The prokaryotic filamentous marine cyanobacteria are photosynthetic microbes that are found in diverse marine habitats, ranging from epiphytic to endolithic communities. Their successful colonization in nature is largely attributed to genetic diversity as well as the production of ecologically important natural products. These cyanobacterial natural products are also a source of potential drug leads for the development of therapeutic agents used in the treatment of diseases, such as cancer, parasitic infections and inflammation. Major sources of these biomedically important natural compounds are found predominately from marine cyanobacterial orders Oscillatoriales, Nostocales, Chroococcales and Synechococcales. Moreover, technological advances in genomic and metabolomics approaches, such as mass spectrometry and NMR spectroscopy, revealed that marine cyanobacteria are a treasure trove of structurally unique natural products. The high potency of a number of natural products are due to their specific interference with validated drug targets, such as proteasomes, proteases, histone deacetylases, microtubules, actin filaments and membrane receptors/channels. In this review, the chemistry and biology of selected potent cyanobacterial compounds as well as their synthetic analogues are presented based on their molecular targets. These molecules are discussed to reflect current research trends in drug discovery from marine cyanobacterial natural products. Lyngbya, Moorea, Symploca and Oscillatoria . Du. Du2) (22)  [30,31,33) and bipyridine analogue, 4, provided potent anticancer and HDAC inhibitory activities (5) or mercaptosulfide , have altered selectivity profile, such as conferring selectivity for HDAC6 or HDAC10  (8)  , was obt7) (8) , the natpound, 9 , was rep9 was found to be 30 times less potent compared to santacruzamate A when tested against HDAC2. In another SAR study by Randino et al., two classes of santacruzamate derivatives having several aromatic amines containing either the ethyl carbamate unit (class I) or oxamic acid moiety (class II) as zinc-binding groups were synthesized [10, 11 and 12  and global HDAC inhibitor, vorinostat (SAHA), to treat hepatocellular carcinoma (HCC) was reported . The com13) and B (14)  and the marine-derived salinosporamide A (16), with IC50 values of 2.7 nM and 1.4 nM, respectively (Carmaphycins A (d B (14)  are pote Curacao . Structut method . The proectively . In addi12), a known proteasome inhibitor, as well as the FDA-approved anticancer drug, carfilzomib. Their irreversible inhibition of proteasome is due to the formation of the morpholino adduct between the epoxyketone warheads with the Thr1 residues in the catalytic sites of the 20S proteasome core. The mechanism involves warhead carbonyl and epoxide undergoing two successive nucleophilic attacks by Thr1 Oγ and Thr1 N, respectively , 17)  over the human proteasome [S. mansoni with 18 resulted in the same phenotypic changes as the FDA-approved drugs, bortezomib and carfilzomib.A number of synthetic analogues of carmaphycin B have been explored as lead molecules for the treatment of protozoan and metazoan parasitic infection as well as cancer. One such synthetic compound is analog 18 (17) , which wactivity . In anotnidazole . Trichomsubunits . Compounoteasome . Treatme19  (21) [22) was reported to inhibit the migration of the highly invasive MDA-MB-231 breast cancer cells [23) and tiglicamides, have also been reported to possess moderate to potent serine protease inhibitory activities (A majority of these serine protease inhibitors are 3-amino-6-hydroxypiperidone (Ahp)-containing cyclodepsipeptides, including lyngbyastatins 4–10  , pompano20) (21) ,76,77,78tivities  [79,80,824 (25)  . This mo20) was carried out in 31 steps by Luo and co-workers [20 demonstrated strong ability in protecting bronchial epithelial cells against elastase-induced antiproliferation as well as negating the elastase-triggered induction of pro-inflammatory cytokine expression [7 was recently developed with improved reaction yields and simplified purification steps [29) and synthetic analogues [30) with enhanced potency  (31)  is one orain 3D7 ,88,89. Hrain 3D7 . Gallina, Panama . At the ida Keys .Plasmodium falciparum-infected red blood cells treated with gallinamide A, at nanomolar concentration, exhibited swollen food vacuole phenotype. Through the use of fluorescent probes based on rhodamine fluorophore-tagged molecules, it was revealed that the natural product is a specific inhibitor of the plasmodial cysteine proteases, falcipains 2, 2′ and 3. In addition, the methoxypyrrolinone unit in gallinamide A is essential for antimalarial activity and modifications to the N-terminal end of the molecule are tolerated [The potent antimalarial activity of gallinamide A (31) led to investigations by Stolze and co-workers on its mode of action and SAR studies based on synthetic and biochemical methods . It was olerated .Plasmodium falciparum cysteine proteases involved in several key processes of the erythrocytic cycle of the malarial parasite, such as the hydrolysis of host hemoglobin, erythrocyte invasion, and rupture [Falcipains are  rupture . These c rupture . The sim rupture .32 and 33  possessed potent inhibitory activity against the P. falciparum falcipain 2 and falcipain 3 as well as cultured chloroquine-sensitive (3D7) and chloroquine-resistant (W2) strains of P. falciparum , tasiamides, and symplocin A, were found to be potent inhibitors of aspartyl proteases, cathepsins D and E (39) were isolated from Lyngbya confervoides and VPG 14-61 collected at Grassy Key, Florida and Cetti Bay, Guam, respectively [39) and B displayed potent inhibitory activity against cathepsins D and E with IC50 values averaging at 16.9 nM and 0.62 nM, respectively. In addition, grassystatin A was able to reduce antigen presentation by dendritic cells [A series of statine-containing linear depsipeptides, including grassystatins  and F (40)  and BACE1 (295-fold) [Tasiamides are grassystatin-related molecules found to possess potent cathepsins D and E inhibitory activity. In particular, tasiamides B (d F (40)  displayeectively . When te D and E . A numbe2 and 43 , have beer BACE1 . In anotACE1 [43  displaye95-fold) . These rSymploca sp. [N,N-dimethylamino and 2-hydroxy acids were employed for absolute stereochemistry determination of this molecule. Symplocin A is a potent inhibitor of cathepsin E with IC50 value of 300 pM, which is comparable to that of pepstatin, a known inhibitor of aspartyl proteases. Taken together, the biological data of these series of statine-containing compounds, including grassystatins, tasiamides, and symplocin A, showed that selectivity can be tuned and these structural scaffolds can serve as a starting point for development of selective aspartic protease inhibitors.Symplocin A (44)  is anothloca sp. . Symploc41)   is a sta 0.19 µM ,109. BAC 0.19 µM ,111. The 0.19 µM . Several5 and 46  showed pectively . Moreovef rodent . Recent f rodent .47) and 15   anmicrobes . Dolastamicrobes . This dibes [48)  in 2011 Lyngbya sp. strains collected from various locations. They possess significant biological activities in the nanomolar range when tested against a panel of cancer cell lines, including HT29, HeLa, and U2OS. To date, a total of nine apratoxin-related compounds have been reported with apratoxin A (49)  and apratoxins S7 (51) to S9 (53), with improved antitumor activity, inhibitory secretion of the angiogenic vascular endothelial growth factor –A (VEGF-A) and tolerability in human HCT116 xenograft mouse model at subnanomolar IC50 concentrations have been reported(49) and E (55), giving rise to the apratoxin A/E hybrid structure, differing in various degrees of methylation at C34 as well as epimeric configuration at C30. From the biological data, it was revealed that the configuration of C-34 is independent of the cytotoxic activity   being th pathway . Due to  reported , revealed the molecule to possess potent antiangiogenic activity by inhibiting the activation of retinal endothelial cells and pericytes through mediating multiple angiogenic pathways [54) (Further studies on apratoxin S4 (pathways . This fiays [54) , was sho49) revealed its ability to interfere with specific cellular signaling pathways as well as protein interactions involved with the formation and maintenance of cancer cells. Using functional genomics approach, apratoxin A was found to exert its antiproliferative property through induction of G1 cell cycle arrest and apoptosis by antagonism of the fibroblast growth factor (FGF) signaling via Signal Transducer and Activator of Transcription 3 (STAT3) [A series of mechanistic studies on apratoxin A ( (STAT3) . In anot (STAT3) . The inh (STAT3) .The specific mode of action of apratoxin A was eventually elucidated in two studies conducted by Paatero et al. and Huang et al. ,132. AprLeptolyngbya sp. [57) was deduced by various 2D NMR spectroscopic experiments, including COSY, TOCSY, multiplicity-edited HSQS, HSQC-TOCSY, HMBC, H2BC, 1H-15N gHMBC, and ROESY as well as mass spectroscopic data. This molecule possesses a high degree of N-methylation with eight out of 11 residues being N-methylated. More importantly, coibamide A displayed potent cytotoxicity against NCI-H460 lung cancer cells and mouse neuro-2a cells, with LC50s reported to be less than 23 nM. The compound was evaluated in the NCI’s panel of 60 cancer cell lines and it exhibited significant activities against MDA-MB-231, LOX IMVI, HL-60(TB), and SNB-75 at IC50 values of 2.8 nM, 7.4 nM, 7.4 nM