Pro shared significant disimilarities against SARS-CoV MPro and MERS-CoV MPro indicating the need for discovering novel drugs. This study has screened the phytochemical cyanin  which may exhibit broad-spectrum inhibitory activity against main proteases of SARS-CoV-2, SARS-CoV and MERS-CoV with binding energies of (−) 8.3 kcal/mol (−) 8.2 kcal/mol and (−) 7.7 kcal/mol respectively. Amentoflavone, agathisflavone, catechin-7-o-gallate and chlorogenin were shown to exhibit multi-target inhibitory activity. Further, Mangifera indica, Anacardium occidentale, Vitex negundo, Solanum nigrum, Pedalium murex, Terminalia chebula, Azadirachta indica, Cissus quadrangularis, Clerodendrum serratum and Ocimum basilicumaree reported as potential sources of phytochemicals for combating nCOVID’19. More interestingly, this study has highlighted the anti-viral properties of the traditional herbal formulation “Kabasura kudineer” recommended by AYUSH, a unit of Government of India. Short listed phytochemicals could be used as leads for future drug design and development. Genomic analysis of identified herbal plants will help in unraveling molecular complexity of therapeutic and anti-viral properties which proffer lot of chance in the pharmaceutical field for researchers to scout new drugs in drug discovery.Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) being a causative agent for global pandemic disease nCOVID’19, has acquired much scientific attention for the development of effective vaccines and drugs. Several attempts have been made to explore repurposing existing drugs known for their anti-viral activities, and test the traditional herbal medicines known for their health benefiting and immune-boosting activity against SARS-CoV-2. In this study, efforts were made to examine the potential of 605 phytochemicals from 37 plant species (of which 14 plants were endemic to India) and 139 antiviral molecules (Pubchem and Drug bank) in inhibiting SARS-CoV-2 multiple protein targets through a virtual screening approach. Results of our experiments revealed that SARS-CoV-2 M It is noV (60%) . Its unihttps://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/). Genomic analysis of nCOVID’19 revealed that it is approximately 30 kb in size (NCBI Accession # NC_045512) and further investigations identified three key genes viz., 1) coronavirus main protease (3CLpro)/papain-like protease (PLpro); 2) RNA-dependent RNA polymerase (RdRp) and 3) spike glycoprotein (S protein) as potential targets for drug designing , lopinavir/ritonavir, chloroquine phosphate, ribavirin, chloroquine, hydroxychloroquine and arbidol is in progress and many of these experiments require pre-clinical and clinical validation . IneffecProtein sequence of SARS-CoV-2 encoding for main protease (PDB ID: 5R81) was used for PSI BLAST (NCBI)  search tPro of SARS CoV (PDB ID: 2GZ9) and MERS CoV (PDB ID: 5C3N) for identifying inhibitors against main protease of all three viruses and inhibitors specific to nCOVID’19.Coronavirus genome was reported to encode for 29 proteins, out of which the main protease is considered to be an important drug target. ORF1ab of the coronavirus genome contains 15 polypeptide chains encoding for non-structural proteins (NS proteins). Another part of the genome encodes for envelope and coat proteins. Availability of X-ray crystal structures for most of the proteins in the coronavirus two genome facilitates virtual screening to search for potential inhibitors. We have performed molecular docking of 744 ligands against seven different target proteins of the nCOVID’19 genome . In addihttp://www.medicinalplants.in/) and Indian Medicinal Plants, Phytochemistry and Therapeutics (https://cb.imsc.res.in/imppat/home) database. Structures of 605 phytochemicals belonging to 37 different herbals and spices used in South Indian Traditional Medicine were also used for virtual screening (Abutilon indicum (Thuthi), Ocimum tenuiflorum (Tulsi), Pedalium murex (Peru nerinji), Anacyclus pyrethrum (Akara), Andrographis paniculata (Nilavembu), Pepper nigrum (Pepper), Azadirachta indica (Neem), Phyllanthus niruri (Keelanelli), Cissus_quadrangularis_L (Perandai), Plectranthus amboinicus , Clerodendrum serratum (Kanduparangi), Solanum torvum (sundakai), Solanum trilobatum  and Terminalia chebula (Kadukai). Phytochemicals from different parts of the plant such as Root, Rhizome, pericarp leaves, Flower, Seed essential oil, Stem and Fruits was curated in order to find bioactive molecule that may have role in the inhibition of SARS CoV two targets.Chemical structures of all the small molecules were retrieved from Dukes database (1992–2016), PubChem  and Drugcreening . Among tcreening  namely, Known active ingredients of eight herbal plants included in the Tamil traditional medicine “Kabasura Kudineer” (meaning water capable of boosting immunity) were also included in the screening. Overall, a total of 744 small molecules/ligands : a-c werPython Prescription Virtual Screening tool (PyRx 0.8) containing AutoDock Vina module (Pro was carried out using