (13%) patients died in the week following the assessment with 856 deaths (52%) following critical care admission and 780 deaths (48%) on the ward without admission. For those recommended for critical care at assessment, there were 862 deaths (19%) of which 663 (77%) received critical care before death and 199 (23%) occurred on the ward without ICU admission.p = 0.702].For those admitted promptly, 90-day mortality was 36.7% (885 deaths) compared to 28.6% (2851) for the watchful-waiting control group. Patients who were admitted promptly had higher physiological severity scores; for example, the ICNARC physiology score was 4.4 ICNARC physiology points [95% CI 4.0–4.7] higher on average. Without risk adjustment, the proportion of patients who died prior to 90 days was higher for prompt critical care admissions with a hazard ratio (HR) of 1.42 (95% CI 1.32–1.54). With risk adjustment , survival was equivalent .For the 4560 patients recommended for critical care, 90-day mortality was 37.2% (837 deaths) for prompt admissions versus 34.4% (794 deaths) for controls. Prompt admissions again had higher physiological severity [2.0 ICNARC physiology points (95% CI 1.6–2.5)]. Before risk adjustment, survival was worse , but again was again equivalent after risk adjustment [HR 0.99 (95% CI 0.85–1.10), p < 0.0001), corresponding upward trend in the median time to admission: 3 h (IQR 1–8), 4 h (IQR 1–12) and 5 h (IQR 2–16) .There were 10,039 (81%) bedside assessments when there were two or more empty beds on the critical care unit, 1353 (11%) when there was just one empty bed and 988 (8%) when the unit was already fully occupied Table . As straF statistic 83, p < 0.0001).Strain varied by time of day, day of the week, and season; however, the relationship between prompt admission and occupancy remained even after adjustment , and there was a strong negative correlation between strain and prompt critical care admission (Kleibergen–Paap p = 0.03). Regardless of timing, the overall probability of receiving critical care also fell .There was evidence that the delay in admission translated into further ongoing physiological deterioration  and median time to admission increased: 2 h (IQR 1–4), 3 h (IQR 1–6) and 4 h (IQR 2–9) (p < 0.0001). The proportion of patients managed without critical care increased from 25%, to 33%, to 50% during low, medium and high strain periods .Within the subgroup recommended, the proportion of prompt admissions also fell as strain increased  which was equivalent to an odds ratio of 0.68 , 30.8% (417 deaths) and 31.6% 312 deaths) for deteriorating ward patients assessed at times of low, medium and high critical care strain respectively  equivalent to an odds ratio of 0.46 .Among those recommended for critical care, unadjusted 90-day mortality was 35.0% (1307 deaths), 41.8% (197 deaths) and 35.9% (127 deaths) for ward assessments at times of low, medium and high strain. For this subgroup, the average reduction in 90-day mortality for prompt admission was 16.2% . Risk adjustment for observed severity is unable to show a benefit for a prompt admission strategy. However, we argue that unobserved differences in baseline risk are part of a clinician’s bedside assessment, and our risk adjustment is likely to be incomplete.We exploit the random variation in critical care strain to additionally allow for unobserved differences in baseline severity. We show that prompt critical care admission is less likely for patients referred during times of strain. Using this natural experimental set-up, we found that prompt admission reduces 90-day mortality without reaching statistical significance. For the subgroup recommended for critical care, the estimated reduction in 90-day mortality is larger and statistically significant. There is a large amount of uncertainty in the precise estimate of these effects, but the weight of evidence favours a prompt admission strategy, especially where the bedside assessor recommends critical care.The prompt admission strategy might confer a survival advantage in two distinct ways: explicitly be delivering critical care to a particular patient more promptly; or implicitly, by increasing the opportunity for critical care in the population. Of note, half of the deaths in the first week occur without admission to critical care, and death without critical care is more common when strain is greater.It is not easy to separate out these two effects. Although previous studies compare the prompt to the ‘delayed’ admission, the delay is defined with hindsight which creates exclusion and survivor bias –13. The Our attempt to minimise treatment selection bias (confounding by indication) relies on the assumption that critical strain acts as a natural randomisation event. We defend this assumption in three ways. Firstly, critical care occupancy at a specific time is unpredictable. It is the net result of all the decisions that lead to admission, discharge and death in a complex, maybe chaotic, system. Secondly, we have included controls where occupancy might be associated with baseline severity (i.e. increased strain during the winter). Thirdly, although strain might translate as a crowding effect thereby impairing the quality of care within the ICU, the evidence for this is conflicting , 26. MorAn important limitation of IV analyses is that they are notoriously weak, and, without very large sample sizes, there is a risk of not detecting a true difference when one exists , 28. ThiOur finding of harm from reduced or delayed access to critical care does not stand in isolation. At a patient level, reducing exposure to critical care by premature discharge rather than delayed admission has also increased mortality . At a poWe also need to acknowledge that in an observational study of this size, there are limitations in the quality of the data recorded. We did not perfectly capture all admissions to critical care in the study database, and we must assume that a proportion of referrals were also missed. However, we tested our findings by raising threshold for judging data capture to 90% so that the median proportion of eligible admissions was 97%. We found no consistent difference in any result other than a fall in precision as the quality threshold increased, and the sample size inevitably fell.Aspects of the study stand independent of these limitations. Regardless of the effect of prompt admission to critical care, we have identified a cohort of hospital patients at very high risk. This risk is heavily front-loaded, and the window for intervention is short. The bedside assessment is an effective but imperfect triage tool, as the mortality in those initially refused admission is high. Given that we already excluded patients with treatment limitations, it is of concern that nearly half of these early deaths occur without a trial of critical care.A substantial proportion of patients recommended for critical care are not offered a bed, and this proportion increases when capacity is limited. Although expanding critical care bed numbers may help, supply quickly saturates this expensive resource . Given tSupplementary material 1 (DOCX 55 kb)Supplementary material 2 (DOCX 7702 kb)Below is the link to the electronic supplementary material.