Fritillaria which are distributed in the temperate zone of the Northern Hemisphere. Increasing attention has been paid to FCB because of its excellent medicinal value such as being antitussive, expectorant, analgesic, anticancer, anti-inflammatory, and antioxidative. During the past years, a large number of research studies have been conducted to investigate the phytochemistry, pharmacology, and pharmacokinetics of FCB. A range of compounds have been isolated and identified from FCB, including alkaloids, saponins, nucleosides, organic acids, terpenoids, and sterols. Among them, alkaloids as the main active ingredient have been illustrated to exert significant therapeutic effects on many diseases such as cancer, acute lung injury, chronic obstructive pulmonary disease, asthma, Parkinson's disease, and diabetes. Due to the excellent medical value and low toxicity, FCB has a huge market all over the world and triggers a growing enthusiasm among researchers. However, there is still a lack of systematic review. Hence, in this work, we reviewed the FCB-based articles published in Sci Finder, Web of Science, PubMed, Google Scholar, CNKI, and other databases in the recent years. The traditional uses, sources, phytochemistry, pharmacology, pharmacokinetics, and toxicity of FCB were discussed in the review, which aims to provide a reference for further development and utilization of FCB.Fritillariae Cirrhosae Bulbus  is a famous folk medicine which has been widely used to relieve cough and eliminate phlegm for thousands of years in China. The medicine originates from dried bulbs of six species of  Fritillaria species [Fritillaria genus used as the botanical origins of FCB, including Fritillaria cirrhosa D. Don, Fritillaria przewalskii Maxim., Fritillaria unibracteata Hsiao et K. C. Hsia, Fritillaria delavayi Franch., Fritillaria taipaiensis P. Y. Li, and Fritillaria unibracteata Hsiao et K. C. Hsia var. wabuensis (S. Y. Tang et S. C. Yue) Z. D. Liu., S. Wang et S. C. Chen  [Fritillaria plants are characterized by poor growth and strict environmental requirements. Also, they are mainly distributed between the altitudes of 3000 and 5000 m in most parts of Tibet, Northwest Sichuan, Northern Yunnan, and Southeastern Qinghai [Fritillariae Cirrhosae Bulbus (FCB) is one of the best known Chinese herbal medicines used to treat respiratory disease and obtained from the  species –3. Accor and FW) . The six Qinghai .Historically, FCB was firstly documented in “Shen Nong Ben Cao Jing”  and has been widely used clinically owing to its remarkable antitussive, expectorant, and antiasthmatic activities . So far,FCB is one of the most ancient Traditional Chinese Medicine (TCM) associated with high economic and medical value , 15. In Fritillaria species, including FC, FP, FU, FD, FT, and FW [Fritillaria species are generally perennial herbs and have strict requirements for the growing environment. Among them, FC commonly grows in high altitude habitats from 3200–4200 m, has a disjunct distribution across the high mountain areas of Southern and Eastern Tibet, northwestern Yunnan, and western Sichuan. FP is mainly distributed at altitudes between 2800 and 4400 m in Southern Gansu, eastern and southern Qinghai, and western Sichuan. FU usually grows at the altitudes between 3200 and 4500 m in northwest of Sichuan and southeastern of Qinghai. FD is chiefly distributed at the altitudes between 3800 and 4700 m in northwestern Yunnan, western Sichuan, southern Qinghai, and Tibet. FT primarily grows at the altitudes between 2400 and 3150 m in Shaanxi (Qinling Mountains and its south), southeastern Gansu, northeastern Sichuan, and northwestern Hubei [Fritillaria genus have been classified as precious, rare, and threatened species by the Endangered Species Scientific Commission of China in 2012 [According to the Chinese Pharmacopoeia (2015), FCB is derived from the dried bulbs of six , and FW   have been extracted and identified from FCB  exhibited inhibitory effects on the proliferation activities of LLC cells, which may partly result from the apoptosis induction mediated by S-phase cell cycle arrest [γ), interleukin- (IL-) 12, caspase-3, B-cell lymphoma-2-associated X apoptosis regulator (Bax) levels, and the decrease of B-cell lymphoma-2 (Bcl-2) production. Moreover, FC-AE (0–100 μg/mL for 48 h) exhibited excellent antiproliferative effects on A549 cells, which was attributable to the accumulation of G2 phase cells and the promotion of Bax, STAT1, and STAT4 activities, as well as the downregulation of Bcl-2 proteins [μg/mL for 48 h) dramatically reduced the viability of LLC cells, and the positive results were accompanied with an accumulation of cells in the S phase and a decrease of the cells in the G0/G1 phase, which subsequently leads to the augmentation of apoptosis. The underlying mechanism was related to the reduction of antiapoptotic protein Bcl-2 and the enhancement of proapoptotic protein Bax and caspase-3. To better understand the role of chuanbeinone on the treatment of lung cancer, an in vivo study suggested that chuanbeinone (10–40 mg/kg/day for 10 days) significantly suppressed the growth of transplantable LLC tumors in mice. The antagonistic effects were attributed to inactivation of CD31-mediated tumor angiogenesis, as well as the promotion of caspase-3-mediated apoptosis [κB (NF-κB) pathway, including phosphoinositide 3-kinase (PI3K) Class III, protein kinase B (Akt), p-Akt, nuclear factor-κB-inducing kinase (NIK), IκB kinase (IKK) α&β, nuclear factor κBα (IκBα), and decreasing the expression of Ki67 , as well as upregulating the levels of caspase-3. Furthermore, imperialine (10 mg/kg for 18 days) prominently blocked the development of non-small-cell lung cancer in the mice model, which involves the downregulation of the levels of inflammatory cytokines, such as IL-1β, IL-6, tumor necrosis factor-α (TNF-α), and the decrease of Ki67 [Currently, lung cancer as the most common cancer is attracting increasing attention . In preve arrest . Besides of Ki67 .μM for 24 h). The positive results may ascribe the elevation of the ratio of LC3B-II/LC3B-I and the upregulation of phosphorylated Unc51-like kinase 1 (p-ULK1), phosphorylated adenosine 5′-monophosphate-activated protein kinase (p-AMPK), caspase-9, and cleaved caspase-3 levels, as well as the damping of phosphorylated mammalian target of rapamycin (p-mTOR), p-Akt, phosphorylated phosphatase, and tensin homolog (p-PTEN) activities [μM for 48 h) could trigger the death of HCT-116 cells by promoting apoptosis and autophagic flux via modulating the production of metabolites, such as glucose, glutamine, oleate, and lignocerate [As a major bioactive constituent of FCB, peiminine has been validated to exert promising antitumor effects. Based on the past studies, peiminine (3 mg/kg/2 days for 14 days) could significantly repress the growth of transplantable HCT-116 tumor in mice through inducing apoptosis and autophagy. The molecular mechanism was related to the enhancement of microtubule-associated protein light chain3B (LC3B) and cleaved caspase-3 levels. Analogously, it was discovered that the cell viability was dose-dependently decreased in colorectal carcinoma HCT-116 cells treated with peiminine (0–400 nocerate .μg/mL for 24 h) conducted the growth suppression effects on HepG2 cells in a concentration-dependent manner. It was simultaneously observed that the number of apoptotic cells increased and the percentage of G2/M phases cells augmented. Moreover, the expression of Bax, cleaved poly-ADP-ribosyl-polymerase (PARP), and caspase-3/8/9 increased, whereas the levels of Bcl-2 and Checkpoint kinase 2 (Chk2) downregulated, which indicates that peiminine triggers apoptosis in HepG2 via both extrinsic and intrinsic apoptotic pathways [An accumulating line of evidence indicates that FCB is a potential antitumor agent. In previous studies, various fractions from FCB, mainly including chloroform extracts, n-hexane extracts, water extracts, petroleum ether extracts, and TFCB, have been demonstrated to exhibit inhibitory activity on the growth of the HepG2 . To furtpathways .μg/mL for 72 h) could markedly repress the growth of endometrial cancer cells through downregulating the transforming growth factor-β (TGF-β)/Smad signaling pathway [κB subunit p50 (NF-κBp50), p-IκBa, cysteine-X-cysteine chemokine receptor 4 (CXCR4), and matrix metalloproteinase-9 (MMP-9) and augmented the expression of p27 and caspase-3. The results given above indicate that FC-AE suppresses progression of endometrial cancer and ovarian cancer by activating apoptotic pathways and cell cycle arrest, as well as abrogating the NF-κB pathways activation [μg/mL for 0–72 h) notably blocked the proliferation of human ovarian cancer cell through inducing apoptosis via increasing G0/G1 phase cell arrest [As research continues, some studies have discovered that the FC-AE has huge therapeutic potential in endometrial cancer and ovarian cancer. For instance, Bokhari et al. illustrated that FC-AE (200  pathway . Additiotivation . Of courl arrest .μg/mL for 3 days) could suppress the proliferation of immortalized and malignant oral keratinocytes through inducing cell cycle arrest and apoptosis. The molecular mechanism was connected with the downregulation of Bcl-2 and retinoblastoma protein (pRb) and the upregulattion of Bax, caspase-3, p53, and p21 protein [Noteworthy, FCB also displays advantageous effects on the treatment of other types of cancers, such as oral cancer and myelogenous leukemia. Based on the published literatures, peimine  establishe mitigative effects in mice with cough [Cough is a crucial defense mechanism which maintains airway patency and eliminates the potential harmful stimuli from the airway and lung . Generalth cough . Additioth cough . Moreoveth cough .