PSI-BLAST (NCBI) (http://www.swissadme.ch)   and MAFFT (NCBI) . Top 10 dme.ch) .pro, also called 3CLpro) is considered as one of the important molecular targets for designing novel drugs against coronaviruses  and bat CoV (76.84% similarity)  . Also itVirtual screening of 744 ligands belonging to small molecules and active compounds from 37 medicinal herbs against seven major protein targets of nCOVID’19 predicted probable SARS- CoV-2 inhibitors. Information regarding the binding site residues predicted using CASTp server is provided in Pro between SARS-CoV (PDB ID: 2GZ9) and SARS-CoV-2 (PDB ID: 5R81), they showed differential binding affinity against different inhibitors  and binding site volume similarity of Mhibitors . VirtualTerminalia chebula, Azadirachta indica and Ocimum basilicum exhibited highest binding affinity value of −9.0 kcal/mol. In case of MERS-CoV main protease (PDB ID: 5C3N), amentoflavone predominantly found in Mangifera indica and Garcinia species showed the maximum binding affinity value of -8.6 kcal/mol. Interestingly, a cytotoxic biflavonoid agathisflavone found in cashew nut  was shown to exhibit significant binding affinity with −8.0 kcal/mol against the SARS-CoV-2 MPro , solanocapsine ) (Solanum nigrum), chlorogenin  (Solanum torvum), Lupeol  (Carica papaya and Azadirachta indica), Cyanin  , 3-O-trans-cafeoyltormentic acid  , Luteolin-7-O-  (Vitex negundo), Agnuside  (Vitex negundo) and Luteolin 7-O-beta-D-glucoside  (Vitex negundo) exhibiting significant binding affinity in the decreasing order against MPro.Out of 744 ligands screened against SARS-CoV main protease, a ligand namely rutin abundantly found in V-2 MPro . AgathisV-2 MPro . VariousV-2 MPro . Also, AV-2 MPro . This coZingiber officinale was found to have higher binding affinity with the main proteases of all three coronaviruses . GLY 496 residue was found to be involved in the formation of hydrogen bond with the 1, 8-Dichloro-9, 10-diphenylanthracene-9,10-diol. Earlier, leaf extracts of Carica papaya was reported to have significant effect in combating dengue virus infection  (Vitex negundo), taraxerol  (Cissus quadrangularis), nimbinene  (Azadirachta indica) and β-amyrin  (Cissus quadrangularis) exhibiting significantly higher level of binding affinity toward spike protein of nCOVID’19. Comparison of hydrogen bond interactions at the binding site for ivermectin (3 hydrogen bonds), amentoflavone (4 hydrogen bonds) and agasthisflavone (3 hydrogen bonds) showed higher number of interactions for amentoflavone involving residues TYR 449, SER 494, GLY 496 and ASP 30 respectively.X-ray crystal structure of spike glycoprotein (PDB ID: 6M0J) was chosen for performing virtual screening. Virtual screening was performed by choosing ACE interacting region as the binding site . 1,8-Dicnfection  and its nfection . This wacal/mol) , amentofcal/mol) , ivermecResidues are colored based on their physiochemical properties.viz., Corilagin  , Agasthisflavone   , Crateogolic acid  (Syzygium aromaticum), 3.8’-biapigenin , cyanin   were found to exhibit significant binding affinity. Interaction analysis showed higher number of vander waals interactions for ivermectin compared to hydrogen bond interactions. Comparison of hydrogen bond interactions exhibited toward ivermectin (3 hydrogen bonds), agasthisflavone (4 hydrogen bond interactions) and amentoflavone .RdRp (PDB ID: 6M71) is responsible for replication of COVID-19 genome inside the host. Among the ligands tested, ivermectin  showed tdentale) , 3-O-traApart from the four major structural proteins , non-structural proteins namely NSP3  (PDB ID: 6W02), NSP9 (ssRNA binding) (PDB ID: 6W4B), NSP10-NSP16 (co-factor in activating replicating enzyme) (PDB ID: 6W4H) and NSP15 (PDB ID: 6W01) involved in the transcription and replication of nCoVID’19 can also serve as potential targets for containing the virus using inhibitory herbal molecules .Mangifera indica) as the top scored ligand with binding affinity of −7.5 kcal/mol (Vitex negundo), rubusic acid  , acteoside  (Clerodendrum serratum), ivermectin , taraxerol acetate  (Cissus_quadrangularis), catechin 7-O-gallate , luteolin 7-O-beta-D-glucoside  (Vitex negundo), agathisflavone   (Vitex negundo) were found to have higher binding affinity next to amentoflavone. It was observed that three inhibitors from Vitex negundo were reported with the highest binding scores. Vitex negundo belongs to Verbenaceae family known for its effects for ailments like ophthalmia, deafness, indigestion, piles and jaundice   and lutejaundice . ResultsVitex negundo and Acorus calamus was also found to exhibit significant binding affinity of −9.6 kcal/mol against NSP9 (PDB ID: 6W4B)  . Even thstresses .Ocimum basilicum and Ocimum tenuiflorum (Syzygium aromaticum), arjungenin  , hederagenin  (Nigella sativa), triterpenoid  (Abutilon indicum), beta-amyrin  (Cissus quadrangularis), friedelin  , catechin 7-O-gallate  (Camellia sinensis) and arjunolic acid  .Virtual screening against NSP15 (PDB ID:6W01) identified oleonolic acid and urosolic acid as