and 7.6 nM, respectively [58 and 59 (Coibamide A (57)  is a strgbya sp. . The comectively . Total sectively ,136,137.8 and 59 , showed 60)–C (62) -3-hydroxy-γ-butyrolactone and 4-chlorocrotonic acid, connected via an ester linkage. These molecules showed inhibition of bioluminescence in Vibrio harveyi BB120 and lipopolysaccharide-stimulated nitric oxide production in the murine macrophage cell line RAW264.7 [Honaucins A –C (62)  are poten Hawaii . The strRAW264.7 . AdditioRAW264.7 . SynthetTwo synthetic analogues, 63 and 64 , were foThis review covers a range of unique marine cyanobacterial natural products as well as their synthetic analogues having potent biological activities, including anticancer and antiinfective properties. A majority of these compounds are nitrogen-containing and products of the modular PKS-NRPS metabolic pathways. Due to their exquisite interference against clinically relevant drug targets, such as proteasomes, proteases and microtubule filaments, they represent important lead compounds for further development into therapeutic drugs.A number of noteworthy drug leads include largazole, apratoxin A, carmaphycins, gallinamide A and the dolastatin class of molecules. Drug candidates, such as the largazole-based synthetic analogue, OKI-179, and apratoxin S4 have been identified from synthetic analogues of these natural drug leads and are currently undergoing or being considered for clinical testing. In addition, a high number of auristatin-based compounds, formulated as ADCs, are undergoing clinical testing as anticancer agents. Unique structural features attributed to marine cyanobacterial compounds could account for their high potency. These include high degree of N-methylation , macrocycles with incorporation of polyketide moiety , presence of heterocycles, such as thiazole, thiazoline, oxazole oxazoline and methoxypyrrolinone , incorporation of D- and non-proteinogenic amino acids  as well as peptide acylation . These structural features contribute to compound permeability and adoption of desired conformation for interaction of cellular drug targets. Moreover, combination of these structural motifs expands the chemical space of marine cyanobacterial natural products.65) and aurilide-class of compounds , antiinfective molecules, almiramides  and janadolide (68), anti-inflammatory molecule, biseokeaniamide A (69) as well as cannabimimetics/CNS modulatory agents, such as mooreamide A and serinolamides    [145,146


Araucaria araucana, plus soil and associated insects, collected along the entirety of its geographic distribution in Chile (January 2017 and 2018).Here, we report the results from PCR and sequencing of bacterial 16S rRNA and fungal internal transcribed spacer 1 (ITS1) genes from needle, branch, trunk, and root samples of  Araucaria araucana, plus soil and associated insects, collected along the entirety of its geographic distribution in Chile (January 2017 and 2018).Here, we report the results from PCR and sequencing of bacterial 16S rRNA and fungal internal transcribed spacer 1 (ITS1) genes from needle, branch, trunk, and root samples of  Araucaria araucana  is an endangered conifer with a fragmented and relict distribution in Chile and Argentina. A. araucana has been historically threatened by logging, wildfires, overgrazing, and extensive human harvesting of its seeds, which has pushed the species to the International Union for Conservation of Nature (IUCN) red list as an endangered species  amplicon sequencing of samples from needle, branch, trunk, root, and soil compartments and associated insects of A. araucana at 10 locations along most of its geographic range , 70% ethanol, 1% sodium hypochlorite, and sterile water to remove epiphytic microbes. Then, plant material was ground manually before being flash frozen for 1 min (liquid nitrogen). After one cycle of tissue disruption in a TissueLyser II for 1 min, samples were frozen in liquid nitrogen again to repeat the disruption step. Fifty milligrams of disrupted plant material was used for DNA extraction using the DNeasy PowerPlant pro (Qiagen) extraction kit. Soil extractions were carried out using the PowerSoil DNA isolation kit (MoBio Laboratories). DNA was quantified by fluorimetry in a Qubit 3.0 instrument (Thermo Fisher Scientific) using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit and stored at −80°C. For sequencing, we targeted the V4 region of the 16S rRNA gene and the ITS1 gene for taxonomic profiling in an Illumina MiSeq instrument  68. SampleProteobacteria was the dominant phylum in all compartments, followed by Actinobacteria in needles, branches, and roots , and Acidobacteria (20.8%) and Verrucomicrobia (14.5%) in soil. Firmicutes and Actinobacteria comprised 10.4% and 10.3% relative abundance, respectively, in trunk samples. For the fungal samples, all compartments were dominated (>50% relative abundance) by Ascomycota, followed by Basidiomycota, with 28.7%, 20.5%, and 15.3% in needles, branches, and roots, respectively. Soil samples were characterized by high levels of Mortierellomycota (19.5%) and Mucoromycota (3.1%) compared with other compartments.There were 16,224,911 reads from 16S rRNA and 14,040,959 reads for ITS1 from 675 and 650 samples, respectively . PRJNA517193.Sequences from this data set are available through NCBI under the accession number


Macrophages, a kind of innate immune cells, derive from monocytes in circulation and play a crucial role in the innate and adaptive immunity. Under the stimulation of the signals from local microenvironment, macrophages generally tend to differentiate into two main functional phenotypes depending on their high plasticity and heterogeneity, namely, classically activated macrophage (M1) and alternatively activated macrophage (M2). This phenomenon is often called macrophage polarization. In pathological conditions, chronic persistent inflammation could induce an aberrant response of macrophage and cause a shift in their phenotypes. Moreover, this shift would result in the alteration of macrophage polarization in some vascular dermatoses; e.g., an increase in proinflammatory M1 emerges from Behcet's disease (BD), psoriasis, and systemic lupus erythematosus (SLE), whereas an enhancement in anti-inflammatory M2 appears in infantile hemangioma (IH). Individual polarized phenotypes and their complicated cytokine networks may crucially mediate in the pathological processes of some vascular diseases (vascular dermatosis in particular) by activation of T cell subsets , deterioration of oxidative stress damage, and induction of angiogenesis, but the specific mechanism remains ambiguous. Therefore, in this review, we discuss the possible role of macrophage polarization in the pathological processes of vascular skin diseases. In addition, it is proposed that regulation of macrophage polarization may become a potential strategy for controlling these disorders. As aMacrophage polarization serves as a crucial role in physiology and disease status, such as embryonic development, individual growth, homeostasis maintenance, immunity defence, inflammation, and trauma. At the stage of embryonic development, fetal macrophages display an M2 phenotype and energetically promote angiogenesis, tissue growth, and organ formation, angiogenesis in particular . With thAlthough the specific mechanism of macrophage polarization in vascular disorders keeps obscure, it is considered that T cell dysregulation, oxidative stress damage, and angiogenesis are probably involved in macrophage polarization-mediated vascular diseases, especially in some vascular skin complaints, e.g., BD, psoriasis, SLE, and IH  [In vascular inflammation site, macrophages and T cells always coexist and interact with each other , 38. Thed IL-26) , 50. Cond IL-26) –53. Worsd IL-26) –56. Justd IL-26) . Besidesd IL-26) , 58, 59.α production promotes M1 macrophage activation, and ROS-stimulated MAPK/NF-κB signals mediate proinflammatory genes to reprogram macrophages towards M1, thereby contributing to OS exacerbation and vascular endothelial cells/tissue injury [ROS and NO, the crucial mediators of vascular inflammation, are mostly derived from macrophages and potently involved in various vascular inflammatory diseases such as BD, SLE, atherosclerosis, and cancer –63. Durie injury –76.in vitro showed that angiogenic factor expression on M2 surpassed those expressions on M1 [in vivo and in vitro, mainly involving the PDGF and FGF signal pathways. Besides, macrophage polarization is closely associated with angiogenesis in tumor growth. Circulating monocytes are recruited into the tumor stroma and then differentiate into tumor-associated macrophages (TAMs). These TAMs release a serial of growth factors  for endothelial cell proliferation and microvessel formation. Most notably, the phenotype of TAMs is greatly similar to M2; once the phenotype deviates from M2, tumor growth would be suppressed [α and IL-1β [As one key member of the angiogenic-promoting cells, M2 dominates in wound healing, oncogenic angiogenesis, and blindness-related aberrant angiogenesis –79. M2 mns on M1 ; M2 instppressed . Therefond IL-1β . M2-domind IL-1β .Growing evidence supports that unbalanced macrophage polarization occurs in some vascular skin diseases, such as BD, psoriasis, SLE, and IH. As a group of typical vascular inflammatory dermatoses, BD, psoriasis, and SLE collectively exhibit same pathological mechanisms involving M1-mediated immune inflammation, T cell dysregulation, and OS damage. Meanwhile, they own the similar microenvironment which provides macrophage polarization with inflammatory cytokines that induce M1 polarization. In addition, M1-produced factors remarkably ascend in these diseases and positively correlate with disease activity. On the other hand, IH is an angiogenesis-related disease, in which polarized M2 and its cytokines substantially appear. Persistent activation of single polarized macrophages with their inflammatory mediators may closely implicate in the occurrence and development of those dermatoses; therefore, we propose that regulation of macrophage polarization direction would be a novel approach to treating these disorders.