β-N-oxide, isoverticine, and isoverticine-β-N-oxide (1.5–4.5 mg/kg for 0.5 h) significantly promoted tracheal phenol red output, which indicated that the expectorant effects of FCB may be related to its ability to increase tracheobronchial mucus secretion [Expectorant activity is another vital function of FCB. Based on the previous data, the alkaloids extracted from FCB, mainly including imperialine, chuanbeinone, peimine, and peiminine , have been demonstrated achieve expectorant effects in mice by enhancing tracheal phenol red output . Analogoecretion . The aboα and IL-6 [γ, TGF-β, connective tissue growth factor (CTGF), extracellular signal-regulated kinase (ERK)1/2, NF-κB, and Fas Ligand (FasL) [α, IL-1β, IL-6, and IL-8 in the LPS-induced acute lung injury mice model. Also, those changes may partly be relevant for the weakening of AKT/PI3K phosphorylation and the attenuation of lipid rafts formation [As the research continues, some studies have reported that FCB is a promising agent to treat acute lung injury. TFCB (15–60 mg/kg/day for 5 days) could markedly attenuate LPS-induced acute lung injury in mice, which was associated with a decrease of TNF-and IL-6 . Furtherd (FasL) . Consistormation .β, IL-6, IL-8, TNF-α, NF-κB p65, TGF-1β, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) [β (p-GSK3β) while increasing the expression of phosphorylated myosin light chain2 (p-MLC2). Increasingly, an FCB-based traditional Chinese medicine formula, Chuan Bei Pi Pa dropping pills (50–200 mg/kg for 35 days), has been demonstrated to block the progression of COPD through decreasing the number of leukocytes [COPD has long been considered as a severe public health problem. It is urgent to find new compounds for minimizing the risk of this disease . Fortunaukocytes .γ production [Asthma is a chronic inflammatory disorder of the airways which has been regarded as a formidable challenge for clinicians. At present, the therapeutic effects of conventional medicine are still unsatisfactory . Noteworoduction  , also have been found establish similar anti-inflammatory effects [α, IL-6, IL-1β, p38, ERK, c-Jun N-terminal kinase (JNK), p65, and IκB and increasing the productions of IL-10 in LPS-induced RAW264.7 macrophages [μM for 24 h) could dose-dependently inhibit the expression of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β, and p–NF–κB p65 in LPS-induced RAW264.7 macrophages [μM for 24 h), and peimine (0–10 μM for 24 h)) could inhibit the productions of NO, TNF-α, IL-1β, and IL-6 and suppress the activation of ERK1/2, p38 mitogen-activated protein kinase (MAPK), and JNK/stress-activated protein kinase (SAPK) in LPS-induced RAW264.7 macrophages [During the past years, the anti-inflammatory effects of FCB have attracted more and more attention. On the grounds of the previous studies, total alkaloid fraction of FCB (0–14 mg/kg/day for 7 days) could significantly attenuate carrageenan-induced paw edema in mice . Consist effects . To furtrophages . Additiorophages . Furtherrophages  ) could inhibit cigarette smoke-induced oxidative stress in RAW264.7 macrophages, which was associated with a decrease of reactive oxygen species (ROS) and an increase of glutathione, heme oxygenase-1 (HO-1), and nuclear erythroid-related factor 2 (Nrf2), the result indicates that FCB could be a potential inhibitor of oxidative stress [Based on a recent study, isosteroid alkaloids from FCB  obviously decreased the expression of the proinflammatory mediators TNF-α, IL-6 and IL-1β, COX-2, and iNOS by inhibiting the phosphorylation of ERK1/2, AKT, and NF-κB p65 in LPS-induced BV-2 cells [α, IL-6, IL-1β, COX-2, iNOS, AKT, NF-κBp65, ERK1/2, and p38. Consistent to the results, peiminine (0–70 μg/mL for 4 h) inhibited the secretion of proinflammatory cytokines in LPS-induced mouse mammary epithelial cells via repressing AKT/NF-κB, ERK1/2, and p38 signaling pathways [μg/mL for 24 h) performed hypoglycemic activities by enhancing the levels of insulin and glucose uptake ability in b-TC6 pancreatic and C2C12 skeletal muscle cells, which implies that peimine has huge therapeutic potential in diabetes [FCB is a natural agent with various pharmacological effects; in addition to the abovementioned pharmacological effects, other biological activities have also been reported in many researches in the last few decades. For instance, peiminine (0–5 mg/kg for 28 days) markedly attenuated behavioural dysfunction and inhibited the loss of dopaminergic neurons and microglial activation in the LPS-induced Parkinson's disease rat model. Moreover, it was ascertained that peiminine (0–50 -2 cells . Additiopathways . Based odiabetes .Cmax), elimination half-life (T1/2), time to reach Cmax (Tmax), oral clearance (CL/F), volume distribution/bioavailability (V/F), and area under the concentration-time curve (AUC) from zero to t and from zero to infinite time.In the past few decades, the pharmacokinetics studies of FCB mainly focused on its alkaloids, such as imperialine, peimisine, and peimine. Imperialine has high oral bioavailability, and a previous study have shown that the bioavailability of administered imperialine at an oral dose of 1 mg/kg, 5 mg/kg, and 10 mg/kg was 31.2%, 53.6%, and 47.4%, respectively . Howeverμg/mL for 72 h) leaded to cytokinesis failure through triggering chromosomal instability via promoting the incidence of binucleated cells in NCM460 cells [Over the past several millennia, it is generally assumed that FCB is a nontoxic or low toxic herb. To date, few studies focused on the toxicity of FCB. Recently, the acute oral toxicity of FCB has been assessed by an in vivo study. The results revealed that the maximum feasible dose value of FCB in mice was 452.14 g/kg. There were no mice dead after administering the FCB extract at the maximum feasible dose for 14 days. In the histological examination, only the liver showed significant pathological changes . Additio60 cells . In summFritillaria species used as the botanical origins of FCB grow extremely slow. Also, they are difficult to meet the increasing market demands [Fritillaria species. Therefore, it is an urgent problem to find out an alternative way to improve the production of FCB. Encouragingly, the emerging of artificial cultivation technique has partly solved the shortage of supplies. Also, the content of total alkaloids in cultivated FCB is higher than that in wild FCB [Fritillaria propagation by seed is low because the growth of seedlings in natural habitats is too weak [Fritillaria species. But, there is a fly in the ointment: the stages of rooting and transplanting are difficult, which may result from dormancy of bulbs [Fritillaria is another point worth taking into consideration. Of course, searching for the substitutes is also a feasible way to offset the shortage of FCB. For instance, Fritillariae Ussuriensis Bulbus possesses pharmacologic function similar to that of FCB and has abundant natural resources [or other Fritillaria herbs could be used as FCB. Overall, it is necessary to explore the better approach to shorten the growth time of medicinal Fritillaria plants, promote the output of FCB, and prevent the overexploitation of wild original species.FCB is a famous TCM with a wide spectrum of pharmacological effects and has been extensively used as a major ingredient in cough syrups with a huge commercial market. At present, FCB is still mainly harvested from the wild fields. However, the wild  demands . In addiwild FCB . But, mawild FCB . Moreovetoo weak . Recentlof bulbs . Hence, esources ,99. So, Fritillaria species [Fritillaria species [In the traditional viewpoint, it is commonly acknowledged that the major bioactive constituents of FCB are alkaloids. Also, a large proportion of pharmacological and chemical studies are focused on them. Moreover, the content of alkaloids has been regarded as an important index to evaluate the qualities of FCB. However, the chemical researches have shown that FCB has the lowest levels of alkaloids compared with other  species . Therefo species , which iIn the past few decades, extensive studies have attempted to assess the pharmacological effects of FCB. To our best knowledge, FCB exerts unexceptionable therapeutic effects on various diseases, including cough, expectoration, acute lung injury, chronic obstructive pulmonary disease, asthma, and some type of cancers, such as lung cancer, colorectal cancer, liver cancer, endometrial cancer, and ovarian cancer. Nevertheless, the underlying mechanisms of its pharmacology actions are still unclear. For instance, the antitussive and expectorant activities have been demonstrated in the previous studies, but the hidden mechanism is not fully understood. Therefore, we should conduct more molecular studies to gain a broader knowledge on the mechanism of FCB's pharmacologic action in future. Additionally, there are not enough studies regarding the pharmacokinetics and toxicity of FCB or its active ingredients. More profound evaluations are required to investigate the pharmacokinetics of other constituents besides alkaloids from FCB. Also, the toxicity studies focusing on molecular levels are still a promising direction.In the recent years, the growing market demand leads to a rapid rise of the price of FCB. The price of FCB was beyond $260 per kilogram in 2014, almost nine-folds of that in 2004, and it is much higher than other Fritillariae Bulbus . In ordeTo sum up, this work is the first to systematically review the traditional uses, sources, phytochemistry, pharmacology, pharmacokinetics, and toxicity of FCB. The recent findings about FCB have been summarized in this work, to provide a reference for further development and utilization of FCB.