molecules exhibiting highest binding affinity values of −9.2 kcal/mol. Oleonolic acid and urosolic acid are known for their anti-cancerous and anti-inflammatory activities . Both thuiflorum . This waCissus_quadrangularis), corilagin  , lupeol acetate  , emetine , chlorogenin  (Solanum torvum), spirostan-3-ol  (Solanum torvum) and 5,7-Dihydroxy-4'-methoxy-8.3'-di-C-prenylflavanone  (Azadirachta indica) were ranked among the top 10 molecules exhibiting highest binding affinity.In the case of NSP10-NSP16 (PDB ID: 6W4H) protein complex, interface of the complex  was chosPhytochemicals showing strong interactions against multiple targets of viruses are expected to confer durable protection to the patients. This will be more beneficial in situations where the virus is developing mutations in one of the targets. Small molecules namely, amentoflavone, agathisflavone, catechin-o-gallate and chlorogenin exhibited significant binding affinity toward multiple targets of nCOVID’19.Torreya nucifera was demonstrated to possess inhibitory activity against SARS-CoV3CLPro  , NSP9 −, NSP3 −, NSP10-NoV3CLPro .Solanum torvum exhibited binding affinity value of −8.2 kcal/mol (NSP9), −7.7 kcal/mol (MPro) and −7.6 kcal mol (NSP16-NSP10) of SARS-CoV-2. Solanum torvum is well known for its pharmacological properties for the treatment of human ailments and its phytoconstituent chlorogenin also has antiviral effect on influenza viruses , SARS-CoV-2 main protease , spike glycoprotein  and NSP 3  . Catechi viruses .viz., 3.8'-biapigenin, 3-O-trans-caffeoyltormentic acid, agnuside, arjungenin, corilagin, crategolic acid, cyanin, friedelin, luteolin 7-O-, N-methylsolasodine, rubusic acid, solanocapsine, spirostan-3-ol, taraxerol and taraxerol acetate were found to possess significant interactions with at least two protein targets of SARS-CoV-2 , agnuside, luteolin-7-o-beta-d-glucopyranoside and friedelin were found to exhibit significant binding affinity against five different protein targets of SARS-CoV-2 namely, spike glycoprotein, SARS-CoV-2 main protease, NSP3, NSP9, NSP15 in the SARS-CoV-2.Among the 605 phytochemicals originating from 37 plant species, 33 (6% approx.) phytochemicals from 22 plants  were fouSolanum nigrum which effectively inhibited three SARS-CoV-2 targets .Solanocapsine, Spirostan-3-ol, N-methylsolasodine, Diosgenin and Solasodine are the phytochemicals reported in Pedalium murex was reported to have Diosgenin (NSP9), Lupeol acetate (NSP16-NSP10), Urosolic acid (NSP15) and Rubusic acid  which might be probable SARS-CoV-2inhibitors. It is noteworthy that maximum of five targets were predicted to be inhibited by the compounds from Pedalium murex. In spite of its role as anti-ulcerogenic, nephroprotective, hypolipidemic, aphrodisiac, anti-oxidant, anti-microbial and insecticidal activities, Pedalium murex has been traditionally used in treating ailments like cough and cold as a regular practice  (20%)  (90.92 ± 4.91 mg/100 g)  (4.39 ± 0.01 (mg/g))  (13.05 mg/g) (Vitex negundo) (123.776 – 127.774 mg/g) (Solanum nigrum) (>50%) (Solanum nigrum) (75.89%)  (0.06%) (Azadirachta indica) (0.016%) (Azadirachta indica) (0.14%) (Azadirachta indica) (1.2%)  (0.17%)  (425.71 ± 2.15 μg/g) (Ocimum basilicum) (179.19 ± 1.93 μg/g)  (20%) , amentofg/100 g) , agathis (mg/g)) , Corilag05 mg/g)  luteolin74 mg/g) , Solanoc) (>50%) , Solasod(75.89%) , Diosgen (0.06%) , Quercet(0.016%) , Rutin ( (0.14%) , Nimbine) (1.2%) , Rutin ( (0.17%) , Rutin (15 μg/g)  and Isoq93 μg/g) .Vitex negundo L. against Chikungunya virus and HIV-1 Reverse transcriptase (RT) have been previously reported by Kothandan and Swaminathan (2014) and Nair and Ramachandran (2012)  . Solanumnfection . Similarnfection . Sivagamnfection  have repn proved . Phytoch viruses . Zingibeal virus . Also, tectively .ADME properties are important to understand the pharmacokinetics and physio-chemical properties of phytochemicals which further provides insight on drug design and formulation research. Analysis on solubility and bioavailability based on the SWISSADME prediction for the screened inhibitors showed different level of classifications as given in Zingiber officinale was screened as the best phytochemical with highest binding energy against main proteases of all the three viruses. Popular fruit tree, mango (Mangifera indica) and, cashew nut  rich in amentoflavone and agathisflavone were showing possible inhibitory activity against multiple targets of SARS-CoV-2. Vitex negundo, Solanum nigrum, Pedalium Murex, Terminalia chebula, Azadirachta indica, Cissus quadrangularis, Clerodendrum serratum and Ocimum basilicum were also found to contain phytochemicals that may have possible inhibitory activity against SARS-CoV-2 proteins. More interestingly, this study has picked up Carica Papaya that may possess inhibitory activity against spike glycoprotein and MPro of SARS-CoV-2 which was known for its protective role against dengue virus in humans , SARS-CoV (PDB ID: 2GCZ9) and SARS-CoV-2 (PDB ID: 5R81) revealed significant differences between the three homologs which were confirmed by differential binding affinity exhibited by 744 phytochemicals/small molecules against the three main proteases. Cyanin from n humans . Overall