α, INF-γ, IL-1β, IL-6) obviously emerged from the serum of active BD patients [in vitro that the serum of BD patients could induce M1 macrophages [α, IL-1b, IL-6, IL-8, and IL-12, are closely associated with the disease activity [BD, a chronic recurrent multisystemic vascular inflammatory disease, is clinically characterized by oral aphthosis, genital ulcers, skin lesions, uveitis, and organ involvements , 91. Patpatients , which frophages ; meanwhirophages . Besidesactivity , 94. Up activity , 95, 96.activity , 97. Theactivity , 99; infactivity . The curα. As a factor primarily derived from M1, TNF-α is considered as the master proinflammatory cytokine and is deemed to be a key candidate gene for the pathogenesis of psoriasis [α, other M1-related proinflammatory cytokines  or chemokines  positively appear in psoriatic serum and facilitate inflammatory cell recruitment [γ, IL-17, and IL-23 [Psoriasis is a chronic immune-mediated inflammatory disorder, histologically featuring as abnormal proliferation/differentiation of keratinocytes, excessive angiogenesis, and inflammatory cell infiltration in the dermis . It has soriasis . Apart fruitment . Althougnd IL-23 . The actnd IL-23 . On the nd IL-23 . Apart fα, GM-CSF, and IFN-γ, significantly elevated in the circulation of lupus patients, which formed a microenvironment to facilitate macrophage polarization towards M1 [α, IL-1, IL-6, CXCL10, etc., obviously increase in SLE and positively stimulate M1 polarization [SLE, a multisystem autoimmune disease based on B cell immunity, involves the skin, connective tissue, and blood vessels . It exhiwards M1 . More imwards M1 , 115; fowards M1 , 117, prwards M1 ; costimuwards M1 , 120. Inrization . On the rization . Althougrization . T cell rization , 124. Ovrization –127; insrization . OS not rization , 130 butrization . Anyhow,rization , 132.in vitro through injecting HemSCs into the subcutaneous of mice, by which the differentiation potential of HemSCs was observed in a polarized M2 macrophage environment [As one of the most common benign tumors in infancy, IH is characterized by abnormal vascular endothelial cell proliferation and excessive angiogenesis; its pathogenesis involves genetic, environmental, and other factors . This anironment . This fiironment . AccordiConsidering the crucial role of dominant M1 or M2 in these dermatoses, various strategies are being explored to regulate the direction of their differentiation or block the transduction of downstream molecules, though most of them are still being in the pilot phase of cell or animal models. Here, we review the latest researches in the treatment of these vascular skin diseases by targeting macrophages, and more vehicles should be developed for modulating macrophage polarization in the future.β, IL-6, and TNF-α produced from M1 in BD patients [β, TNF-α, and IL-6) in murine psoriatic skin and serum [α inhibitor could decrease the ratio of M1 to M2 macrophage in patients with psoriasis [α and IL-1β, produced from M1, would be a potential excellent option to IH through inducing endothelial-to-mesenchymal transition and accelerating hemangioma regression [in vitro; it was also shown that normal murine monocytes preferentially differentiated into M2 rather than M1 after weekly PAM3 treatment in the lupus-prone NZB/W mice (a mouse model of SLE whose symptoms and gender bias are similar to human) [in vitro and in human psoriasis plaques has demonstrated that metallic polyphenol-enriched nanoparticles could not only reduce M1 but also suppress the production of proinflammatory cytokines, especially M1-derived TNF-α and IL-12; this new biological material, meanwhile, was found to powerfully repress NF-κB  activation in macrophages in vitro [Currently, many natural plant extracts or Chinese herbal ingredients have been widely studied on controlling macrophage polarization-mediated dermatosis owing to their multiple powerful activities, like anti-inflammation, immunoregulation, antioxidation, antiangiogenesis, and antitumor. It was reported that curcumin, a natural polyphenolic pigment derived from turmeric, could inhibit M1 polarization and decrease the level of IL-1patients ; what wapatients . Anotherpatients . Similarnd serum . In addind serum . Althougsoriasis , while tsoriasis . In the gression . Moreoveo human) . Thus, iin vitro .κB), the level of M1 would decrease. Generally, polarized M1 can produce a lot of inflammatory mediators in serum . These inflammatory mediators from M1 are closely implicated in the occurrence and development of above dermatoses, so the exogenous intervention such as an addition of factor receptor antagonists may be a promising alternative. But in M2-dominant skin disorders, IH as the representative, the signal pathways involved in M2 polarization and the pathogenic molecules released from M2 are critical therapeutic targets for these diseases. Additional antagonists to hemangioma tissue contribute to the reversal of polarization state of M2 in IH via inhibition of M2 polarization-related signaling pathways  and downregulation of M2-related angiogenic factors .According to the different phenotype macrophages and their respective roles in the dermatosis, some critical targets are proposed and summarized for the treatment of skin diseases via regulating macrophage polarization shown in . In the In conclusion, the effect of macrophage in different vascular dermatosis is variable owing to their M1/M2 phenotypes. Here, we present the evidence of macrophage polarization in M1/M2-mediated vascular skin diseases and discuss the possible pathological role of individual polarization types in these dermatoses. Typically in BD, psoriasis, and SLE, there is an enhancement in the ratio of M1/M2 and M1 plays a leading role in the continuous development and vicious cycle of inflammatory reactions, whereas in IH, the ratio of M1/M2 drops and the effect of M2 on angiogenesis are amplified. Targeting macrophage polarization to shift macrophages to M1 or M2 would be a novel strategy for control of vascular dermatosis; however, the mechanism of macrophage polarization in these diseases is not yet fully clarified and further studies in this field will be required. Besides, more therapeutic vehicles need to be developed and applied in clinical practice in the future.


Severe acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown.We evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis. We also assessed the presence of association with inflammatory biomarkers and the 28-day mortality.Lymphocytopenia was present in 51 of 63 (80.9%) patients, with a median value of 720 lymphocytes/µl . This reduction was mirrored also on CD8+ , natural killer  and natural killer T  cells. Monocytes were preserved in total number but displayed among them a subpopulation with a higher forward and side scatter properties, composed mainly of cells with a reduced expression of both CD14 and HLA-DR. Patients who died in the 28 days from admission , when compared to those who did not, displayed lower mean values of CD3+  and CD4+ cells  and an higher percentage of CD8+/CD38+/HLA-DR+ lymphocytes .The early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2-like lymphocytes and monocytes with altered immune profile, which include atypical mononuclear cells. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new beta coronavirus identified in China in December 2019 which is responsible of coronavirus disease 2019 (COVID-19) , a concomitant decrease in interferon gamma (IFN-γ) production and an association with elevated inflammatory markers –5. An exA better knowledge of the immune response against the virus is mandatory, since it has been postulated that severe forms of the disease are associated with a cytokine storm, where monocytes/macrophages play a central role –12. As aIn attempt to provide our contribution to the field, we designed the present study describing PBMC’s characteristics and basic inflammatory markers values in a cohort of Italian patients who were diagnosed of SARS-CoV-2 infection and were hospitalized.We enrolled patients consecutively admitted to the Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, a University Hospital in Milano, Italy, diagnosed with COVID-19 in the period from March 17 to April 3, 2020. Diagnosis of COVID-19 was defined by the presence of compatible signs/symptoms  and a positive nasopharyngeal swab for SARS-CoV-2 detected through real-time reverse transcription-polymerase chain reaction (rRT-PCR). Serum levels of inflammatory biomarkers were assessed as standard clinical practice. Flow cytometry analysis was performed on fresh blood left-over of samples collected at hospital admission for clinical purposes. We analyzed only blood samples collected in the first 48 h since diagnosis. Samples of fresh blood left-over from local blood donor bank were also employed as healthy controls. The study was approved by the Institutional Review Board Milano Area 2 (#358_2020) and was conducted in accordance with the Helsinki Declaration.Procalcitonin (PCT), ferritin and interleukin-6 (IL-6) were measured with electro-chemiluminescent immunoassays (ECLIA) on a Roche Cobas e801 instrument . C-reactive protein (CRP) was measured with an immunoturbidimetric method and lactate dehydrogenase (LDH) with the International Federation of Clinical Chemistry (IFCC) optimized method on a Roche Cobas c702 instrument .2)/fraction of inspired oxygen (FiO2)], was calculated at the admission to the hospital. The 28-day mortality was collected from electronic medical records.For each patient, the P/F ratio [partial pressure of oxygen , RNA extraction with STARMag Universal Cartridge kit on Nimbus instrument  and amplification with Allplex® 2019-nCoV assay, while the second employed a GeneFinder® COVID-19 Plus RealAmp Kit  on ELITech InGenius® instrument . Both assays identify the virus by multiplex rRT-PCR targeting three viral genes .Fresh peripheral blood samples were processed within 24 h from collection for the following evaluations: i) classical lymphocyte subpopulations count, ii) CD4+ T-cells polarization –16, iii)p value <0.05 was deemed statistically significant. All the analysis was performed with GraphPad Prism 8 .Descriptive statistics were performed for all the variables assessed in the study population. The Spearman test and linear regression were used to examine correlations. The Friedmann test followed by Dunn’s correction for multiple comparisons and multiple t test followed by Holm-Sidak correction for multiple comparisons were employed to assess differences among groups. Parametric tests were employed for continuous variables, whereas non-parametric test for those not-normally distributed (Kolmogorov-Smirnov test). A Sixty-three patients with confirmed SARS-CoV-2 infection were enrolled, on average 5.3 days (SD 2.9) after symptoms onset. The median P/F ratio at admission was 170 mmHg. Hypoxemia was severe (P/F < 100 mmHg) in 13 patients (20.6%), moderate (P/F 100–200 mmHg) in 13 (20.6%) and mild (P/F 200–300 mmHg) in 23 (36.5%), while the remaining 14 patients had a P/F > 300 mmHg. Overall, the 28-day mortality rate was 15.9% (10/63). Demographic and clinical characteristics and inflammatory biomarker values are shown in At diagnosis of SARS-CoV-2 infection, median values of white blood cell (WBC), monocyte and neutrophil were within the reference intervals employed in our Centre. Instead, median lymphocyte count was below the lower reference limit. Overall, lymphocytopenia was found in 51 of the 63 patients (80.9%). Consequently, the NLR was oriented toward high values, well above the reference intervals of 0.78–3.53 Table 2 brightCD16- (immature) and CD56dimCD16+ (mature) NK cells among total lymphocytes between patients and healthy donors (CTR) , intermediate (CD14+CD16+), and classical monocytes (CD14+CD16-), respectively. Reference intervals for the above monocyte subpopulations were 7.7% (IQR 5–10), 5.3% (IQR 4–7), and 82.6% (IQR 80–84) for non-classical, intermediate and classical monocytes, respectively. Finally, the median percentage of HLA-DR+ monocytes was 83.4% (IQR 75.2–92.3), below our internal control reference value of 96% (IQR 94–98).The median frequencies of monocyte subpopulations are displayed in dim cell population with high FSC and SSC . Regarding HLA-DR, we found a significant reduction on COVID-19 patients’ monocytes with normal scatter properties (p<0.05). For a better characterization of these atypical cells, we compared their CD14 and HLA-DR expression with normal monocytes and granulocytes of the same patient. As shown in Moreover, to better understand the differences in the phenotypic signature between normal and high-SC monocytes in COVID-19 patients and in healthy donors (n=10), we compared the Median Fluorescence Intensity (MFI), a parameter proportional to antigen density, of CD14, CD16, and HLA-DR in these monocyte populations. Overall, the MFI of CD14 and HLA-DR on high-SC monocytes was lower than those of healthy donors .  were below the lower reference limits. Moreover, activated CD8+ T cells were increased, whereas CD4+ lymphocytes were polarized toward a Th2-like phenotype. Intriguingly, monocytes, although not modified in cell number and distribution, displayed morphological and phenotypical alterations. IL-6 levels were not correlated to monocyte counts. Finally, patients who died within 28 days from admission presented lower counts of CD3+ and CD4+ cells and a higher percentage of activated CTLs.Our results are in line with those recently reported by several groups regarding a profound reduction of lymphocytes in the context of a preserved total WBC count , 10, 18.Regarding monocytes, Zhang and Zhou reported in their studies , 23 a reMonocytes, considered the crucial players in the pathogenesis of lung damage caused by the cytokines storm, showed substantial phenotypical alterations already in the early phase of the disease. The mechanism leading to monocytes alteration is unclear, but a direct viral role can be suspected. Indeed, Zhang et al  showed hConcerning lymphocyte compartment, Zhou and colleagues observed that CD4+ T lymphocytes were rapidly activated to become pathogenic Th1 cells, secreting proinflammatory cytokines. The resulting environment induced inflammatory CD14+CD16+ monocytes with high expression of IL-6 . In contFinally, we observed some peculiar results stratifying the variables according to 28-day mortality. Indeed, in addition to the clinical variables which are expected to be linked with the disease outcome , we observed lymphocytopenia and CTLs activation status more evident in those patients who died. It is hard with the current knowledges to understand the significance of these data. Both lymphocytopenia and lymphocyte activation could be the physiologic consequence of an ongoing viral infection, particularly evident in those with a severe outcome. However, it could be interesting to assess whether lymphocytes of these patients are able to control activated innate immunity and modulate inflammatory response. Indeed, in mice, Kim et al. demonstrated that after a viral infection, an unleashed innate immune response, due to the absence of residential T cells, can also be a direct cause of death  and helper lymphocytes. These changes, which are more pronounced in patients with adverse outcome, could represent the first steps in the cascade leading to the uncontrolled activation of monocytes in the context of cytokines storm. It is possible to speculate that an early initiation of an immunomodulatory treatment might abrogate the immune system alterations leading to monocytes activation-dysfunction and to the consequent lung damage. Nevertheless, further prospective studies are needed to provide a precise picture of the immunologic profile during the course of the disease in order to define better therapeutic approaches and to improve the clinical management of COVID-19 patients.Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico. Scientific Direction: Silvano Bosari, Luigia Scudeller, Giuliana Fusetti, Laura Rusconi, Silvia Dell’Orto; Department of Transfusion Medicine and Hematology (Biobank): Daniele Prati, Luca Valenti, Silvia Giovannelli; Infectious Diseases Unit: Andrea Gori, Alessandra Bandera, Antonio Muscatello, Davide Mangioni, Laura Alagna, Giorgio Bozzi, Andrea Lombardi, Riccardo Ungaro, Teresa Itri, Valentina Ferroni, Valeria Pastore, Roberta Massafra, Ilaria Rondolini; Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa: Flora Peyvandi, Roberta Gualtierotti, Barbara Ferrari, Raffaella Rossio, Elisabetta Corona, Nicolò Rampi, Costanza Massimo; UOC Internal Medicine, Immunology and Allergology: Nicola Montano, Barbara Vigone, Chiara Bellocchi, Elisa Fiorelli, Valerie Melli, Eleonora Tobaldini; Respiratory Unit and Cystic Fibrosis Adult Center: Francesco Blasi, Stefano Aliberti, Maura Spotti, Edoardo Simonetta, Leonardo Terranova, Francesco Amati, Carmen Miele, Sofia Misuraca, Alice D’Adda, Silvia Della Fiore, Marta Di Pasquale, Marco Mantero Martina Contarini, Margherita Ori, Letizia Morlacchi, Valeria Rossetti, Andrea Gramegna, Maria Pappalettera, Mirta Cavallini, Annalisa Vigni; Cardiology Unit: Marco Vicenzi, Irena Rota. Emergency Unit: Giorgio Costantino, Monica Solbiati, Ludovico Furlan, Marta Mancarella, Giulia Colombo, Giorgio Colombo, Alice Fanin; Acute Internal Medicine: Valter Monzani, Angelo Rovellini, Laura Barbetta, Filippo Billi, Christian Folli; Rare Diseases Center: Marina Baldini, Irena Motta, Natalia Scaramellini; General Medicine and Metabolic Diseases: Anna Ludovica Fracanzani, Rosa Lombardi, Federica Iuculano; Geriatric Unit: Matteo Cesari, Marco Proietti, Laura Calcaterra. Istituto di Ricerche Farmacologiche Mario Negri IRCCS: Alessandro Nobili, Mauro Tettamanti, Igor Monti.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comitato Etico Milano Area 2, Fondazione IRCCS Ca’ Granda, Francesco Sforza 28, 20122 Milano. The patients/participants provided their written informed consent to participate in this study.AL, LP, AB, and AG conceived the study. VC, EP, GL, GC, VS, GG, RG, and MM enrolled the patients. LP, AC, ET, MT, and FC performed the immunologic and biochemical analysis. AL, LP, and AB analyzed the data. AL and LP wrote the first draft. All authors contributed to the article and approved the submitted version.The grant “COVID-19 Biobank” was provided by the Scientific Direction, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy.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.