This article comments on:Shang Y, Yuan L, Di Y, Jia Y, Zhang Z, Li S, Xing L, Qi Z, Wang X, Zhu J, Hua W, Wu X, Zhu M, Li G, Li C. 2020. A CYC/TB1 type TCP transcription factor controls spikelet meristem identity in barley. Journal of Experimental Botany 71, 7118–7131. Genetic diversity for inflorescence architecture has helped improve the yields of our modern cereals. In barley, enhanced fertility of lateral spikelets has underpinned the generation of higher-yielding six-rowed cultivars, relative to their two-rowed progenitors. The genetic basis of lateral spikelet fertility has been well-characterised; however, very little is known of genes controlling other aspects of barley inflorescence architecture. Now, et al. (2020)Shang  BRANCHED AND INDETERMINATE SPIKELET 1 (BDI1), which encodes a CYC/TB1 type TCP transcription factor that represses inflorescence branching. In partnership with reports in rice and maize, these findings provide insights into genes controlling the diverse inflorescence architectures of cereals.report on the identification of VRS1, VRS2, VRS3, VRS4 and VRS5: VRS1 encodes a homeobox transcription factor that is expressed exclusively in lateral spikelets to suppress their development , encodes a TCP transcription factor that is orthologous to TEOSINTE BRANCHED1 (TB1) from maize – VRS5 suppresses development and outgrowth of the lateral spikelets  mutant, where a mutation in an AP2/ERF transcription factor prevents termination of the spikelet meristem  in rice and BRANCH ANGLE DEFECTIVE1 (BAD1) from maize, which were discovered by studying mutants that influence palea formation and development of lateral branches on the tassel, respectively , show that barley has potential to link the function of this TCP transcription factor in controlling palea formation and inflorescence branching across cereals  In wild-type barley, the developing inflorescence consists of one central determinate spikelet (CS), which contains a lemma  and a palea  that enclose floral organs. The bdi1 mutant forms an indeterminate CS with extended branches, and thickened cell walls form within the palea ((B) In rice, a mutation in OsREP1 forms a smaller palea ((C) In maize, a mutation in ZmBAD1 causes defects in tassel branch angles, causing a clumped branching phenotype  produced palea with increased cell size, cell wall thickness and more vascular bundles, relative to wild-type  is down-regulated in bdi and com1, relative to wild-type (SRA3 and its association with inflorescence branching are consistent with the reduced spikelet meristem determinacy of ramosa3 and tpp4 mutants of maize (The transcriptome analysis performed by In conclusion, the study by


Assessment of hematotoxicity from environmental or xenobiotic compounds is of notable interest and is frequently assessed via the colony forming unit (CFU) assay. Identification of the mode of action of single compounds is of further interest, as this often enables transfer of results across different tissues and compounds. Metabolomics displays one promising approach for such identification, nevertheless, suitability with current protocols is restricted. Here, we combined a hematopoietic stem and progenitor cell (HSPC) expansion approach with distinct lineage differentiations, resulting in formation of erythrocytes, dendritic cells and neutrophils. We examined the unique combination of pathway activity in glycolysis, glutaminolysis, polyamine synthesis, fatty acid oxidation and synthesis, as well as glycerophospholipid and sphingolipid metabolism. We further assessed their interconnections and essentialness for each lineage formation. By this, we provide further insights into active metabolic pathways during the differentiation of HSPC into different lineages, enabling profound understanding of possible metabolic changes in each lineage caused by exogenous compounds. Assessment of toxic properties from environmental or xenobiotic compounds remains a major field of research. Mammalian laboratory animals are currently considered as the “gold standard” in toxicology . However6 cells per replicate), making metabolomics mostly incompatible for large screening approaches with the aforementioned hematopoietic models . H. H+ HSPCpositive A. Nevertpositive  52]. In. In+ HSPpulation . Howeverassessed . In brie+ dendritic cells , MRC1 and C1QC by macrophages and DC, while EPX is expressed by eosinophils, the expression patterns indicate, that the main lineages formed are erythrocytes, DC as well as neutrophils [We therefore continued differentiation of the progenitor population into erythroid , megakaric cells  and CD66trophils  were fortrophils . As HBB trophils ,58,59,60Taken together, these data demonstrate the multi-lineage potential of the generated progenitor population as it was possible to form erythroid, DC, neutrophil and eosinophil lineages. Since all populations reside in the myeloid branch of hematopoiesis, we assume that the progenitor population either consists of restricted myeloid progenitor cells or of a mixture of further differentiated progenitors. Indeed, transcriptomics data also indicate that other lineages are formed within each population, however, the main lineages formed are erythrocytes, DC as well as neutrophils, as verified via flow cytometry.Based on the successful identification of each main lineage formed, we performed global targeted metabolome profiling, testing for 514 metabolites in total. We totally identified 173 metabolites being altered from the progenitor population in the mature lineages A. In thip value in order to avoid an increase of the type II error  [As we sought to identify the underlying changes on metabolic pathway levels, we analyzed the expression of enzymes, related to the relevant metabolites. Whole pathway activity is often regulated by a small number of key enzymes , therefore, whole pathway activity can often be derived from the expression of these in combination with metabolite concentrations . Furthergatives) . By usinSLC2A1, SLC2A5 and SLC2A6) was either upregulated or unchanged  via the PPP is vital for erythroid differentiation [FBP1 catalyzes the back conversion of fructose 1,6-bisphosphate to fructose 6-phosphate (F6P), acting as the rate-limiting enzyme in gluconeogenesis. As F6P readily can be converted to G6P, we assume that both lineages primarily use glucose for nucleotide biosynthesis and ROS production from NADPH via PPP, which is also supported by findings of other groups [As the energy metabolism seems to play a crucial role during HSPC maintenance and differentiation, we focused on glycolysis, FAO and glutaminolysis, as these display the three major pathways for energy transformation . Hexosesnchanged , indicatntiation . Howeverr groups ,65.Several acylcarnitines were raised in erythrocytes and DC, 18 of which could be defined as changes common to both lineages . Only thCPT1a lowered in the neutrophil lineage  and triglycerides (TG). FAs are further catabolized via FAO in neutrophils, while erythrocytes and DC seem to utilize FAs for anabolic processes. Erythrocytes, however, preferably elongate C16:0 and C16:1 FAs , which lIn context to glutamine metabolism, we found uniquely lowered levels of the products Glu, Asn, Asp, Ala and Pro in neutrophils . As it iME1 and IDH1 raised in DC, both resulting in NADPH generation from Krebs cycle intermediates  reduced erythroid expansion rates and HBB expression , a specific inhibitor of OAT [HBB independent of Gln , we also focused on GLP metabolism. GLP need to be further subdivided in acyl-acyl species  and acyl-alkyl species , as the former are involved in glycerophospholipid metabolism and the latter are involved in ether lipid metabolism. Interestingly, the different GPL classes , phosphatidylethanolamine (PE) and phosphatidylserine (PS)) followed the same trend in all lineages (not shown). However, erythrocytes displayed increased aGPL levels, DC showed lowered aGPL levels and higher EL levels, while neutrophils exhibited reduced aGPL levels, in comparison to the progenitor population . FurtherPLA2G15, PLA2G16, LPCAT3, PISD, PHOSPHO1 and LPIN1 were affected distinctively in the corresponding populations. Furthermore, the activity of PLBD1, PLD3 and PISD has been reported as negligible, which was also reflected by the corresponding metabolic changes [PLA2G15 and PLA2G16 [FAR1/2, the major regulator of EL biosynthesis, uses C16 and C18 acyl-CoA species as substrates [PLA2G16 additionally reduces EL biosynthesis by inducing peroxisomal dysfunction, which results in the lack of finding of uniquely raised EL, despite their higher percentual content [We found several changes in the expression of enzymes, related to glycerophospholipid and ether lipid metabolism ; however changes ,87,88,89 PLA2G16 ,91. The bstrates ,93. SincSPTSSB, well-known to stimulate serine palmitoyltransferase (SPT) activity, was raised in DC and erythrocytes [SPT uses C18-CoA substrates, we conclude that de novo ceramide synthesis is enhanced in DC, due to higher availability of sphingosine, resulting from higher C18-CoA availability.In line with this, we found higher levels of C16, C18 and C18:1 ceramide in DC B. Furthehrocytes . As SPT PLA2G15 or PLA2G16, resulting in the described unique metabolic signatures.Taken together, our data strongly indicate that parts of the cellular lipidome analyzed here are all regulated by FA levels, in combination with the expression of Cellular metabolism is an essential feature of living organisms; various metabolites are involved in specialized cellular tasks, ultimately defining the function of each cell. In the context of hematopoietic stem cells, it is fully appreciated that the activity of metabolic pathways not only regulate quiescence, but also regulate commitment ,95. InhiThe total cellular metabolism plays a fundamental role in the cellular response towards different stimuli. In the context of hematopoiesis, several different compounds are known to exert distinct effects on the different blood cell populations ,97,98,99We, therefore, established an HSPC differentiation model, suitable for omics-based screening approaches, and performed metabolic and transcriptomic comparison of three myeloid lineages, regarding to the initial progenitor population. Generated populations, in fact, were not purified further, potentially leading to overlapping metabolic signatures. However, by identifying distinctively altered metabolites, we found several lineage-related active metabolic pathways, some of which have also been identified previously . StemPro-34 SFM, IMDM and valproic acid were from Fisher Scientific . Cytokines were purchased from GeneScript . BML-210 and stemregenin 1 (SR-1) were from ApexBio Technology LCC . L-Glutamine was from Carl Roth GmbH + Co.KG . Etomoxir was from VWR International GmbH . Elfornithine (DFMO) and Human male AB serum was from Sigma-Aldrich Chemie GmbH . The 5-Fluoromethylornithine (5-FMO) was from Chemspace Europe . All antibodies were purchased from Beckton Dickinson GmbH .Human CB CD34All flow cytometry data were acquired on a CyFlow Cube 8 flow cytometer (Sysmex) and analyzed using FCS Express software (De Novo Software). Cells were washed twice with PBS and blocked with 10% human male AB serum for 15 min at 4 °C. The following are the antihuman antibodies used: CD34-allophycocyanin (APC), CD38-fluorescein isothiocyanate (FITC), CD45RA-APC, CD135-phycoerythin (PE), CD71-FITC, CD235a-APC, CD3-APC, CD94-FITC, CD41-APC, CD42a-FITC, CD90-FITC, CD66b-FITC, CD14-Pacific Blue (Pac Blue) and CD1a-APC, FITC-, PE-, APC- or Pac Blue-conjugated isotype-matched antibodies served as controls.