CNKSR1 functions as a scaffold protein for activation of an EGFR phosphatase, PTPRH, at the plasma membrane through the exclusive interaction with RhoB-GTP which is constitutively degraded by the CUL3/KCTD10 E3 complex. Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL3/KCTD10 E3 complex, enabled CNKSR1 to interact with PTPRH at the plasma membrane resulting in inactivation of EGFR phosphatase activity. Depletion of CUL3 or KCTD10 led to the accumulation of RhoB-GTP at the plasma membrane followed by its interaction with CNKSR1, which released activated PTPRH from CNKSR1. This study suggests a mechanism of PTPRH activation through the exclusive binding of RhoB-GTP to CNKSR1. Inactivation of receptor tyrosine kinases is mainly achieved through their dephosphorylation by protein tyrosine phosphatases. Thus, protein tyrosine phosphatases generally act as tumor suppressors by terminating signal transduction from phosphorylated oncogenic receptor tyrosine kinases . ExceptiHuman epidermal growth factor receptor (EGFR) 2 (HER2) is a member of EGFR tyrosine kinase family. Unlike other EGFR family members , HER2 recognizes no known ligands and forms heterodimers with each other member, leading to the transduction of various cellular signals . EspeciaCullin-3 (CUL3) is a scaffold protein for cullin/RING-type E3 ubiquitin ligase complexes. In such complexes, a substrate recognition receptor, specifically a BTB domain-containing protein (BTBP), recruits substrates leading to their ubiquitination . One BTBIn this study, we found that depletion of CUL3 or KCTD10 reduced the phosphorylation of EGFR and HER2, as well as cell proliferation of HER2-positive breast cancer cells. Making use of a human protein array  and a human phosphatase array (171 phosphatases) produced in a wheat cell-free protein synthesis system, we found that connector enhancer of kinase suppressor of Ras1 (CNKSR1) directly interacted with RhoB-GTP and protein tyrosine phosphatase receptor type H (PTPRH). In cells depleted of CUL3 or KCTD10, accumulated RhoB-GTP released an EGFR phosphatase, PTPRH, from a scaffold protein, CNKSR1, via its exclusive binding to the PH domain of CNKSR1 at the plasma membrane, resulting in PTPRH activation. Our results suggest a unique model of phosphatase activation by protein–protein interactions at the plasma membrane.1068 in EGFR and Tyr1221/1222 in HER2 was detected in a HER2-positive breast cancer cell line, SKBR-3 cells, without the addition of any ligands of the HER family  (2 did not inhibit the interaction between RhoB (Q63L) and CNKSR1 in vitro , and a dominant negative RhoB mutant (T19N) using a wheat cell-free protein synthesis system , 2008 F. The bio2)  Fig 2H)2)  and 4,212 human protein array.Table S1 We then validated the interaction between RhoB-GTP and CNKSR1 in SKBR-3 cells. In control SKBR-3 cells in which RhoB-GTP is undetectable , CNKSR1 CNKSR1 gene in SKBR-3 cells. CNKSR1 knockdown reduced the phosphorylation of EGFR P2, localized at the plasma membrane in CUL3- or KCTD10-knockdown SKBR-3 cells as was seen in control cells P2-independent manner.The PH domain of CNKSR1 interacts with PIPmembrane . The in brane +, Ki-67low, HER2−), luminal-B , HER2-positive , basal , and claudin-low  . We also found that low mRNA expression of RhoB significantly correlated with poor prognosis for HER2-positive/EGFRhigh breast cancer patients . In contrast, high mRNA expression of RhoB significantly correlated with poor prognosis for HER2-positive/EGFRlow breast cancer patients . Low mRNA expression of RhoB also significantly correlated with poor prognosis for HER2high and HER2high/CNKSR1high breast cancer patients . Taken together, these data suggested that RhoB might function as a tumor suppressor in HER2/EGFR-double positive breast cancers.Breast cancers can be classified based on their gene expression into five subtypes, namely, luminal-A (estrogen receptor (ER)udinlow) . Among tudinlow) . We nextudinlow) . These dudinlow) . AlthougWe next examined the protein expression of CNKSR1 in HER2-positive human breast cancer tissues. In total, 20 individual primary tumor tissues from patients with HER2-positive breast cancer were subjected to immunohistochemistry. CNKSR1 was highly expressed in 80% of HER2-positive breast cancer tissues . Of note493 residue in the PH domain were required for CNKSR1-mediated EGFR/HER2 phosphorylation and cell proliferation, although the protein expression of RhoB remained low  mutant-expressing SKBR-3 cells, as seen in control and wild-type CNKSR1-expressing SKBR-3 cells  in vitro , RhoB (Q63L) and human phosphatases.Table S2 We then sought to identify the responsible regions of PTPRH for the interaction with CNKSR1. For this aim, we generated seven deletion mutants of PTPRH-cyto, which delete 50 or 39 sequential amino acids in the cytosolic region of PTPRH . As showOur result indicated that the depletion