MicroRNAs play vital roles in the development of endometriosis. It is reported that miR-200b-3p is downregulated in endometriosis, although its mechanisms in this disease remain still unclear. Therefore, the purpose of this study was to explore the function and potential regulatory network of miR-200b-3p in endometriosis through database analysis.  The endometriosis gene expression profiles were downloaded from the GEO database to screen differentially expressed genes (DEGs). The predicted and validated target genes of miR-200b-3p were obtained from miRWalk and miRTarBase database. Then, a comparison was performed between miR-200b-3p target genes and DEGs. GO enrichment and KEGG pathway analysis of the target genes was performed using clusterProfiler package. STRING was used to predict the protein-protein interaction among the proteins encoded by the target genes. Then, TransmiR, LncBase, StarBase, PROMO, and AnimalTFDB were employed to identify interactive transcription factors and lncRNAs of miR-200b-3p.  miR-200b-3p was associated with the transcription factors DNMT1, EZH2, HNF1B, JUN, MYB, ZEB1, and ZEB2 during the pathogenesis of endometriosis. The downstream 110 target genes were involved in the biological processes of positive regulation of MAPK cascade, muscle cell proliferation, organ growth, vasculogenesis, and axon development. KEGG analysis revealed that the main pathways related to miR-200b-3p were microRNAs in cancer, PI3K-Akt signaling pathway, colorectal cancer, and tight junction. In addition, four lncRNAs such as MALAT1, NEAT1, SNHG22, and XIST interacted with miR-200b-3p and were associated with transcription factors FOXP3 and YY1.  The predicted target genes and molecular regulatory network of miR-200b-3p in endometriosis not only revealed its biological function but also provided a valuable guideline for further research. Endometriosis is a benign gynaecological disease characterized by endometrial glands and stroma located outside the uterine cavity. It affects 10–15% of women in their reproductive age and results in a significantly affected quality of life . EndometMicroRNAs (miRNAs) are small, single-strand noncoding RNA molecules containing ~22 nucleotides, which regulate the expression of target genes and control a variety of cellular functions, including apoptosis, proliferation, migration, and invasion . FurtherTherefore, in this study, an integrated analysis of gene expression profile datasets from GEO database was performed to screen differentially expressed genes (DEGs) in endometriosis. Then, the target genes of miR-200b-3p and the network it regulates were investigated using bioinformatics tools. Altogether, our results showed that miR-200b-3p might play critical roles in the pathology of endometriosis.http://www.ncbi.nlm.nih.gov/geo/). GSE7305 microarray data contained 10 endometriosis tissues and 10 normal endometrial tissues [p value (FDR) <0.05 and fold change >1.5. DEGs that appeared in both sets of data were selected for subsequent analysis.Two endometriosis microarray data sets were downloaded from the GEO website  [The miRWalk 2.0 tool  , which irwalk2/)  and combThe clusterProfiler package  was usedhttps://string-db.org/) [Target genes were submitted into STRING (db.org/)  to analydb.org/) .TransmiR v2.0 is a database for TF-miRNA regulations, which covers 623 TFs and 785 miRNAs and contains 3730 literature-curated TF-miRNA regulations . TherefoLncBase Experimental v2 is an online software for experimental validation and computational derivation of the relationship between miRNA and lncRNA, which was used to predict lncRNAs associated with miR-200b-3p . The StaPROMO is a program that predicts TF binding sites in related sequences from a species through the TRANSFAC database . AnimalTOn the basis of the genomic information that was collected, the potential regulatory network of miR-200b-3p in endometriosis was constructed.As regards DEG analysis, GSE7305 contained 4223 upregulated probes and 3830 downregulated probes in endometriosis, while GSE7307 contained 2360 upregulated probes and 2200 downregulated probes. After comparison, 2105 common upregulated probes corresponding to 1264 genes and 1867 common downregulated probes corresponding to 1269 genes were obtained.A total number of 482 predicted miR-200b-3p target genes that appeared in the four prediction databases were obtained. A total of 186 validated target genes associated with miR-200b-3p were obtained from the mirTarBase. The two sets were taken together, and 634 candidate target genes of miR-200b-3p were obtained and compared with the DEGs obtained from the microarray data analysis. Finally, 110 target genes of miR-200b-3p in endometriosis, including 56 upregulated genes and 54 downregulated genes, were obtained .GO analysis showed that the target genes were mainly enriched in biological processes such as positive regulation of MAPK cascade, muscle cell proliferation, organ growth, vasculogenesis, and axon development . In addiSTRING was used to extract the proteins expressed by the target genes that can interact with others. A total of 63 target genes were filtered into the PPI network, containing 63 nodes and 155 edges . The NetA total of 75 TFs associated with miR-200b-3p were obtained from the TransmiR v2.0 database. Then, DNMT1, EZH2, HNF1B, JUN, MYB, ZEB1, and ZEB2 were found as TFs related to endometriosis by interacting with target genes of miR-200b-3p.The lncRNAs associated with miR-200b-3p were predicted by LncBase Experimental v2 and StarBase databases, and the lncRNAs overlapping in both the databases were selected, such as MALAT1, NEAT1, SNHG22, and XIST. Thus, the mutual regulation of these four lncRNAs and miR-200b-3p might play an important role in the development of endometriosis.PROMO and AnimalTFDB were used to predict the TFs that could be associated with four lncRNAs  in the upstream 2 kb promoter sequence. The predicted intersections resulted in two TFs, such as FOXP3 and YY1.From the above prediction results, a miR-200b-3p network regulating endometriosis was constructed .Although endometriosis is a benign inflammatory disease, some of its characteristics are comparable to the ones of malignant tumours, such as abnormal cell migration, implantation, invasion, and angiogenesis. Several studies indicated that miR-200b-3p is decreased in endometriosis, but its specific pathogenesis in this disease is not yet clear. In this study, the miR-200b-3p network that regulates endometriosis was constructed by integrating microarray data sets and the target pair information from several interactome databases. The four relationships considered in this network were the following: (1) the regulatory pair of miR-200b-3p and target genes, (2) the regulatory pair of miR-200b-3p and TF genes, (3) the regulatory pair of miR-200b-3p and lncRNA genes, and (4) the regulatory pair of lncRNA genes and TF genes. A total of 110 target genes, 7 TF genes, and 4 lncRNAs associated with miR-200b-3p were identified in endometriosis. The functions of the target genes were analysed, and the enriched GO terms were positive regulation of MAPK cascade, muscle cell proliferation, organ growth, vasculogenesis, and axon development. These results suggested that the identified miR-200b-3p target genes in endometriosis and the associated regulators (TFs and lncRNAs) might play an important role in the induction of endometriosis.By integrating the information from gene expression profiles, the target genes of miRNA can be found more accurately; thus, the genes regulated by miR-200b-3p in endometriosis can be also discovered. A total of 110 target genes of miR-200b-3p were screened, and many of them play important roles in the development of endometriosis. They included FOXP1, GATA6, JUN, and EGFR involved in muscle cell proliferation –28; NTRKmiRNAs not only regulate the development of several diseases by interacting with protein-coding genes but also promote or inhibit cell proliferation, apoptosis, and metastasis by interacting with lncRNAs. The lncRNAs associated with miR-200b-3p were predicted by LncBase and StarBase databases and identified 4 overlapping lncRNAs . Therefore, these four lncRNAs might have a mutual regulation with miR-200b-3p. Liang et al. found that the expression of MALAT1 is significantly upregulated in ectopic endometrial tissue and is negatively correlated with the expression of miR-200c , suggest+FOXP3+Treg cells is significantly increased in the peritoneal fluid of women with endometriosis [The common predicted TFs of the four lncRNAs were FOXP3 and YY1. FOXP3 is essential in the development and function of regulatory T cells. Ectopic Foxp3 expression can phenotypically convert effector T cells to regulatory T cells . A recenetriosis . Furtheretriosis . YY1 is etriosis . YY1 binetriosis , and losetriosis . Furtheretriosis . In addietriosis . Han et etriosis .In conclusion, based on the results of various bioinformatics methods, our hypothesis is that miR-200b-3p is associated with TFs such as DNMT1, EZH2, HNF1B, JUN, MYB, ZEB1, and ZEB2 in the occurrence and development of endometriosis, regulating downstream target genes, and it is also regulated by four lncRNAs. Furthermore, these four lncRNAs interact with the TFs FOXP3 and YY1. Although the prediction of the molecular regulatory network of miR-200b-3p by bioinformatics needs further experiments to be confirmed, these predictions may represent a valuable guideline in the mechanism triggering endometriosis.