+ cells were thawed according to the vendors protocol and expanded in Stempro-34 SFM media containing 2 mM L-glutamine, 100 ng/mL SCF, 100 ng/mL Flt-3L, 50 ng/mL IL-6, 40 ng/mL TPO, 1 µM SR-1, 0.1 µM BML-210 and 0.2 mM valproic acid for 13 days [v/v). CD34 13 days . Until d 13 days . Cells wTotal RNA isolation was performed using the NucleoSpin RNA XS Kit (Macherey-Nagel GmbH & Co. KG). Purification of poly-A containing mRNA molecules followed by mRNA fragmentation, random primed cDNA synthesis and single read 50 bp sequencing, as well as transcriptome alignment and determination of differential expression levels were done by Eurofins GATC Biotech GmbH  .HBB: fw: 5′-CTCGCTTTCTTGCTGTCCA-3′, rv: 5′-CAAGGCCCTTCATAATATCCCC-3′; ELANE: fw: 5′-CTGCGTGGCGAATGTAAACG-3′, rv: 5′-CGTTGAGCAAGTTTACGGGG-3′; GPNMB: fw: 5′-CTGATCTCCGTTGGCTGCTT-3′, rv: 5′-CTGACCACATTCCCAGGACT-3′; S100A8: fw: 5′-GATAAAGATGGGCGTGGCAG-3′, rv: 5′-TGCAGGTACATGTCCAGGG-3′; RPLP0: fw: 5′-TGGCAATCCCTGACGCACCG-3′; rv: 5′-TGCCCATCAGCACCACAGCC-3′.7 cells/ml in −20 °C cold ethanol containing 15% (v/v) 10 mM K2PO4, pH 7.5, sonicated for 3 min and frozen in liquid nitrogen for 30 seconds. The sonication-freeze cycle was repeated twice, afterwards, samples were centrifuged at 31,500 rcf at 2 °C for 5 min and the supernatant was stored at −80 °C until measurement. The metabolome analyses were carried out using the AbsoluteIDQ p180 Kit (Biocrates Life Science AG), according to the manufacturer’s instructions. Analysis of additional lipids, covering 162 glycerophospholipids, 33 sphingomyelins and 131 ceramides was done by Biocrates Life Sciences AG .Metabolite measurement was performed using a 4000 QTRAP mass spectrometer (ABI Sciex), connected to a NexteraXR HPLC (Shimadzu). Cell pellets were washed twice with ice-cold PBS and the pellets were resuspended at 1 × 102 transformed prior to statistical analysis, resulting “-Infinitive” values from metabolites below the LOD were removed from the data frame. Absolute log2 fold changes (logFC) above 1, compared to the progenitor population, with a corresponding FDR-adjusted p value below 0.05 where considered relevant. From these, unique metabolites were defined by a ∆logFC > 1 to both other lineages. As ∆logFC, we defined the absolute difference in the logFCs from each lineage compared to the progenitor population. Common metabolites of two lineages were defined as owning a ∆logFC < 1, whilst both owning a ∆logFC compared to third lineage > 1, from the relevant set of metabolites. Common metabolites of all lineages were defined as ∆ logFC < 1 between all lineages from the relevant set of metabolites. Gene Ontology enrichment analysis (Overrepresentation Test) was done by using the PANTHER Software (http://pantherdb.org), using the Fisher’s Exact test type in combination with Holm adjusted p value [p value smaller than 0.05 was considered as significant. For metabolic pathway visualization, using KEGG pathways as template, the software PathViso [Statistical analysis of metabolomics data using an empirical Bayes approach was done by using the statistical software R  and the  p value ,111. SigPathViso ,112 was RNA sequencing datasets generated are available at GEO under the accession number GSE129993. A list of identified significant altered metabolites can be found in the


This study aimed to evaluate the factors associated with death in patients with coronavirus disease 2019 by clarifying the clinical characteristics and immune responses.The clinical characteristics and laboratory findings, including cytokine and lymphocyte subsets, were obtained from the electronic medical records of patients in Wuhan Tongji Hospital.This study included 836 patients with confirmed COVID-19. In total, 699 (83.6%) were cured and discharged, and 137 (16.4%) died. Our analysis revealed that age ≥ 65 years, male sex, malignancy, chronic obstructive pulmonary disease, dyspnea, dizziness, respiratory rate > 20 bpm, heart rate > 100 bpm, systolic blood pressure < 90 mmHg, neutrophils > 6.3×109/L, lymphopenia, thrombocytopenia, D-dimer ≥ 0.5 mg/L, lactate dehydrogenase > 250 U/L, aspartate aminotransferase > 40 U/L, total bilirubin > 26 μmol/L, albumin < 35 g/L, blood urea nitrogen > 9.5 mmol/L, estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, N-terminal pro-brain natriuretic peptide ≥ 900 pg/ml, C-reactive protein ≥ 25 mg/L, procalcitonin ≥ 0.05 ng/ml and ferritin > 400 μg/L were associated with death in patients with COVID-19. The multivariate logistic regression analysis revealed that an estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, C-reactive protein ≥ 25 mg/L and procalcitonin ≥ 0.05 ng/ml were predictive of mortality. Regarding immune responses, IL-2R, IL-6, IL-8, IL-10, and TNFα were remarkably higher in the deceased group at admission, and the levels of IL-2R, IL-6, IL-8, IL-10, and TNFα in the deceased group showed a rapid increase; the dynamics of these cytokines were highly consistent with disease deterioration. Lymphocyte subset analysis revealed that the deceased patients showed significant decreases in lymphocyte counts, especially helper T cells, suppressor T cells and NK cells.This study identified that an estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, C-reactive protein ≥ 25 mg/L and procalcitonin ≥ 0.05 ng/ml were predictors of mortality in COVID-19 patients. Elevated cytokine levels and a continued increasing trend, including in IL-2R, IL-6, IL-8, IL-10 and TNFα, and a decrease in lymphocyte subsets, especially helper T cells, suppressor T cells and NK cells, were associated with a poor prognosis. A new type of coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was discovered to be the cause of unexplained pneumonia cases in Wuhan, China, in late December 2019 . On FebrThe World Health Organization has recently declared COVID-19 a public health emergency of international concern. Here, we performed a comprehensive exploration of the clinical features of 836 patients with confirmed COVID-19 who were transferred or admitted to the isolation ward of a Wuhan hospital. Furthermore, we evaluated 137 patients who died and 699 patients who recovered; our hope is that this study can aid clinicians in the early identification of patients with a poor prognosis.This retrospective study was conducted in Wuhan Tongji Hospital. This study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (Record number: QYFY WZLL 25751). All patients were diagnosed based on the Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment (7th edition) by the National Health Commission . When anThe epidemiological, clinical, laboratory, and radiological characteristics, treatment and outcome data were obtained from electronic medical records using data collection forms. The recorded information included demographic data, medical history, exposure history, underlying comorbidities, symptoms, signs, laboratory findings, chest computed tomographic (CT) scans, and treatment measures . The date of the disease onset was defined as the day when the first symptom was noticed.The descriptive statistics included a frequency analysis (percentages) of the categorical variables and median and interquartile range (IQR) analysis of the continuous variables. Regarding the laboratory results, we also assessed whether the measurements fell within the normal range. The comparisons were performed by Student’s t-test for the continuous variables as appropriate and χ  test forThe study population included 836 hospitalized patients with confirmed COVID-19. The median age was 64 years , and 439 (52.5%) patients were men Table 1.The deceased patients were significantly older  than the recovered patients. The male sex was more predominant among the deceased patients  than the recovered patients . The deceased patients were more likely to have underlying comorbidities, including chronic obstructive pulmonary disease (16 [11.7%] vs 32 [4.6%]) and malignancy (15 [10.9%] vs 22 [3.1%]). The proportion of deceased patients with hypertension, diabetes and coronary heart disease was higher, but there was no significant difference between the two groups. Compared with the recovered patients, the deceased patients were more likely to have reported dyspnea and dizziness. There were no marked differences in the median time from the onset of symptoms to hospital admission between the two groups. The median time from admission to death was 10 days, and the median time from admission to discharge was 23 days Table 1.The vital signs were measured at admission. Although the heart rates in the deceased patient group (100 beats per min (bpm)) and recovered patient group (90 bpm) were statistically significantly different, this difference was not clinically significant. Regarding the respiratory rates, both groups of patients were tachypneic, and the respiratory rates in the deceased patient group (24 bpm) were higher than those in the recovered patient group (20 bpm). The deceased patients developed tachycardia and tachypnea (respiratory rate ≥20 bpm) (49.2% and 70.4%) more often than the recovered patients (28.0% and 39.9% and 281 (33.3%)) Table 1.There were numerous abnormal results in the laboratory findings Table 2,We observed substantial differences in the laboratory findings between the patients who died and those who recovered within the available data Table 2.Of 761 patients with available chest CT, all patients had abnormal findings Table 2.In total, 733 patients (87.7%) received oxygen therapy. In total, 157 (19.0%) patients received non-invasive ventilation Table 3.In total, 699 enrolled patients (83.6%) were discharged, and 137 patients (16.4%) died. More deceased patients than recovered patients received systemic corticosteroid therapy (150 [75.4%] vs 345 [40.9%]) and intravenous immunoglobulin therapy (95 [47.7%] vs 171 [20.3%]). Significantly more deceased patients received non-invasive mechanical ventilation (114 [83.8%] vs 43 [6.2%]) and invasive mechanical ventilation (62 [45.6%] vs 2 [0.3%]) Table 3 The common complications observed in the deceased patients included type I respiratory failure (106 [77.4%]), heart failure (87 [63.5%]), hyperkalemia (55 [40.1%]), acute kidney injury (52 [38%]), disseminated intravascular coagulation (44 [32.1%]), arrhythmia (29 [19.0%]), pancreas injury (21 [15.3%]), shock (15 [10.9%]) and sepsis (12 [8.8%]) Table 4.Of all demographic data, clinical characteristics and laboratory findings 2, we evCOVID-19 patients have high levels of circulating interleukin 2 receptor (IL-2R), IL-6, IL-10 and tumor necrosis factor α (TNFα) Table 7.In our study, lymphopenia was common in the patients with COVID-19 (65.5%), indicating an impairment of the immune system during the course of infection. We further analyzed different subsets of lymphocytes. Decreases in the total number of T cells (CD3+CD19-), helper T cells (CD3+CD4+), suppressor T cells (CD3+CD8+) and NK cells (CD3−/CD16+CD56+) were also observed in the COVID-19 patients Table 8.In this retrospective study, we report 836 patients confirmed to have COVID-19 and provide the detailed clinical characteristics of this cohort of patients, including 137 fatal cases. We further comprehensively describe the major differences in the clinical features and immune responses between the deceased patients and those who recovered. We hope that this study could help clinicians identify patients with a poor prognosis early by increasing awareness of some characteristics indicative of a higher risk, realizing effective patient risk stratification and helping appropriately deploy health care resources.We found that older age, male sex, baseline diseases such as hematological and neoplastic disorders and COPD, the presence of dyspnea and