of CUL3 or KCTD10 caused the accumulation of RhoB protein in HER2-positive breast cancer cells. Physiologically, a microRNA, miR-101, induced by hypoxia targets to the 3′ untranslated region of CUL3, which down-regulates protein expression of CUL3 in human umbilical vein endothelial cells resulting in the up-regulation of VEGF and promotion of angiogenesis both in vitro and in vivo . The hydWe noticed that the depletion of CUL3 or KCTD10 increased the protein expression of EGFR and reduced the protein expression of HER2 in SKBR-3 cells  and 6F. RhoB mRNA expression and prognosis of HER2-positive breast cancer patients were reversed dependent on expression level of EGFR mRNA , which is essential for phosphatase activities, resulted in the loss of capacity to interact with CNKSR1 , HepG2 cells , nor MIA-PaCa-2 cells  in the steady-state condition. These data suggest that EGFR is not activated by endogenously produced ligands in those cancer cells. Knockdown of CUL3, but not KCTD10, increased the protein expression of RhoB in A431 cells  , rabbit anti-HER2 antibody , rabbit anti-phosphorylated HER2 antibody (Tyr1221/1222) , rabbit anti-HER3 antibody , rabbit anti-phosphorylated HER3 antibody (Tyr1222) , rabbit anti-HER4 antibody , rabbit anti-phosphorylated HER4 antibody (Tyr1284) , rabbit anti-RhoA antibody , rabbit anti-RhoB antibody , rabbit anti-RhoC antibody , mouse anti-CUL3 antibody , rabbit anti-KCTD10 antibody , rabbit anti-CNKSR1 antibody , rabbit anti-Akt antibody , rabbit anti-phosphorylated Akt antibody (Ser473) , rabbit anti-ERK-1/2 antibody , rabbit anti-phosphorylated ERK-1/2 antibody (Thr202/Tyr204) , mouse anti-β actin antibody , rabbit anti-PTPRH antibody , mouse anti-HA antibody , mouse anti-GAPDH antibody , mouse anti-Myc antibody , mouse anti-FLAG antibody , goat Cy3-conjugated anti-rabbit IgG antibody , goat Alexa 488–conjugated anti-mouse IgG antibody , HRP-conjugated anti-mouse IgG antibody , and HRP-conjugated anti-rabbit IgG antibody .The following antibodies were purchased from the indicated manufacturers: rabbit anti-EGFR antibody , rabbit anti-phosphorylated EGFR antibody . 20 ml of solution containing 0.5 μl of a biotin-tagged bait protein and 19.5 μl of the AlphaScreen buffer was dispensed into reaction plate by using automated multichannel pipet Viaflo and Viaflo Assist system (Integra). Next, 1 μl of each FLAG-GST tagged protein array protein was transferred from 384-well stock plate to the reaction plate using Janus automated dispensing workstation (PerkinElmer) and Nanohead, a 384-well micro syringe head (PerkinElmer). Then 9 μl of detection mixture containing 0.02 μl of anti-DYKDDDDK tag monoclonal antibody (Wako), 0.06 μl of streptavidin-conjugated AlphaScreen donor beads and 0.06 μl of protein A–conjugated AlphaScreen acceptor beads (PerkinElmer) in the AlphaScreen buffer were added to the reaction plates using FlexDrop dropper. After incubation at 25°C for 1 h in a dark incubator, AlphaScreen signal was detected by Envision multilabel reader (PerkinElmer).Transcription and translation for the production of biotinylated or FLAG-tagged recombinant proteins were performed using the WEPRO 7240 Expression Kit (Cell Free Science) as reported previously . For pur2 (Sigma-Aldrich) or PHT-7.3  was added to the protein mixtures followed by incubation for 1 h.The in vitro binding assays were performed as previously described using an AlphaScreen IgG (Protein A) detection kit (Perkin Elmer)  with sliSKBR-3 cells, A431 cells, HepG2 cells, MIA-PaCa-2 cells, and HEK293T cells were maintained at 37°C with 5% CO2 in DMEM (Wako) supplemented with 10% FBS, 20 U/ml penicillin, and 100 μg/ml streptomycin. MCF-7 cells were maintained at 37°C with 5% CO2 in EMEM (Wako) supplemented with 10% FBS, 20 U/ml penicillin, and 100 μg/ml streptomycin. MDA-MB-231 cells and MDA-MB-453 cells were maintained at 37°C without CO2 in Leiboviz’s L-15 medium (Wako) supplemented with 10% FBS, 20 U/ml penicillin, and 100 μg/ml streptomycin. To inhibit neddylation, cells were treated with 1 μM MLN4924 (Boston Biochem) at 37°C for 24 h. Cells were treated with 1 μg/ml Rho inhibitor I  at 37°C for 24 h, or with 50 μM PHT-7.3  at 37°C for 24 h.For transfection of plasmids into SKBR-3 cells and HEK293T cells, Lipofectamine 3000 (Invitrogen), and GeneJuice (Millipore) was used according to the manufacturer’s instructions, respectively. At 48 h post-transfection, the cells were processed for subsequent experiments. Transfections of siRNAs (10–25 nM) into breast cancer cell lines were performed using RNAimax (Invitrogen) according to the manufacturer’s instructions. Subsequent experiments were performed at 72 h post-transfection.Transient or stable expression of Myc-CNKSR1 , Myc-PH, PTPRH-HA  in SKBR-3 cells was induced through lentiviral infection as previously described . The CSIThe following validated siRNA duplex oligomers were purchased and used for knockdown experiments: GAGUGUAUGAGUUCCUAUU , GGAUCGCAAAGUAUACACAUAUGUA , GAAUGAGCGUCUAAAUCGU , GUAACAACAAAUACUCAUA , GCAUCCAAGCCUACGACUA , CUAAGAUGGUGUUAUUUAA , AGGCUACAGACAGAGAACCUGCAAA , CAGAUCUGAGCAUGUGGGUGCGUCA , GAGACAAACUAAUUGAGAA , GAGAAGAAGUAUUACUUAU , GCAUCAAAACUUAAUCAGA , CAUGCAGCCUUAAGCCUCA , GAACCGCUACAAGAACAUU , ACCUCUCCCUGACCCUGUA , CCAUUGUCACGGGCUUGUA , CAGACUUAUUGUAACAAGA , GGAAGAACCGCUACAAAGA , AGAGCAGAUUCAAGAAAGA , GGCUUGUCGCGAAUCUUCA , CCCUAAAUCUGCUUCCGCA , GGAUAAGAGGCAUGAGGAA , AGAGAGAGCUUCAAAAUUA , AGAUGAACGAUGACCACAA , GGCUCAACUAUGACCGGAA , AGAAAGGCCUGUUCAACUA , AGUUCGAGGAAGACCUAAA , GGUACGAGAUGACAAGACA , GGUCGUUUUAGAUCAAUGA , AGUCCAACAUCUCGCCCAA , ACAAGUUUCAAACAGAGUA , GGGUCCAACGUAUCUCUUA , CAUUCGAAACCUUGUCUUA , GGACUACACCUACUGGGUA , GACUGAGGCUCAGUACGUA , GCCUAAUUCCUAUUACCCA , GGAAUAUCAUUGCAUACGA , GCACGUAUGACAAAGCGAU , CAAGGACCUUUACCGAACA . Control siRNAs were purchased from Sigma-Aldrich (SIC-001).5 SKBR-3 cells were seeded into a six-well plate in triplicate. Cells were treated with siRNA on the next day. After 48 h, 0.5 × 105 cells were replated into a 12-well plate in triplicate. Cells were then treated with the same siRNA again on the next day. The cells were counted at 72 h post-siRNA transfection.A total of 1 × 10Western blotting and immunoprecipitation were performed as previously described . The biog for 10 min at 4°C. The resulting supernatants were incubated with purified His-tagged RhoB-GTP recombinant proteins for 2 h at 4°C followed by incubation with prewashed TALON resin beads (Clontech) for 1 h at 4°C. The beads were washed three times with IP buffer.To detect GTP-Rho forms, SKBR-3 cells plated into a six-well plate were transfected with the indicated siRNAs. After 72 h, cells were lysed and subjected to pull-down assay to detect active GTP form of Rho using the RhoA Pull-down Activation Assay Biochem Kit . To detect the interaction between RhoB-GTP and CNKSR1, SKBR-3 cells expressing Myc-CNKSR1  were lysed in IP buffer  containing cOmplete protease inhibitors (Roche). After incubation of cell lysates on ice for 10 min, the cell lysates were centrifuged at 10,000Total RNAs were extracted from SKBR-3 cells using ISOGEN II (Nippon Gene) according to the manufacturer’s protocol. The total RNA (1 μg) was used for cDNA synthesis using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). Real-time PCR was carried out  on the ABI 7500 Real-Time PCR system (Applied Biosystems) using the following pairs of primers:EGFR sense primer)5′-GTCTGCCATGCCTTGTGCTC-3′ (EGFR antisense primer), 5′-AGCCTTGCCCCATCAACTG-3′ (HER2 sense primer), 5′-AATGCCAACCACCGCAGA-3′ (HER2 antisense primer), 5′-CATGAGAAGTATGACAACAGCCT-3′ (GAPDH sense primer), 5′-AGTCCTTCCACGATACCAAAGT-3′ (GAPDH antisense primer).5′-CTTGTCCACGCATTCCCTGC-3′  with 60 × 1.27 Plan-Apochromat water immersion lens. Images were analyzed with FIJI (NIH). FRAP were performed on an A1R laser confocal microscope (Nikon), and FRAP analysis of EGFR-GFP was performed as described previously .Immunohistochemical staining of HER2-positive breast cancer tissues was carried out as described previously  with sliAll human HER2-positive breast cancer samples were collected from the archives of the Division of Diagnostic Pathology, Ehime University Hospital. Clinical and pathological information was obtained from the pathology records at Ehime University Hospital. This study was approved by the Institutional Review Board of Ehime University Hospital (approved IRB protocol number: 1701015). Informed consent was obtained from all subjects. All experiments were performed by following the approved study plan and relevant guidelines.EGA00000000083) deposited in the European Genome-phenome Archive (EGA) database were subjected to survival analysis. Survival analysis was performed by Kaplan–Meier method with overall survival status in R (ver. 3.6.3) as reported previously  (RNA-seq datasets of HER2-positive breast cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia (SE36133) . ExpressStatistical comparisons were made using the one-way ANOVA followed by Turkey’s post hoc test. The log-rank test in survival analysis was performed by the “survminer” package in R (ver. 3.6.3.). Cox test in correlational analysis was performed in R.


Convincing data has suggested that four and a half LIM domain 2 protein (FHL2) serves a key function in cancer cell metastasis and that microRNA (miR)-340-5p can regulate cancer cell migration. The current study hypothesized that targeting FHL2 expression by miR-340-5p in colon cancer may attenuate colon cancer cell migration and invasion. FHL2 expression was therefore assessed in colon cancer microarray datasets using Qlucore omics explorer as well as in HT-29 and AZ-97 colon cancer cell lines via reverse transcription-quantitative PCR (RT-qPCR). Colon cancer cell migration and invasion were evaluated in the presence of miR-340-5p mimic, mimic control or mimic with a target site blocker. Confocal microscopy and RT-qPCR were subsequently performed to assess FHL2, E-cadherin (E-cad) protein and mRNA expression in colon cancer cells. Microarray dataset analysis revealed that FHL2 expression was lower in primary colon cancer cells compared