Myl9, a gene encoding the SM myosin regulatory light chain (SM RLC). We find that global Myl9-deficient bladders contracted with an apparent sustained phase, despite no initial phase. This sustained contraction was mediated by NM myosin RLC (NM RLC) phosphorylation by myosin light chain kinase (MLCK). NM myosin II was expressed abundantly in the uterus and young mice bladders, of which the force was accordingly sensitive to NM myosin inhibition. Our findings reveal distinct roles of SM RLC and NM RLC in SM contraction.Both smooth muscle (SM) and non-muscle (NM) myosin II are expressed in hollow organs such as the bladder and uterus, but their respective roles in contraction and corresponding physiological functions remain to be determined. In this report, we assessed their roles by analyzing mice deficient of  Smooth muscle (SM) cells line the walls of hollow organs, including the gastrointestinal tract, vasculature, uterus and bladder, and they maintain homeostasis and responses to imposed signals . SM contMyl12a and Myl12b encode NM RLCs, Myl9 encodes SM RLC . This observation suggests the existence of SM RLC-independent contraction in the bladder. A substantial analysis demonstrated that the sustained force was mediated by NM RLC through phosphorylation by MLCK, and this NM RLC-mediated contraction was necessary for tethering fluid tension in some hollow organs, such as the bladder and uterus. As NM RLC did not functionally or biochemically compensate for SM RLC, our results revealed independent and distinct roles of SM RLC and NM RLC in SM contraction as well as hollow organ functions.Myosin II is one of the most important superfamilies of motor proteins, consisting of six subunits, including two MHCs, two RLCs, and two essential light chains ELCs; . SM exprs SM RLC , Myh9 an kinases . The pho kinases . General kinases . There a kinases ; pharmac kinases ; the res kinases ; and NM  kinases . HoweverMyl9 flox mice were generated by the bacterial artificial chromosome (BAC) retrieval method and ES cell targeting as previously reported  and the AceQ qPCR SYBR Green Master Mix kit (Vazyme) following the manufacturer’s instructions. Primers used were as follows: Myl9, forward, 5'-CTGAACCGTAAGACCCACAG-3'; reverse, 5'-GGGGATGCTGGGAATTAGTG-3'; Myl12a, forward, 5'-GGGCCCTAGACTTTGTCTTT-3', reverse, 5'-AACACAGCGAAATCTGTCCTT-3'; Myl12b, forward, 5'-CTGCGTGCCACTTAGTTTCT-3', reverse, 5'-ACAACATTGTCCCCCGTTC-3'; Myl6, forward, 5'-GACCGTGGCCAAGAACAA-3', reverse, 5'-CAGCCATTCAGCACCATCCG-3'; Myh9, forward, 5'-CCGGCAAGGTGGACTATAAA-3', reverse, 5'-GTCTTGAAGGCACCAGGTAG-3'; Myl10, forward, 5'-GGGAAGAAACGCCATGAGAT-3', reverse, 5'-CGTTCAAGTTCCCCAGGAAT-3'; Myl11, forward, 5'-ACAGAGAGCAATGAGGCCAT-3', reverse, 5'-CCGAGCGTCCATTTCTTCTT-3'; and Actb, forward, 5'-CTTTGCAGCTCCTTCGTTGC-3', reverse, 5'-CCTTCTGACCCATTCCCACC-3'.Total RNA was extracted from fresh isolated tissues using Trizol reagent  following the manufacturer’s instructions. cDNA was reverse-transcribed using the HiScript Q RT SuperMix for qPCR (+gDNA wiper) kit  following the manufacturer’s instructions. PCR was performed to determine the expression level of Measurement of the isometric contraction of intestinal SM, bladder SM, uterine SM, and aortic SM was performed as described . The for2 at 37°C. The resting tension was set to 0.1–0.3 g, and 87 mM KCl and 100 μM acetylcholine  were used to induce SM contraction.Jejunum SM contraction: A freshly isolated 6 mm jejunum ring was mounted in a longitudinal orientation in an organ incubation chamber  containing H-T buffer  with bubbling O2 at 37°C. The resting tension and corresponding diameter were determined by measuring the circumference-pressure curve. Then, 124 mM KCl and 10 μM U46619  were applied to induce aortic SM contraction.Aortic SM contraction: A 2 mm aortic ring was mounted in a myograph chamber  containing H-T buffer with bubbling O4, 2.5 mM CaCl2, 20 mM NaHCO3, 1.18 mM KH2PO4, and 11 mM glucose). For P28 mice, the cleaned bladder SM layer was cut into 3 mm long and 1 mm wide pieces in the longitudinal direction. For E18.5 mice, the bladder SM layer was cut into 2.8–3 mm long and 1 mm wide pieces in the circular direction. One end of the SM strips was tied to a steel rod, and the other end was fixed on a force transducer. Then, they were placed into an organ incubation chamber at 37°C and bubbled with gas containing 95% O2 and 5% CO2. The resting force was set to 0.1 g, and 124 mM KCl and CCh  were used to induce bladder SM contraction.Bladder SM contraction: The bladder was isolated and immersed in Krebs buffer  were applied to induce contraction.Uterine SM contraction: One side of the uterus was cut along the longitudinal axis. After carefully removing the endometrium, 1 cm long and 1 mm wide strips were cut. The strips were incubated at 37°C in Krebs buffer bubbled with gas containing 95% OFresh SM tissues were stimulated as described in the main text; then, they were snap-frozen in liquid nitrogen and quickly immersed into an acetone solution containing 10% trichloroacetic acid (TCA) and 10 mM DTT and stored at −80°C overnight. The samples were homogenized in a water solution containing 10% TCA and 10 mM DTT. The proteins were pelleted and subsequently washed once with acetone and twice with diethyl ether, dissolved in a urea sample buffer, and loaded onto urea/glycerol-PAGE gels. The urea/glycerol-PAGE gel can separate non-phosphorylated and phosphorylated NM RLC proteins, both of which can be detected by a single antibody to NM RLC. The separated proteins were transferred to PVDF membranes, fixed with glutaric dialdehyde, blocked, and incubated with Myl12a antibody .17 , mouse anti-β-actin , and mouse anti-SMA  at 4°C overnight. After extensive washes with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The bands were imaged using an AI600 imager , and the densities were quantified using NIH ImageJ software (v1.48).To analyze protein expression in SM tissues, proteins were extracted using the TCA method as described above. The lysates were supplemented with Laemmli buffer and boiled at 95°C for 10 min; then, they were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% skimmed milk and incubated with the following primary antibodies: rabbit anti-SM-MHC , rabbit anti-SM RLC , rabbit anti-NMIIA , rabbit anti-NMIIB , rabbit anti-LCFresh isolated SM tissues, including the jejunum, bladder, and aorta, were fixed in 4% paraformaldehyde, dehydrated with an ethanol series, and embedded in paraffin. The tissues were sectioned at 6 μm, dewaxed, rehydrated and stained with H&E according to standard histological procedures. Images were captured using Olympus BX53 light microscope .n represents the number of mice used. Data are presented as means ± SEM. The statistical analysis was performed using Prism 6.05 software. Student’s t-test was used as indicated in the figure legends. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.In all experiments, Myl9 gene was inserted with two loxP sites flanking exon 2 (Myl9 knockout line. These KO mice (Myl9−/−) died within 1–4 days after birth -Cre mice  were used as CTR mice, while the homozygous  mice were used as knockout mice. Similar to the CTR and WT mice, the knockout mice followed the expected Mendelian ratio for their births. At postnatal day 14 (P14), the knockout mice showed normal appearance, physiological behaviors, body weights and activities. At P21, the MYL9SMKO mice had a similar body weight to the CTR mice  in the airway SM cells, but not in vascular SM cells . Upon stimulation with 100 μM ACh, contraction in the mutant jejunum SM was abolished at both the initial and sustained phases . In addition, the peristaltic amplitude of the mutant jejunum muscle was significantly inhibited , the mutant muscle showed no initial peak force, which was consistent with the inhibited initial phase observed before. But to our surprise, the mutant muscle had a higher sustained force than the control  and its downstream target, the LIM domain kinase (LIMK), were reported to regulate RLC phosphorylation both directly and indirectly . To inveSM cells  and otheSM cells  and reguSM cells , we alsod by CCh ,G, suggeMyl9−/− bladder SM with a urea/glycerol PAGE-based Western blot assay. Upon treatment with 30 μM CCh, KO bladder SM started to generate NM RLC phosphorylation at 10 s after stimulation, peaked (approximately 70% RLC phosphorylation) at 30 s, and then decreased gradually , two types of RLC encoded genes (Myl12a and Myl12b) and an ELC encoded gene (Myl6), were unchanged, whereas the mRNA level of Myh11, a gene encoding SM MHC, was increased by two-fold in contrast to that in the control  and hence SM myosin-mediated contraction is abolished. On the functional side, the NM myosin II is up-regulated after Myl9 deletion to compensate for the inhibited force. Due to the variation of NM myosin II abundance, this compensation also varies. Identification of these distinct roles has important physiological implications for hollow organ physiology, e.g., in the bladder, abundant NM myosin II expression facilitates the production of the persistent force necessary for urine elimination, particularly for the young bladder when SM is not well developed. In the uterus, NM myosin II is highly expressed in the SM layer, which is useful for producing sufficient force to adapt the expanded uterus. In addition, it would be helpful to understand the alterative contraction properties of remodeling SM, particularly during abnormal NM myosin II expression in the vascular system.Hollow organs display diverse contractile behaviors, including peristalsis, tension maintenance and spasms, which are primarily conferred by different SM compositions. The SM myosin II and NM myosin II proteins within the SMs are usually co-expressed and thus potentially co-regulate contractile properties . Howeverutations . This fiutations . We thusutations . Combineutations . Under pvia phosphorylated by protein kinase C , and MLCK . We founIt has been well documented that SM contraction is triggered by crossbridge movement of thin and thick filaments. However, NM myosin II does not incorporate into the thin/thick filaments, even in the absence of SM myosin II, and it mediates contraction possibly through other contractile mechanisms such as cytoskeleton reorganization . HoweverThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Model Animal Research Center of Nanjing University.JS, TT, and M-SZ conceptualization and writing-review and editing. JS, Y-NQ, TT, WZ, L-SW, Y-QL, WW, YW, Y-YZ, XC, and X-NZ investigation. JS and TT visualization. JS, Y-NQ, and M-SZ methodology. JS and M-SZ writing-original draft. H-CP resources. M-SZ supervision and funding acquisition. 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.