dizziness, deterioration of vital signs, evidence of increased acute inflammation and end organ damage  at admission were associated with an increased risk of mortality due to COVID-19 infection, which is consistent with the results of previous clinical studies . The mulWe found a decreased estimated glomerular filtration rate in 48.2% of the COVID-19 patients, and this finding is strongly associated with mortality despite the significant correlation between chronic kidney disease and poor prognosis in our analysis. We found that 36.6% of the deceased patients developed acute kidney injury (AKI) during hospitalization. The etiology of AKI in COVID-19 cases has not been fully elucidated . The cloAlthough we did not find that coronary heart disease is associated with a poor prognosis, other reports have shown that cardiovascular disease may increase susceptibility to COVID-19 and its severity . HoweverCoinfection with SARS-CoV-2 and other viruses has been described in other studies, but the reported frequency is variable ranging from 0.02% to 2.6% . In our In addition, SARS-CoV-2 can infect host cells by interacting with membrane-bound angiotensin-converting enzyme 2 (ACE2) in cardiomyocytes . TreatmeMixed bacterial infection is an important contributor to mortality during the COVID-19 epidemic period . The mulMost patients with COVID-19 exhibit substantially elevated serum levels of pro-inflammatory cytokines, including IL-6, IL-2R, IL-8, IL-10 and TNFα, which is characterized as cytokine storms. High levels of pro-inflammatory cytokines may lead to shock and tissue damage to the lungs, heart, liver and kidneys, which can be confirmed by our data. These increases lead to massive infiltration of neutrophils and macrophages, diffuse alveolar damage with the formation of hyaline membranes and diffuse thickening of the alveolar wall .Lymphocyte subsets play an important role in cellular immune regulation . We founCOVID-19 is associated with both immunodeficiency and hyperinflammation, and T cell numbers are negatively correlated with serum IL-6, IL-10 and TNF-α. As the virus can directly infect T cells, T cell infection may result in cell death by apoptosis or necrosis . In addiStudies have shown that type-I interferon (IFN) is critical for preventing viral replication and triggers subsequent impairment and hyperactivation of the immune system, including T cell exhaustion and cytokine storm . TreatmeWe identified many candidate variables for risk stratification that may serve as clinical predictors of fatal COVID-19 and explored the immunodeficiency and hyperinflammation state during the course of COVID-19. Our research has some limitations, and some potential bias may arise. First, this study was a retrospective and single-center study, and some data were missing; a larger cohort study should be conducted to evaluate the immune response after COVID-19 infection. Second, the patients diagnosed with COVID-19, especially those with severe cases and bacterial coinfection, might affect the results of the immune response. Most patients presented increased procalcitonin, which was more evident in the severe cases. Despite these limitations, our study demonstrated several indicators suggesting that because of the dysregulated immune response in COVID-19 patients, SARS-CoV-2 might mainly act on T lymphocytes, inducing a cytokine storm in the body and generating inflammatory damage; thus, surveillance of lymphocyte subsets and serum levels of pro-inflammatory cytokines is helpful for the early screening of critical cases, risk stratification and management strategy adjustment.To evaluate the factors associated with death in COVID-19 patients, we collected clinical characteristics and laboratory findings, including cytokines and lymphocyte subsets, from 836 laboratory confirmed patients, including 137 deceased patients and 699 survivors. In conclusion, this study identified four predictors, i.e., an estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, C-reactive protein ≥ 25 mg/L and procalcitonin ≥ 0.05 ng/ml, of mortality among COVID-19 patients. We further found that elevated and a trend of a continued increase in cytokine levels, including IL-2R, IL-6, IL-8, IL-10 and TNFα, and decreased lymphocyte subsets, especially helper T cells, suppressor T cells and NK cells, were associated with a poor prognosis of COVID-19. Hence, paying attention to patients with renal insufficiency, myocardial injury, bacterial co-infection, impaired T-cell immune function, and dynamic increase in cytokines could help clinicians identify patients with a poor prognosis.The original contributions presented in the study are included in the article/The study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (Record number: QYFY WZLL 25751). Written informed consent from the participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.RY, CQ, and JZ collected the epidemiological and clinical data. NC and JZ summarized all the data. NC and RY drafted the manuscript. CQ and JZ revised the final manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Medicine and Health Technology Development Plan Project of Shandong Province (Grant NO. 2019WS377), Traditional Chinese Medicine Research Project of Qingdao City (Grant NO. 2020-zyy059), and the Affiliated Hospital of Qingdao University Youth Research Fund (Grant NO. 2019030).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Novel coronavirus 2019 (COVID-19) is a severe respiratory infection leading to acute respiratory distress syndrome (ARDS) accounting for thousands of cases and deaths across the world. Several alternatives in treatment options have been assessed and used in this patient population. However, when mechanical ventilation and prone positioning are unsuccessful, venovenous extracorporeal membrane oxygenation (VV-ECMO) may be used. We present a case of a 41-year-old female, with no significant medical history and no recent history of exposure to sick contacts, presented to the emergency department (ED) with fever, severe shortness of breath, and flu-like symptoms with a positive COVID-19 test. Ultimately, she worsened on mechanical ventilation and prone positioning and required VV-ECMO.The use of VV-ECMO in COVID-19 infected patients is still controversial. While some studies have shown a high mortality rate despite aggressive treatment, such as in our case, the lack of large sample size studies and treatment alternatives places healthcare providers against a wall without options in patients with severe refractory ARDS due to COVID-19. The novel coronavirus 2019 (COVID-19) is a respiratory tract infection that has resulted in a pandemic, infecting more than 1,250,000 humans and claiming the lives of over 75,000 in less than six months [3, hemoglobin of 11.6 g/dL, potassium of 2.8 mEq/L, aspartate aminotransferase (AST) of 85 IU/L, alanine aminotransferase (ALT) of 66 IU/L, lactate of 1.4 mmol/L, and procalcitonin 1.57 ng/mL. Polymerase chain reaction (PCR) tests for influenza A and B, metapneumovirus, adenovirus, parainfluenza, respiratory syncytial virus, and coronaviruses HKU1, NL63, 229E, and OC43 were all negative. Given the current pandemic, COVID-19 was suspected, and a nasal swab was sent to be tested. A computed tomography scan of the chest was obtained and showed bilateral infiltrates  with a worsening dry cough, shortness of breath, and chest tightness, followed by fever, chills, and myalgias for four days duration. Other reported symptoms included a mild sore throat and watery diarrhea. Vital signs on admission were a temperature of 104.2°Fahrenheit , a heart rate of 120 beats per minute, a blood pressure of 130/62 mm Hg, respiratory rate of 32 breaths per minute, and oxygen saturation of 89% on room air . Physical examination was pertinent for ill appearance and rhonchi over the left lung base. Blood tests showed a white blood cell count of 11.7 cells/mm3, lymphopenia of 700 cells/mm3, white blood cell count was 26.1 cells/mm3, fibrinogen level < 35, and D-dimer 116,193. She was started on Lovenox, 1 mg/kg, due to a severely elevated D-dimer; however, her platelet count decreased by greater than 50%, and she was switched to Argatroban. Heparin-induced thrombocytopenia (HIT) panel was sent and returned negative, but she remained on Argatroban for anticoagulation due to the significant drop in her platelet count on heparin products. She began to develop ischemia in her fingers and toes bilaterally but was continued on Levophed for hemodynamic stability and VV-ECMO. Four days after the initiation of VV-ECMO, the patient developed an asystole rhythm and ultimately passed away.The patient was started empirically on intravenous (IV) vancomycin, piperacillin-tazobactam, azithromycin, and hydroxychloroquine. Over a one-day period, the patient’s respiratory status progressively deteriorated, and she was subsequently intubated. On the following day, the COVID-19 test came back positive, and the patient was continued on 400 mg daily of hydroxychloroquine and 500 mg twice daily of azithromycin. She was also started on high-dose vitamin C at a rate of 6 grams IV twice daily, and 220 mg of zinc sulfate via orogastric tube once daily. Despite aggressive management, she developed severe acute respiratory distress syndrome (ARDS) and was requiring higher mechanical ventilation settings (100% fraction of inspired oxygen and 16 of positive end-expiratory pressure). The decision was also made to begin prone positioning of the patient for 18 hours a day for a ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio) of < 100. Liver enzymes continued to trend up (AST 274 and ALT 300), and the patient developed acute kidney injury due to decreased organ perfusion. She was started on Levophed for hemodynamic stability and to maintain a mean arterial pressure > 65. She was also given one dose (8 mg/kilogram body weight) of tocilizumab, an anti-interleukin-6 receptor monoclonal antibody, in order to help control her cytokine storm. Despite this, she continued to decompensate, and the patient was started on continuous venovenous hemodialysis (CVVHD) for renal failure and on venovenous extracorporeal membrane oxygenation (VV-ECMO). Prior to VV-ECMO, an echocardiogram was performed which showed an ejection fraction of 60% - 65%, moderate pulmonary hypertension, and grade 1 (mild) diastolic dysfunction. Two days after starting VV-ECMO, the patient lymphocyte count was 0 cells/mmExtracorporeal membrane oxygenation (ECMO) has remarkably progressed over recent years and became a reliable tool in severe cardiac and pulmonary dysfunction -4. Venov3 and she did not respond to VV-ECMO and passed away. While most studies lack a significant sample size, this case adds to the concern on the use of ECMO in COVID-19 patients. In patients with severe ARDS unresponsive to mechanical ventilation, prone positioning, and other alternatives, the need for further studies and understanding the role of ECMO in respiratory failure need to be assessed. The use of ECMO in COVID-19 patients is still controversial and has mixed results. Li et al. reported seven COVID-19 infected patients with P/F ratios < 100 on VV-ECMO and was able to successfully wean three patients thus far; however, they had a mortality rate of 50% . Yang etVV-ECMO use in patients with severe refractory ARDS due to COVID-19 infections is still controversial. While some studies have shown a high mortality rate despite aggressive treatment, such as in our case, they lack sufficient sample sizes. Due to limited alternatives and treatment options for patients with severe refractory ARDS, studies evaluating the use of ECMO in COVID-19 are desperately needed.