with normal colonic mucosa. It was revealed that the expression of miR-340-5p and FHL2 were inversely related in serum-grown and low-serum conditions in HT-29 and AZ-97 cells. Short-time serum exposure to low-serum grown cells induced FHL2 expression. Transfection of HT-29 cells with miR-340-5p mimic not only decreased serum-induced expression of FHL2 but also decreased cancer cell migration and invasion. Bioinformatics analysis revealed that FHL2 mRNA had one putative binding site for miR-340-5p at the 3-untranslated region. Blocking of the target site using a specific blocker reverted miR-340-5p mimic-induced inhibition of FHL2 expression and cancer cell migration and invasion. Confocal microscopy confirmed that the reduction of FHL2 expression by miR-340-5p mimic also reversed serum-induced E-cad disruption and that the target site blocker abrogated the effect of miR-340-5p. The current results suggested that miR-340-5p could be used to antagonize colon cancer cell metastasis by targeting the FHL2-E-cad axis. Colon cancer  is the second most frequent cause of cancer-related death in the world . The oveFour and a half LIM domain 2 protein (FHL2) is a multifunctional oncoprotein involved in cancer progression and metastasis ,8. FHL2 E-cadherin (E-cad) is an important epithelial cell adhesion molecule and loss of its function is associated with epithelial cancer cell migration and invasion . E-cad smiRNAs are 21–22 base pair long non-coding RNAs that post-transcriptionally down-regulate mRNA by targeted degradation or blocking translation initiation . AccumulBased on the considerations above, we hypothesized that miR-340-5p might regulate colon cancer cell migration and invasion via targeting FHL2-E-cad axis. For this purpose, we used metastatic human colon cancer cell lines to evaluate miR-340-5p-mediated suppression of colon cancer cells migration and invasion.Four human colon cancer-related gene microarray datasets  were downloaded from the Gene Expression Omnibus (GEO) database of NCBI . GDS4382 and GSE115313 were used to compare FHL2 expression between normal colon mucosa and colon cancer tissue. GDS4393 and GDS4516 were used to compare FHL2 expression between primary colon cancer, metastatic cancer and metastatic recurrence samples. GDS4382 dataset contains 17 colon cancer tumors and 17 adjacent non-cancerous tissues. GSE115313 data set contains 42 paired tumor and normal colon mucosa samples from a cohort study of 42 colon cancer patients. GDS4393 dataset contains 33 primary and 21 metastatic lesions from patients with unresectable colon cancer. GDS4516 dataset contains 66 non metastasized tumors, 20 metastasized tumors and 18 metastatic recurrence tumors of colon cancer patients. GDS4382, GDS4393 and GDS4516 datasets were from GPL570: [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. GSE115313 dataset was from GPL16686: [HuGene-2_0-st] Affymetrix Human Gene 2.0 ST Array [transcript (gene) version] platform. Qlucore Omics Explorer version 3.6  was used2. miR-340-5p mimic and mimic-Ctrl were purchased from Life Technologies. TransIT-TKO transfection reagent (Mirus) was used to evaluate the role of miR-340-5p. To study the biological function of miR-340-5p, a target site blocker (TSB) was purchased from Exiqon A/S (Vedbaek). The miRCURY LNA_TSB was designed to specifically compete with the miR-340-5p.The human epithelial colorectal cancer cell line HT-29 was obtained from American Type Culture Collection . HT-29 cell line was characterized to have metastatic properties ,33. AZ-96 cells were seeded in a 6-well culture plate. Cells were then transfected with miR-340-5p mimic (50 nM) or mimic-Ctrl (50 nM) for 24 h by using Mirus transfection reagent in Opti-MEM reduced serum media according to manufacturer's instructions. After 24 h, cells were harvested and expression of miR-340-5p and FHL2 was analyzed by RT-qPCR. To evaluate the effect of short serum exposure to serum starved cells, transfected cells were cultured in Opti-MEM reduced serum media for 24 h and then exposed to 10% BSA for 30 min. RNA samples were extracted by using TRIzol  and purified using Direct-zol RNA extraction kit (Zymo Research) according to manufacturer's recommendations. cDNA was synthesized using total RNA (0.4 µg) by Mir-XTM miRNA First-Strand Synthesis Kit. Expression of miR-340-5p, FHL2 and E-cad mRNA were quantified using miR-X™ miRNA RT-qPCR SYBR® kit (Clontech). The PCR primers used were as follows; hsa-miR-340-5p specific sense 5′-GGCTTATAAACGAATCACAGTCATTAAAA-3, FHL2 mRNA sense; 5′-GAAACTCACTGGTGGACAAGC-3′, antisense; 5′-GTGGCAGATGAAGCAGGTCT-3′, E-cad mRNA sense; 5′-ACAGCCCCGCCTTATGATT-3′, antisense; 5′-TCGGAACCGCTTCCTTCA-3′, U6 sense; 5′-GCTTCGGCAGCACATATACTA-3′, U6 antisense; 5′-CGAATTTGCGTGTCATCCTTG-3′, β-actin sense; 5′-AGAGCCTCGCCTTTGCCGATCC-3′, antisense; 5′-CACATGCCGGAGCCGTTGTCG-3. Expression of miR-340-5p relative to U6 snRNA and expression of FHL2 as well as E-cad relative to β-actin were determined using 2−ΔΔCq method.HT-29 and AZ-97 colon cancer cells were cultured to 