Amomum villosum (Zingiberaceae) was reported in this study. The A. villosum genome was 163,608 bp in length, and comprised a pair of inverted repeat (IR) regions of 29,820 bp each, a large single-copy (LSC) region of 88,680 bp, and a small single-copy (SSC) region of 15,288 bp. It encoded 141 genes, including 87 protein-coding genes (79 PCG species), 46 tRNA genes (28 tRNA species), and 8 rRNA genes (4 rRNA species). The overall AT content was 63.92%. Phylogenetic analysis showed that A. villosum was closely related to two species Amomum kravanh and Amomum compactum within the genus Amomum in family Zingiberaceae.The first complete chloroplast genome of  Amomum villosum is one species of the genus Amomum (Zingiberaceae), which distributes predominantly in Fujian, Guangdong, Guangxi and Yunan provinces in China , Guangdong Academy of Agricultural Sciences, Guangzhou, China. Total chloroplast DNA was extracted from about 100 g of fresh leaves of A. villosum using the sucrose gradient centrifugation method .A. villosum was 163,608 bp in length, and comprised a pair of inverted repeat (IR) regions of 29,820 bp each, a large single-copy (LSC) region of 88,680 bp and a small single-copy (SSC) region of 15,288 bp. It was predicted to contain a total of 141 genes, including 87 protein-coding genes (79 PCG species), 46 tRNA (28 tRNA species), and 8 rRNA (4 rRNA species). Twenty species genes occurred in double copies, including eight PCG species , eight tRNA species  and all four rRNA species . All these 20 species genes were located in the IR regions. The ycf1 gene crossed the bounders of SSC-IRa and SSC-IRb regions, respectively, while the rps12 gene was located its first exon in the LSC region and other two exons in the IRs regions. In addition, 10 PCG genes  and 6 tRNA genes  had a single intron, while two other genes (ycf3 and clpP) possessed two introns. The nucleotide composition was asymmetric  with an overall AT content of 63.92%. The AT contents of the LSC, SSC, and IR regions were 66.30%, 69.94%, and 58.85%, respectively.The complete chloroplast genome of Costus viridis, Costus pulverulentus, and Canna indica as outgroup taxa. The SNP arrays were obtained as a previously described method  arrays from available 15 species chloroplast genomes using


Acute myeloid leukemia (AML) is a heterogeneous malignancy with the most common genomic alterations in NPM1, DNMT3A, and FLT3. Midostaurin was the first FLT3 inhibitor FDA approved for AML and is standard of care for FLT3 mutant patients undergoing induction chemotherapy . As therex vivo drug sensitivity screen on primary and relapsed AML samples with corresponding targeted sequencing and RNA sequencing. We observed a correlation between FLT3-ITD mutations and midostaurin sensitivity as expected and observed KRAS and TP53 mutations correlating with midostaurin resistance in FLT3-ITD negative samples. Further, we identified genes differentially expressed in sensitive vs. resistant samples independent of FLT3-ITD status. Within FLT3-ITD mutant samples, over-expression of RGL4, oncogene and regulator of the Ras-Raf-MEK-ERK cascade, distinguished resistant from sensitive samples. Overall, this study highlights the complexity underlying midostaurin response. And, our results suggest that therapies that target both FLT3 and MAPK/ERK signaling may help circumvent some cases of resistance.We performed an  Acute Myeloid Leukemia (AML) is a heterogeneous malignancy, most commonly affecting individuals ≥60 years of age , 4. Techde novo AML cases, of which, 25% are ITD mutations and 5% are tyrosine kinase domain (TKD) point mutations . Mut. Mutp <  N = 14; .p = 0.09, Outside of FLT3-ITD, we observed an increase in resistance to midostaurin for samples with pathogenic KRAS and TP53 mutations . When exde novo primary AML samples (N = 193) with RNA-Sequencing which provided a cohort of 170 patients of both FLT3-ITD mutated and non-mutated samples. We identified 47 differentially expressed genes between sensitive and resistance cohorts  and resistance samples were samples above the 80th percentile (N = 34). Midostaurin sensitive samples were enriched for FLT3-ITD mutant samples, however there remained 16 / 34 sensitive samples that were FLT3-ITD wild-type.Next, we sought to evaluate the impact of gene expression on drug sensitivity. We selected all p = 0.0125) [Differentially expressed genes can be interpreted in two groups: those that have a change in expression in samples that are both mutant FLT3-ITD and sensitive and those that have a change between sensitive and resistant regardless of FLT3-ITD status. Within the FLT3-ITD mutated, sensitive cohort we observe known genes found to be over-expressed in FLT3-ITD mutated samples including SOCS2, TRIM16, MIR155HG, and C10orf128  and 3B –30. Howe 0.0125) . Additio 0.0125) –34. Furt 0.0125)  . We sought to identify expression changes within the mutant cohort that stratified the patient by midostaurin response . PerformTo confirm these findings, RGL4 expression was measured in 21 FLT3-ITD positive independent samples. Indeed, RGL4 over-expression within these samples correlated with increased midostaurin resistance .Our research explored the multi-targeted nature of midostaurin and suggested a number of molecular mutational patterns that correlated with midostaurin drug sensitivity and resistance in both FLT3-ITD mutated and FLT3-ITD wild-type AML patient samples. In line with its known mechanism and previous reports, we observed that FLT3-ITD mutated patients had increased sensitivity to midostaurin though we did not identify an association with FLT3-TKD possibly due to the limited number of FLT3-TKD samples in our cohort. Further, we noted specific point mutations and gene expression patterns that may better explain the range of response to midostaurin treatment.https://www.cancerrxgene.org/), RAS mutations were also seen to confer resistance in AML cell lines with NRAS mutations significantly mutated in midostaurin resistant cell lines (p = 0.003) [Within the FLT3-ITD positive cohort, an increased expression of RGL4, an oncogene and upstream regulator of the Ras-Raf-MEK-ERK cascade, correlated with a decrease in midostaurin response. Additionally, within the FLT3-ITD negative cohort, KRAS mutations correlated with a poorer midostaurin response. We did not have sufficient samples to investigate the impact of KRAS within FLT3-ITD AML. Leveraging the Genomics of Drug Sensitivity database (= 0.003)  Combined= 0.003) , 40 and = 0.003) , 40. Fur= 0.003) , 42. Herp = 0.0335) from the Genomics of Drug Sensitivity database [Within our cohort we initially suggested TP53 as a marker of resistance, however were unable to confirm such correlation in a similar demographic validation set. This may have been due to a smaller effect size or larger variance than initially predicted. Additionally, given the limited number of samples in the validation set, there may have been non-annotated characteristics that influenced the results. To further investigate this mutation, we identified TP53 mutations as significantly mutated in midostaurin resistant AML cell lines . Sample collection protocols received approval and guidance from the institutional review boards at OHSU, University of Utah, University of Texas Medical Center (UT Southwestern), Stanford University, University of Miami, University of Colorado, University of Florida, National Institutes of Health (NIH), Fox Chase Cancer Center and University of Kansas (KUMC).ex vivo drug sensitivity assays within 24 hours of draw (described below).Briefly, peripheral blood, bone marrow, and leukapheresis samples were extracted from all AML patients. Mononuclear cells (MNCs) were isolated by Ficoll gradient centrifugation and cell pellets were snap frozen in liquid nitrogen for subsequent DNA isolation . Freshly pelleted cells were lysed immediately in GTC lysate for subsequent RNA isolation , and freshly isolated mononuclear cells were plated into Skin punch biopsies were collected at the site of Jamshidi needle insertion for subsequent bone marrow biopsies and genomic DNA was isolated for use as matched normal controls for exome sequencing .via a tetrazolium-based colorimetric assay .The extracted MNCs were exposed to escalating dose concentration gradients of small-molecule inhibitors – including midostaurin – at a concentration of 0.014, 0.041, 0.123, 0.37, 1.11, 3.33, and 10 μm. The cells were then incubated for 72 hours at 37° C in 5% CO2. Cell viability was then measured by determining the relative number of remaining MNCs Sequencing on a gene panel of 42 genes was performed as part of standard clinical care through the CLIA certified Cancer Diagnostics Laboratory at OHSU (GeneTrails). The custom capture panel of 42 genes is a set known to play a role in leukemia pathogenesis, prognosis, or response to therapy and include: ABL1, ASXL1, BCOR, CBL, CBLB, CEBPA, CREBBP, CSF3R, DNMT3A, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF1, IL7R, JAK1, JAK2, JAK3, KDM6A, KIT, KRAS, MPL, NOTCH1, NPM1 NRAS, PAX5, PTPN11, RUNX1, SF3B1, SRSF2, STAT3, SUZ12, TET2, TP53, U2AF1, WT1, and ZRSR2.Genomic DNA was extracted and purified from blood or bone marrow, and sequenced by next-generation sequencing (NGS) using multiplexed PCR (AmpliSeq primers) and emulsion PCR, followed by semiconductor-based sequencing on an Ion Torrent PGM. Gene segments that were not easily covered by NGS are covered instead by Sanger dideoxy sequencing methods.The minimum detection for the GeneTrails assay is 5% to 15% mutant allele fraction depending on sequence read depth, with a minimum sequence coverage depth of 100 ×.A subset of samples within the Beat AML dataset possessed whole exome sequencing as well. In brief, Illumina Nextera RapidCapture Exome capture probes and protocol were used, giving coverage of 37 Mb of DNA coding regions. Libraries were run on a Hiseq protocol  with five or six lanes per capture group .For