Actinidia melanandra has been characterized from Illumina pair-end sequencing. The complete cp genome was 156,124 bp in length, containing a large single-copy region (LSC) of 88,006 bp and a small single-copy region (SSC) of 20,332 bp, which were separated by a pair of inverted repeat (IR) regions of 23,893 bp. The genome contained 131 genes, including 84 protein-coding genes, 39 tRNA genes, and 8 ribosomal RNA genes (4 rRNA species). Most genes occur as a single copy, while 17 gene species are duplicated. Phylogenetic analysis revealed that A. melanandra is closely related to the species of A. deliciosa and A. chinensis.The whole chloroplast (cp) genome sequence of  Actinidia melanandra is a kind of dioecious climbing species belonging to the family Actinidiaceae. It is an endemic species to China and distributed in broad-leaved forests, mountain forests, or moist places of central and southern China   and Voucher herbarium specimens (ZA16015) were deposited at the Herbarium of Shangluo University. Genomic DNA was extracted from the fresh leaves using the CTAB method Doyle . Total DA. melanandra was 156,124 bp in size, and comprises a pair of inverted repeat (IR) regions of 23,893 bp each, a large single-copy (LSC) region of 88,006 bp, and a small single-copy (SSC) region of 20,332 bp. The chloroplast genome contained 131 genes including 84 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. In these genes, 16 genes contained one intron and two genes contained two introns. The majority of the gene species are single copy; however, 17 gene species in the IR regions are totally duplicated, including 5 protein-coding genes, 8 tRNA genes, and 4 rRNA genes. Out of these 17 gene species, rps12 are partially located within the IR regions, while all the others completely within the IR regions. The overall GC content of A. melanandra chloroplast genome is 37.2%.The circular chloroplast genome of Clematoclethra scandens subsp. hemsleyi and Sladenia celastrifolia were used as outgroup. As shown in the highly resolved NJ phylogenetic tree  method with 1000 bootstrap replicates based on 10 complete chloraplast genome sequences of Actinidiaceae, of which tic tree , all the


The soil bioavailability of phosphorus (P) is often low because of its poor solubility, strong sorption and slow diffusion in most soils; however, stress due to excess soil P can occur in greenhouse production systems subjected to high levels of P fertilizer. Silicon (Si) is a beneficial element that can alleviate multiple biotic and abiotic stresses. Although numerous studies have investigated the effects of Si on P nutrition, a comprehensive review has not been published. Accordingly, here we review: (1) the Si uptake, transport and accumulation in various plant species; (2) the roles of phosphate transporters in P acquisition, mobilization, re-utilization and homeostasis; (3) the beneficial role of Si in improving P nutrition under P deficiency; and (4) the regulatory function of Si in decreasing P uptake under excess P. The results of the reviewed studies suggest the important role of Si in mediating P imbalance in plants. We also present a schematic model to explain underlying mechanisms responsible for the beneficial impact of Si on plant adaption to P-imbalance stress. Finally, we highlight the importance of future investigations aimed at revealing the role of Si in regulating P imbalance in plants, both at deeper molecular and broader field levels. Silicon (Si) is the second most abundant element in the Earth’s crust, and its content within plants ranges from 0.1% to 10% depending on species. Although still not proven to be a plant-essential element, Si is widely recognized as a beneficial factor for plant growth and development. Si can alleviate biotic stresses, such as plant pathogens and insect pests, and abiotic stresses, such as drought, heat, cold, lodging, salinity, ultraviolet radiation, metal toxicity and nutrient imbalance ,4,5,6,7.Phosphorus (P) is a key element that greatly influences plant growth and productivity. Available P is very low in soils for two reasons: P fixation into organic forms, and binding or adsorption of P either by aluminum and iron (Fe) oxides or calcium minerals, depending on soil pH . Critica4SiO4), which is ultimately irreversibly precipitated as amorphous silica in cell walls and extracellular spaces by transpirational flow. The Si content of various plant species differs depending on their Si absorption capabilities and Si loading capacities from roots to xylem [All plants contain Si . In someto xylem .−10 m·s−1 [Oryza sativa), cucumber (Cucumis sativus) and tomato (Solanum lycopersicum) were found to be correlated with the abundance of Si transporters on the root plasma membrane, which was highest and lowest in rice and tomato, respectively [Although the cytoplasmic membrane of plant roots may provide a channel for the absorption of silicic acid by non-ionic diffusion, the permeability coefficient of silicic acid through the plasma membrane is only 1010 m·s−1 . This pr10 m·s−1 . Absorpt10 m·s−1 . Si upta10 m·s−1 ,30,31. POsLsi1 and OsLsi2) in rice was a landmark discovery that accelerated the development of the field of plant Si research [Lsi1, Lsi2, Lsi3 and Lsi6) have now been identified and functionally validated in numerous plant species, both monocots, such as rice, maize (Zea mays) and barley (Hordeum vulgare), and dicots, such as pumpkin (Cucurbita moschata), cucumber, tomato and horsetail (Equisetum arvense)   [32,33,3arvense) . The tisOne of the most essential mineral elements, P is not only a component of many important compounds in plants, such as nucleic acids, proteins and phospholipids, but also plays an important role in photosynthesis, respiration and many enzymatic reactions . P homeoArabidopsis thaliana), rice, wheat (Triticum aestivum), maize, barley, soybean (Glycine max), sorghum (Sorghum bicolor), tomato and potato (Solanum tuberosum) [ZAT6 can bind to various cis-elements, such as MYCS, P1BS and W-box elements, in the promoter region of plant PHT1 family genes [PHT1 gene expression. PHT1 family genes are also affected by arbuscular mycorrhiza [P often exists in soil in two chemical forms: organic and inorganic . Pi acquisition, utilization and homeostasis depend on complicated transport processes mediated by Pi transporters belonging to five phosphate transporter (PHT) families: PHT1 to PHT5 ,54,55,56berosum) . Most PHberosum) ,58,59,60ly genes ,63,64,65corrhiza ,67. Thiscorrhiza ,72,73,74corrhiza ,76,77,78+ symport or Pi/OH− antiport [PHT4 genes play important roles in various biological processes, including Pi translocation in plastids and the Golgi apparatus [PHT1 proteins, which generally function in the acquisition of Pi from soil, have received far more attention than other PHT family genes. PHT2, PHT3, PHT4 and PHT5 family members, which play vital roles in maintaining Pi distribution and homeostasis in plants, are localized in plastids ,80, mitoantiport ,85. The pparatus ,86, carbpparatus ,88, pathpparatus , and salpparatus . SYG1/PHpparatus ,91.The first evidence of Si-alleviated P deficiency was obtained from a long-term field experiment conducted at the Rothamsted Experimental Station. In that experiment, which compared barley yields in two fields not subjected to P fertilizer application, the yield of barley fertilized with Si was higher than that of a field without Si amendment . The benSi is commonly applied as calcium or sodium silicate, both of which increase soil pH. In acidic soils, aluminum (Al) toxicity is the most important factor limiting crop growth and production. At soil pH values at or below pH 5, Al ions are solubilized into the soil solution and dramatically inhibit root growth and function, in turn severely impairing water and nutrient acquisition by roots and, thereby, leading to a significant reduction in crop yields ,106,107.TaPHT1;1 and TaPHT1;2) in wheat roots at low P [Plants have evolved diverse strategies to cope with P deficiency. One effective strategy to counteract P-deficiency stress is the secretion of organic acid anions  by plant roots . Currentat low P . Despiteat low P . Si-decrat low P . Given tat low P , the int−1 in greenhouse systems  [Excess-P stress rarely occurs in natural soils. In greenhouse production systems, however, famers apply high levels of P fertilizer, which results in large amounts of P accumulated in the soil . Soil Old soils) ,113,114.d soils) . For exad soils) ,118,119.d soils) ,121. In d soils) . FurtherFragaria vesca) [In several plant species, Si alleviation of excess P damage has been attributed to a decrease in P uptake  that reda vesca) . Furthera vesca) .2 content of shoots exceeds 10% of the dry matter weight [One possible explanation for the aforementioned observations is that Si deposition in endodermal cells of plant roots may contribute to decreased P uptake and alleviation of excess-P stress. This effect has been attributed to the formation of apoplastic barriers to P permeability across roots caused by Si deposition in roots, which decreases excessive uptake of P ,123. Anor weight . A positr weight . Except OsPHT1;6 in roots [OsPHT1;6 expression was decreased by Si accumulation in shoots, resulting in decreased Pi uptake in rice [lsi1, defective in Si uptake. Compared with the wild type, Oochikara, lsi1 accumulated a similar level of Si in roots but exhibited much less accumulation in shoots [A number of studies have suggested that Si improves the growth of plants under abiotic stress by regulating the expressions of genes directly associated with the uptake and translocation of stress factors. We recently investigated the effect of Si on P uptake and accumulation under high P concentrations in both relatively long- and short-term experiments. We found that Si decreased both P uptake and accumulation in rice by downregulating the P transporter gene in roots . A split in rice . In thisn shoots . By takilsi1 [lsi1 [In cereal crops, P stored in seeds accounts for 60–85% of total plant P at maturity ,127,128.lsi1 . This rei1 [lsi1 . This fi3+ in acid soils, increasing the exudation of both malate and citrate, upregulating P transporter genes and enhancing internal P utilization by decreasing Fe and Mn uptake With the development of multi-omics methods, such as metabolomics, ionomics, transcriptomics and proteomics, the molecular mechanisms of Si mitigation of plant biotic and abiotic stresses can be explored in depth. These investigations should provide new insights and opportunities for dissecting the underlying mechanisms of Si mediation of P balance in plants under P-deficiency or excess-P stress.(2)TaPHT1;1, TaPHT1;2 and OsPHT1;6), but the associated detailed regulatory mechanisms and signaling pathways have not yet been fully determined.Recent studies have demonstrated experimentally the direct effect of Si on the expressions of P transporter genes ((3)lsi1 is a rice mutant defective in Si uptake. Although Si accumulation in the roots of lsi1 is similar to that of the wild type, the level of Si accumulated in shoots is much lower in the mutant. This characteristic can be applied to help researchers distinguish the different roles of Si accumulation in shoots and roots in Si alleviation of P-imbalance stress.Si-mediated tolerance mechanisms against P-deficiency or excess-P stress should continue to contribute to the improvement of P-imbalance stresses in diverse crop plants. Given currently published results, however, research on the improvement of plant P balance via Si has mainly focused on physiology and biochemistry, with deeper molecular regulatory mechanisms remaining to be fully revealed. Therefore, our recommendations regarding future research on Si alleviation of P-imbalance stresses and research prospects are as follows:The adaptive mechanisms of plant response to P-imbalance stress are complex. The regulatory effects of Si on P-deficiency or excess-P stress may constitute a complex system involving various biochemical and physiological processes as well. Consequently, systematic studies of Si-mediated alleviation of P-imbalance stress in plants with a focus on molecular and genetic levels should provide a theoretical foundation for practical applications of Si to agricultural production.


Adipose tissue, as the largest endocrine organ, secretes many biologically active molecules circulating in the bloodstream, collectively termed adipocytokines, which not only regulate the metabolism but also play a role in pathophysiological processes. C1q tumor necrosis factor (TNF)-related protein 3 (CTRP3) is a member of C1q tumor necrosis factor-related proteins (CTRPs), which is a paralog of adiponectin. CTRP3 has a wide range of effects on glucose/lipid metabolism, inflammation, and contributes to cardiovascular protection. In this review, we comprehensively discussed the latest research on CTRP3 in obesity, diabetes, metabolic syndrome, and cardiovascular diseases. Adipose tissue (AT) is widely known as an endocrine organ and a sensor/regulator of energy homeostasis . RecentlCTRPs are a family of adipocytokines. Due to the same modular organization, CTRPs members and adiponectin are classified into the C1q/TNF superfamily . CTRP3, CTRP3 was named as CORS26 (collagenous repeat-containing sequence of 26-kDa protein) when it was first discovered by Maeda T. et al. . Since tRecently, it has been reported that the possible receptors of CTRP3 are lysosomal-associated membrane protein 1 (LAMP-1) and lysosome membrane protein 2 (LIMP II), both of which are widely expressed in many different tissues . HoweverPreviously, the putative transcription binding sites of the promoter region of the CTRP3 gene were analyzed, including the locus of the following transcription factors: activator protein 1 (AP-1), POU domain, class 1, peroxisome proliferator-activated receptor (PPAR), transcription factor 1 (Pit-1a), specificity protein 1 (SP-1), Fos proto-oncogene (c-FOS), c-Jun N-terminal kinase (c-Jun), similar to myelocytomatosis viral oncogene (c-Myc), cAMP response element-binding protein (CREB), etc. However, only a few transcription factors regulate CTRP3 expression , b, 2004It is reported that many cell lines express CTRP3, such as mouse chondrogenic progenitor (N1511) , mouse pExcessive fat accumulation can lead to obesity, which is a pandemic social problem worldwide. Obesity can change the endocrine function of AT  and causIn vivo experiments have shown that diet-induced obesity (DIO) leads to decreased CTRP3 levels and increased TNF and IL-6 levels  and healthy subjects (n = 20), including decreasing the levels of MCP-1, CC-chemokine ligand (CCL)3/macrophage inflammatory protein (MIP)-1, CCL4, and macrophage migration inhibitory factor (MIF)  . Besidesor (MIF) . In vivoresponse , and furresponse . Additioresponse  and inflresponse . OverallIn addition to its role in inflammation, CTRP3 affects adipocytes differentiation and adipogenesis. Knocking down CTRP3 in adipocytes reduces the size of lipid droplets and triglyceride (TG) in cells, indicating that CTRP-3 deficiency appears to induce the dedifferentiation of mature adipocytes . ConsistSome studies investigate the association between obesity and circulating CTRP3 levels in patients. The level of CTRP3 in the tissue is about one-tenth of the level of circulating adiponectin, and the level of CTRP3 in obese patients is further reduced . Indeed,Based on the current data, the results of most studies are consistent. CTRP3 is reduced in the blood of obese patients, and it decreases the production of inflammatory factors, inhibits the activation of inflammatory signals, and reduces lipid formation. Overall, these findings suggest that CTRP3 is a potential target in the treatment of patients with MS or obesity.in vivo experiment indicates that an acute administration of rCTRP3 decreases serum glucose for up to 8-h, but not influences insulin levels in rodents. Intriguingly, the chronic transgenic overexpression of CTRP3 doesn’t influence glucose levels, demonstrating the existence of a potential compensatory mechanism , which has caused huge economic losses . Researcechanism . Transgeechanism .Although CTRP3 plays a beneficial role in the regulation of IR and serum glucose, the discrepancy for CTRP3 levels still exists in different studies. Several cross-sectional human studies show that the correlation between CTRP3 and some metabolic indicators appears to evolve dynamically from the period of pre-diabetes to the clinical diagnosis of diabetes. In nondiabetic Korean adults, Choi et al.  demonstr1.in vitro and in vivo (In vitro experiments show that CTRP3 doesn’t affect glucose uptake and AKT pathway in 3T3-L1 adipocytes and muscle cells (L6 myotubes)  . The met2.in vitro data show that pre-treatment with CTRP3 increases total oxygen consumption in H-4-II-E hepatoma cells under the condition of an excess of free fatty acids. In vivo data showed that the respiratory exchange rate of transgenic mice overexpressing CTRP3 was reduced compared with the control group. These data imply that CTRP3 enhances the hepatic fat oxidation, which plays a role in liver lipid metabolism, but the underlying mechanism is unclear. Moreover, we know that the expansion of AT causes insulin resistance , thereby reducing liver lipid content and improving hepatic insulin resistance induced by a high-fat diet . In the sistance . CTRP3 rsistance .3.in vitro and in vivo data have clarified that CTRP3 decreases the levels of IL-6, MCP-1, TNF, MIF, CCL3/MIP1, and CCL4 in either adipocytes or AT-infiltrated inflammatory cells through inhibiting the binding of LPS to TLR4. Conversely, CTRP-3 knockdown can reduce the release of anti-inflammatory factors and cause adipocytes to dedifferentiate, thereby having a stronger pro-inflammatory ability and lower insulin sensitivity  and negatively related to HOMA-IR , fasting plasma glucose (FPG), 1-h PG, 2-h PG, and fasting plasma C-peptide in the gestational diabetes mellitus patients (the specific cases are unknown) . However6.Lastly, Currently, the information shows that CTRP3 inhibits the synthesis of glycogen and triglycerides in the liver, increases lipid oxidation in the liver, reduces fat synthesis in adipose tissue, and inhibits systemic inflammation, thereby participating in the regulation of blood sugar homeostasis, reducing insulin resistance and prevention the development of diabetes complications.In vivo, the level of circulating CTRP3 decreased significantly one day after MI, reached the lowest point, and then slowly recovered. On the first day after MI, the level of CTRP3 mRNA in adipocytes decreased significantly, and then gradually recovered  . rCTRP3 promotes VSMCs proliferation in a dose-dependent manner by activating ERK1/2 and MAPK signaling. By contrast, rCTRP3 doesn’t alter VSMCs migration , correlative parietal cells (such as VSMCs), and other cell types (such as immune cells). According to the report, CTRP3 promotes cell proliferation and migration via activating ERK1/2 in mouse endothelial cells (MSS31 cells) . Peterso (HUVEC) . Interes (HUVEC) . Howeverigration . On the igration . Additioigration . CTRP3 rigration , cell prigration . In addiigration . Althougigration , it playigration .In vitro, CTRP3 directly induces hypertrophy in cardiomyocytes and the underlying mechanism is that CTRP3 exacerbates cardiac hypertrophy via the TGF β-activated kinase 1 (TAK1)-c-Jun N-terminal kinase (JNK) axis which is activated by PKA  . CTRP3 sd by PKA . Regardld by PKA  and ameld by PKA .Considering the above evidence, CTRP3 not only promotes angiogenesis and improves inappropriate cardiovascular remodeling after MI, but also ameliorates the function of damaged endothelial cells and cardiomyocytes. However, the role of CTRP3 in cardiovascular disease needs further studies to support it.Adipokines secreted by AT contribute to various biological processes. CTRP3 is a new type of adipokine that can affect obesity-related cardiovascular diseases , which pL-QY conceived and revised the manuscript. BG, TZ, FX, XL, S-KS, FW, J-YZ, FL, YW, M-HZ, and Q-SX analyzed the data. BG wrote the manuscript. TZ, Q-SX, and UE revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, this review will focus on the most recent aspects of autophagy and tumor immune environment. We describe the dual role of autophagy in modulating tumor immune responses and discuss some aspects that must be considered to improve cancer treatment.The tumor microenvironment (TME) is complex, and its composition and dynamics determine tumor fate. From tumor cells themselves, with their capacity for unlimited replication, migration, and invasion, to fibroblasts, endothelial cells, and immune cells, which can have pro and/or anti-tumor potential, interaction among these elements determines tumor progression. The understanding of molecular pathways involved in immune escape has permitted the development of cancer immunotherapies. Targeting molecules or biological processes that inhibit antitumor immune responses has allowed a significant improvement in cancer patient’s prognosis. Autophagy is a cellular process required to eliminate dysfunctional proteins and organelles, maintaining cellular homeostasis. Usually a process associated with protection against cancer, autophagy associated to cancer cells has been reported in response to hypoxia, nutrient deficiency, and oxidative stress, conditions frequently observed in the TME. Recent studies have shown a paradoxical association between autophagy and tumor immune responses. Tumor cell autophagy increases the expression of inhibitory molecules, such as PD-1 and CTLA-4, which block antitumor cytotoxic responses. Moreover, it can also directly affect antitumor immune responses by, for example, degrading NK cell-derived granzyme B and protecting tumor cells. Interestingly, the activation of autophagy on dendritic cells has the opposite effects, enhancing antigen presentation, triggering CD8 TME is composed of stromal cells , extracellular matrix (ECM), and immune cells, such as natural killer (NK) cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and T and B lymphocytes. During cancer progression, tumor cells display genetic and phenotypic diversity, changing cellular metabolism and, consequently, the TME , 6.Generally, TME displays low levels of oxygen and nutrients, and high production of reactive oxygen species (ROS), crucial factors for autophagy activation. Autophagy is a natural cellular survival process, usually activated to maintain cellular homeostasis , 8. DespIn this review, we will present the major mechanisms by which the immune system interferes in the TME, and how autophagy can influence it. Then we will focus on the modifications of cancer immune responses in TME influenced by autophagy and how it can affect cancer therapy.Besides the TME elements already mentioned, soluble molecules as cytokines, metabolites, and inflammation mediators also contribute to the interaction among the cellular elements. These biochemical signals orchestrate cell death, proliferation, survival, and other cells recruitment. Leukocyte activity is essential in cancer progression  1 and Th17 lymphocytes are considered