genotyping, AML paired/skin biopsies were realigned together and then somatic point mutations were identified with Mutect v1.1.7 and insertions/deletions were called using Varscan2 v2.4.1 , 48. FurMutations were combined with GeneTrails by prioritizing those calls identified by GeneTrails and then augmenting any samples without GeneTrails sequencing with the mutations identified by exome sequencing. While the Beat AML dataset included exome-wide mutations, this study focused on those genes mutated in at least 5% of the cohort.Due to the challenge of identifying FLT3-ITD and the common NPM1 four-base pair insertion using the GeneTrails NGS protocol, FLT3-ITD and NPM1 mutation status was confirmed using an internally run PCR assay and capillary electrophoresis as described previously .FLT3-ITD and NPM1 mutations identified using the internal capillary PCR test (described above) were prioritized over the CLIA/CAP laboratory sequencing (GeneTrails) when available. When GeneTrails results disagreed with the internal testing, samples underwent manual review.The Beat AML dataset was leveraged for RNA-Sequencing across a subset of the samples in the cohort. Briefly, RNA-Sequencing was performed using the Agilent SureSelect Strand-Specific RNA Library Preparation Kit, and sequenced on an Illumina HiSeq 2500 as described previously . The finRNA-Seq analyses were postprocessed using EdgeR v3.7 . Counts Ex vivo functional drug screens were performed on freshly isolated mononuclear cells from AML samples as previously described [escribed . The tetDrug sensitivity was quantified as area under the curve (AUC) with the concentration on log-scale, and calculated via average cell viability across all concentrations. AUC was calculated as the area under the fitted probit curve (via direct integration) using all seven doses as x-values and cell viability with limits from 0 to 100% as the y-value, and then normalizing the AUC values to a 0 to 100 scale.H test was used to determine significance of differences in drug sensitivity measures between mutant and wild-type groups. P-values were corrected for multiple hypothesis testing using FDR. Statistical analysis was performed in R v.3.4.0.Kruskal–Wallis


Eicosanoids are a class of functionally bioactive lipid mediators derived from the metabolism of long-chain polyunsaturated fatty acids (PUFAs) mediated by multiple enzymes of three main branches, including cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450s (CYPs). Recently, the role of eicosanoids derived by COXs and LOXs pathways in the control of physiological and pathological processes associated with cancer has been well documented. However, the role of CYPs-mediated eicosanoids, such as epoxyeicosatrienoic acids (EETs), epoxyoctadecenoic acids (EpOMEs), epoxyeicosatetraenoic acids (EpETEs), and epoxydocosapentaenoic acids (EDPs), as well as hydroxyeicosatetraenoic acids (HETEs), in tumorigenesis and cancer progression have not been fully elucidated yet. Here we summarized the association of polymorphisms of CYP monooxygenases with cancers and the pleiotropic functions of CYP monooxygenase-mediated eicosanoids  in the tumorigenesis and metastasis of multiple cancers, including but not limited to colon, liver, kidney, breast and prostate cancers, which hopefully provides valuable insights into cancer therapeutics. We believe that manipulation of CYPs with or without supplement of ω-3 PUFAs to regulate eicosanoid profile is a promising strategy to prevent and/or treat cancers. Eicosanoids, a class of bioactive lipid mediators, are the metabolites of long-chain n-3 and n-6 polyunsaturated fatty acids (PUFAs) mediated by three primary enzymatic systems, cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450s (CYPs) enzymes. The common PUFAs include arachidonic acid , linoleic acid , γ-linolenic acid , α-linolenic acid , eicosatetraenoic acid  and docosahexaenoic acid . Eicosanoids are synthesized rapidly in response to multiple factors  and act putatively through their cognate receptors in local cells. Although some eicosanoids exhibit immediate and short-lasting activity, they play an important role in many chronic diseases, including asthma, allergy, autoimmune diseases, and malignancies since they have pleiotropic functions, such as pro-inflammation, anti-inflammation, vasodilation, analgesia, and hyperalgesia . Interes2, corticosterone, retinoic acid, vitamin D3, and some PUFAs, like AA and LA -hydroxyeicosapentaenoic acids (19- and 20-HEPE) by CYP ω-hydroxylases, and five regioisomeric epoxyeicosatetraenoic acids  by CYP epoxygenases  Van Rol. DHA canThe CYP superfamily comprises 57 functional CYP genes and 58 pseudogenes in humans . The CYPIn humans, CYP2C and CYP2J are the predominant epoxygenases that metabolize PUFAs. CYP2C and CYP2J are widely distributed in the human body, including but not limited to the cardiovascular system, kidney, lung, brain, gastrointestinal tract, cerebral cortex, hippocampus, fetal nasal mucosa, and many other tissues . The CYPIn mammals, the CYP4 family primarily mediates the ω-hydroxylation of PUFAs, which includes 12 genes and 13 enzymes, such as, CYP4A, CYP4B, CYP4F, CYP4V, CYP4X, and CYP4Z. CYP4A, CYP4B, CYP4X, and CYP4Z are located on chromosome 1, while CYP4F and CYP4V on chromosome 19 and 4, respectively . The CYPOther CYPs, such as CYP1A1, CYP 1A2, CYP1B1, CYP2D6, and CYP3A4, can detoxificate carcinogens regardless of whether they are expressed in the liver or kidney.In addition to many studies investigating the expression of CYP epoxygenases in multiple organ tissues, a few studies summarized the expression of CYP epoxygenases in some specific cell types. Almost all the CYP epoxygenases were found in peripheral blood cells, vascular endothelial cells, and vascular smooth muscle cells . In addi.Recently, the expression of various CYP genes has been proved to be closely related to malignant tumors. Polymorphisms of CYPs have been suggested to influence susceptibility to cancers for many years. Genetic polymorphisms in CYPs have been reported to be associated with individuals variations in drug metabolism and disease susceptibility . Here, w.CYP1A1 is a hepatic and extrahepatic enzyme that is regulated by the aryl hydrocarbon receptor signaling pathway. It has been always associated with the metabolism of pro-carcinogenic compounds to highly carcinogenic metabolites. For CYP1A1, four common variants  were widely studied for the susceptibility to various cancers . T3801C CYP1A2, which is similar to CYP1A1, can metabolize a broad range of foreign compounds and drugs. An SNP C>A (rs762551) was found in intron one of CYP1A2 , which iCYP1B1 not only mediates the metabolisms of xenobiotics, e.g. theophylline, ethoxyresorufin, and caffeine  but alsoCYP2A6 is an essential hepatic enzyme involved in the metabolism of drugs, is responsible for a major metabolic pathway of nicotine. The first polymorphism identified of CYP2A6 was a nonsynonymous polymorphism (L160H) (rs1801272) which leads to no enzyme activity . There aCYP2C9 metabolizes about 15% of clinically administrated drugs. Two common non-synonymous polymorphisms of CYP2C9, R144C, rs1799853 (CYP2C9*2), and I359L, rs1057910 (CYP2C9*3), have been reported to be highly frequent in Caucasian populations . These tAmong the CYP2C subfamily, CYP2C19 is the most polymorphic . CYP2C19CYP2D6 is one of the most studied enzymes in the field of pharmacogenetics. It exhibits large interindividual variability on drug metabolism. The polymorphism of CYP2D6 causes different metabolizer genotypes, including poor, intermediate, efficient, or ultra-rapid ones . The numCYP3A4 is the best-studied gene polymorphism in the CYP3A family. A meta-analysis comprising 55 separate studies including 22,072 cancer cases and 25,433 controls found a significant association between CYP3A4*1B and cancer risk especially leukemia in the overall population . In the EETs were discovered in the early 1980s . EETs ha3-kinase/AKT pathway and the STAT3 pathway (CYP-mediated biosynthesis of four EETs  has been implicated in tumor growth and angiogenesis, as well as suppression of inflammation in murine models of cancers . Compare pathway . More re pathway . In trip pathway . In addi pathway .EETs are also anti-inflammatory , which mRecently, EETs were discovered to act the biological function in a receptor-dependent manner. However, the receptors of EETs have not been identified. Some research suggested that the actions of EETs are in part mediated via G protein-coupled receptor (GPCR) signaling. Five G protein-coupled receptors of the prostaglandin receptor family  may be the EET receptors .3K/Akt /dextran sodium sulfate (DSS)-induced colon tumorigenesis in vivo , the metabolite of 19 (20)-EDP mediated by sEH, did not have any effect on tumor growth, indicating that the anticancer effect was from 19 (20)-EDP but not its diol metabolite (EDPs are the metabolites of DHA mediated by CYP enzymes. They are suggested to be responsible for some of the beneficial effects of n-3 PUFAs and n-3 PUFA-rich diet . Evidenctabolite . The putIncreasing studies supported the important role of CYP-derived eicosanoids in the progression of cancer and the resolution of inflammation. The action of EETs and EpOMEs has been investigated for decades while EEQs and EDPs are less-studied. Further studies are suggested to pay more attention to EEQs and EDPs since increasing the supplement of EPA and DHA with a focus on the biological activities of these eicosanoids and underlying mechanisms.The additional complexity of the regulation of eicosanoids on inflammation, pain, angiogenesis, and cancer is the sEH enzyme, which can rapidly metabolize many CYPs-derived eicosanoids to corresponding fatty acid diols . sEHs maYL and JYL designed the paper frame; YL wrote the draft; JYL critically revised and finalized the paper. YL and JYL approved the final 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.