cytotoxic responses. These cells can control tumor growth through either directly killing tumor cells, as NK and CD8+ T cells, or indirectly, in the case of CD4+ T cells, which secrete cytokines capable of activating other effector leukocytes , which recognize human leukocyte antigen (HLA) class I molecules and CD94/NKG2A, which specifically binds to the non-classical HLA-E molecule. The last one causes NK inhibition to ensure that normal cells cannot be lysed. However, transformed cells that downregulate the HLA-I surface molecules are not able to inhibit NK cells. The stimulatory receptors bind to stress-inducible molecules in the target cell surface, as sialic acid, Fcγ, adhesion molecules, and others, to trigger cytotoxic activity. Displaying a different strategy for target recognition, CD8+ T lymphocytes activation depends on TCR (T cell receptor) binding to specific antigens presented by classical HLA-I molecules in target cells  activation, through CD40L signaling, and cytokine secretion as IL-2, or activate other effector cells, such as macrophages, neutrophils, NK cells through IFN-γ and tumor necrosis factor-alpha (TNF-α). Moreover, TNF-α, through its receptor, can trigger cell death, and IFN-γ and cytokines secreted by Th17 cells, through activation of stromal cells, can stimulate ROS production and neutrophils, enhancing the cytotoxic effects on cancer cells , well-characterized suppressors of effector T lymphocytes function. Moreover, it is well known that cancer cells display reduction in antigen presentation potential, decreasing tumor cell recognition by CD8 T lymphocytes. One classic example, from a virus associated cancer is the HPV E7 oncoprotein, which binds to interferon regulatory factor 1 (IRF1) in the IFN type I (IFN-I) signaling pathway, and recruits histone deacetylase (HDAC) to the promoter sequences responsive to IRF1, repressing genes that otherwise would be transcribed in response to the virus . IFN-I aOncogenes also drive the reprogramming of tumor cell metabolism, the so-called Warburg effect. Tumor cells display different metabolic strategies to maintain energy production and catabolism at a rate to allow continuous cell proliferation. Some cells use glycolysis almost exclusively, while others also required amino acids and fatty acids as well, and keep the Krebs cycle and oxidative phosphorylation active. In either case, tumor cells usually increase the glucose uptake and secrete lactate in higher concentrations than other cells in the body . Both thvia CD80 or CD86, to cytotoxic T lymphocyte-associated protein 4 (CTLA-4) expressed by Treg. The signal triggered by this interaction promotes indoleamine-2,3-deoxygenase (IDO) expression in DCs. This enzyme, which physiological function is the protection of immune-privileged tissues, catabolizes the reaction that converts the essential amino acid tryptophan in kynurenine, which through the aryl hydrocarbon receptor, promotes regulatory phenotype in T lymphocytes  and cancer cells  is also an inhibitory molecule expressed upon IFN-er cells . The rec T cells . PD-1 cohe tumor . These cg others . The PD-g others .As mentioned before, CTLA-4 binds to the co-stimulatory molecules CD80 and CD86. It competes with the activating receptor CD28, which also binds to these molecules, but with lower affinity. CD28 signaling is essential for T lymphocyte activation since it triggers the PI3K/Akt pathway and causes stabilization of the antigen activation signal. Not only CTLA-4 competes for biding to co-stimulatory molecules, but also, through a process called trans-endocytosis, this biding removes CD80 and CD86 from the APC surface, eliminating the possibility of CD28 activation, and consequently preventing T cell activity .Many of these mechanisms can happen simultaneously in the TME, in a dynamic process that varies through time. To add to this complex situation, other factors should also be considered. Some tumors are very immunogenic, and tumor antigen-specific T lymphocytes can be found in the TME, where it can also be observed evasion mechanisms and chronic antigen signaling that can, eventually, inhibit anti-tumor responses. When immune responses persist, it results in chronic inflammation leading to cancer progression. Other tumors are less immunogenic and recruit mainly myeloid cells, which display a tolerogenic phenotype, helping cancer cells meet their metabolic demands, and promoting angiogenesis. M2 macrophages, for example, display arginase activity, causing conversion of arginine to ornithine, which is a substrate to the synthesis of polyamines, necessary for catabolism and cell proliferation .Autophagy is a survival cellular process in which organelles and other cytoplasmic components are directed to the lysosomes for degradation , 36. ThiMacroautophagy is the main type of autophagy, therefore it will be referred to just as autophagy. In response to hypoxia and oxidative stress, hypoxia-inducible factors (HIF) 1 and 2 bind to hypoxia response elements (HREs), leading to the transcription of several genes that are involved in angiogenesis, metastasis, cell survival, immune escape, and autophagy pathways. Activation of HIF-1 subunit-1 leads to an increase in BCL2 interacting protein 3 (BNIP3) and BCL2 interacting protein 3 like (BNIP3L) expression levels. These factors are responsible for breaking the connection between Beclin1 (BECN1) and B-cell lymphoma 2 (Bcl-2), an inhibitory complex that prevents autophagy . AnotherBeclin1, on MCF7 human breast carcinoma cells, contributed to tumorigenesis in nude mice, indicating that autophagy could inhibit tumor growth  context by DCs   was a competent tumor antigen carrier, which stimulated antigen cross-presentation mediated by DCs to CD8+ T cells and stimulated CD8+ T cell proliferation in an autophagy-dependent fashion. Overall, these findings demonstrated a new mechanism of immune activation by α-TEA, which stimulated tumor cell autophagy and antigen cross-presentation to CD8+ T cells  and melanoma . Autophagy initiates when TIM-4 binds to AMPK-α1. This activation promotes the degradation of TAA through the lysosomal pathway, which led to a decrease in antigen presentation and, consequently, in specific anti-tumor CD8oduction . Moreovemulation . Togetheγ can induce autophagy in gastric cells, thus inhibiting carcinogenesis  displayed protection against gastric dysplasia in comparison to controls. The authors observed that gastric cells displayed lower proliferation rates and T cell apoptosis dependent on IFN-γ expression. Furthermore, transgenes resulted in increased levels of LC3-II and Beclin-1 mRNA and protein, in the stomach, indicating autophagy activation. Additionally, transgenic animals showed higher apoptotic T cells, concurrently with inhibition of IL-6, IL-1β; and TNF-α production, and presented less chronic inflammation  in myeloid cells increased the co-stimulatory molecule CD86 and reduced CD206, a mannose receptor associated with M2-like phenotype. In this model, the reduction on M2-like TAMs improved CD8+ and CD4+ T cell IFN-γ production, increased IFN-I, and IL-1β, although no quantitative alteration in the frequency of TILs was observed , in TME was demonstrated using Cre-lox based ablation of several genes in myeloid cells of immunocompetent mice (C57BL/6). The ablation of  pathway . In the observed , suggestcreAtg5fl/fl mice, in which myeloid cells lacked Atg5 expression, there were less Treg TILs, tumor cell proliferation rate reduction, and a significant increase in both MHC-II expression and IFN-γ production  and clinically healthy controls’ peripheral blood. MDSCs from melanoma patients, and also from mice with melanoma, showed higher levels of functional autophagy. In LysModuction . Again, γ), expression of T cell exhaustion markers, and quantitative changes in TILs.TME is a hostile environment where autophagy can lead to degradation of cytotoxic molecules . Engraftment of PC from Lamp2a knockout mice (PC-KO) into GB grown in mice led to central-memory  CD4+ and CD8+ T TILs and better CD4+/CD8+ ratio of 2:1 than in tumors engrafted with wild type (WT) PC. Moreover, only 2% of TILs expressed PD-1 and CTLA-4 and there was a lower GB cell proliferation rate than in GB/PC-WT control mice. Compared to GB/PC-KO, GB/PC-WT mice displayed increased IL-2 levels, tumor proliferation, FoxP3+PD-1+CTLA-4+ TILs, higher Tgfβ, and Il10 mRNA expression levels, and a CD4+/CD8+ ratio of 4:1. Collectively, these results demonstrated that CMA activity in PC favored an immunosuppressive environment in response to GB cells  cells induced GB cells .+ T cell exhaustion phenotype, and also resistance to targeted therapy and autophagy inhibitors. PD-L1+ A375 melanoma cells showed resistance to BRAF inhibitor Vemurafenib and CQ, but not to nitrobenzoxadiazole (NBD). Both CQ and NBD inhibit autophagy, however, NBD acts at multiple levels, targeting not only the late stages of autophagy but also different apoptotic pathways  released lower amounts of ATP in response to MTX chemotherapy. The lower ATP release resulted in DCs recruitment impairment and consequent lack of T cell priming to elicit an anti-tumor immune response  or competent tumors were observed in the B16 melanoma mouse model, even after Doxorubicin treatment  treated with ICD inducer chemotherapy agents did not recruit effector cells and it possibly contributed to chemotherapy (CT) resistance  and had no effect on apoptosis and proliferation rates. However, the treatment with a combination of enzalutamide and autophagy inhibitors  impaired tumor growth, indicating that the combined treatment may be a potential strategy to avoid enzalutamide-resistant bladder cancer (FOXM1). Thus, the knockout of ATG7, Beclin-1, or treatment with CQ restored the antitumor effect of docetaxel, demonstrating that either autophagy or FOXM1 may be potential targets for combined therapies with docetaxel to treat metastatic prostate cancer patients  improved overall survival of metastatic pancreatic cancer patients treated with gemcitabine hydrochloride and nab-paclitaxel was performed . Autophar cancer . A similpatients . On the patients , 83. OxaBecn1± or Atg4b-/-) mice, although the MTX toxicity was higher on Atg4b-/- mice   .Atg5 or Beclin1 depletion with specific single guide(sg)RNA, generated larger tumors with reduced CD4+ and CD8+ TILs and IFNγ+ T cells in Balb/c mice, when compared to autophagy competent 4T1 cells. Moreover, the antitumor effect of the anti-PD1 antibody was limited in autophagy-deficient tumors, while a significant reduction in tumor volume and increased cytotoxic activity of TILs was observed in the control group  for the scholarship provided to LG (141955/2020-1) and fellowship to AL (307841/2018-9), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship provided to AS (88882.382114/2019-01). This work was partially funded by LIGH-FUNPAR Alliance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.