70–80% confluency. After that cells were serum starved (0.1% serum) overnight. On the next day 1×10http://www.targetscan.org/). To evaluate the function of the binding site, 20 nucleotides long target site blocker (TSB) was designed to specifically compete with the mir-340-5p in the 3′-UTR of FHL2 mRNA. The miRCURY LNA_TSB The blocker was synthesized as fully phosphorothiolated Locked Nucleic Acids (LNA) in the DNA sequences to increase their affinity and selectivity for the target. Under serum starved conditions, the target site blocker TSB_FHL2_miR-340-5p; 5′-TTATAAAGTAGTTACAGCCT-3′ were co-transfected with the miR-340-5p mimic (50 nM). FHL2 and E-cad mRNA and proteins levels were quantified using RT-qPCR and confocal imaging, respectively.Target Scan prediction tool was used to predict binding site for miR-340-5p at the 3′-UTR of FHL2 mRNA . Data are expressed as the mean number of migrated cells per high power field.Migration and invasion assays were performed using 24-well cell migration chambers with 8 µm pore size inserts (Corning Coster) as described previously . For invFor immunofluorescence imaging of E-cad and ki67, cancer cells were grown to 60–70% confluency and then cells were transfected with either miR-340-5p mimic (50 nM) alone or mimic-Ctrl (50 nM) alone or miR-340-5p (50 nM) in the presence of TSB or TSB-Ctrl for 24 h in Opti-MEM serum reduced media as on glass coverslips as described above. Next day, cells were exposed to 10% BSA for 30 min. Cells were then fixed with 2% formaldehyde and permeabilized with 0.2% Triton X-100 for 20 min. After fixation and permeabilization, cells are then washed two times with PBS containing 2% fetal bovine serum. Samples were then incubated with primary antibodies: Fluorescein isothiocyanate (FITC) conjugated anti-ki67 antibody  and rabbit anti-human FHL2 antibody  in PBS containing 2% BSA overnight. In a separate experiment for E-cad staining, samples were first incubated with rabbit anti-human E-cad  primary antibody in PBS containing 2% BSA overnight. After washing two times, all samples were incubated with rat anti-rabbit allophycocyanin (APC) conjugated secondary antibody  for 1 h. After immunostaining, coverslips were rinsed with PBS twice and then stained with Hoechst 33258  for 10 min. ProLong Diamond Antifade Mountant  was added on all coverslips before putting on the slides. Confocal z-stakes images were taken using LSM 800 confocal microscope (Carl Zeiss) and orthogonal projection images were created using all slices for a total height of ~10 µm. Images were taken by using a ×63 oil immersion objective (numeric aperture=1.25) and processed later using ZEN2012 (Carl Zeiss) software.in vitro experiments were performed using GraphPad Prism 8 software. For multiple comparisons, we used one-way analysis of variance (ANOVA) followed by the Tukey's post hoc test. For comparison between two groups, we used two-tailed t-test. P<0.05 was considered to indicate a statistically significant difference.Statistical analyses for Knowing that FHL2 expression is different in different tissues, we first compared FHL2 expression between normal colon mucosa and colon cancer in different datasets. Datasets GDS4382 and GSE115313 analysis revealed that colon cancer tissue had significantly lower levels of FHL2 expression than matched normal non-cancerous colonic tissue . We alsoExpression of miR-340-5p and FHL2 were assessed in HT-29 and AZ-97 colon cancer cell lines in low-serum (to mimic stress condition of tumor microenvironment) and serum-grown culture conditions by RT-qPCR. It was found that expression of FHL2 mRNA was significantly higher in serum-grown HT-29 and AZ-79 cell lines compared to low-serum condition . FurtherTo investigate whether FHL2 oncogene had any binding site for miR-340-5p, we utilized bioinformatics based target site prediction tool (TargetScan). Our analysis revealed that there is one potential binding site for miR-340-5p at 3′-UTR of FHL2 mRNA where it contains complementary sequences of perfect 8′mer base-pair match to the seeding region of miR-340-5p . We desiShort-time (30 min) serum stimulation increased FHL2 mRNA expression and transfection of miR-340-5p mimic was found to reduce the expression of FHL2 mRNA . MoreoveTranswell migration and invasion assays were performed using 10% FBS as a chemoattractant in the lower chamber of the wells. It was found that miR-340-5p regulates colon cancer cell invasion and migration by targeting FHL2. Serum stimulant significantly increased invasion and migration of HT-29 cells . InteresTo further examine the downstream target protein of FHL2 we examined E-cad expression, which is known to regulate cancer cell metastasis. Short-term serum stimulation significantly decreased E-cad mRNA expression in HT-29 cells . The inhMetastasis of colon cancer is a major cause of colon cancer-related deaths all over the world. Accumulating data suggest that FHL2 oncogene plays an important role in cancer cells migration and invasion. However, the role of FHL2 in colon cancer migration and invasion has not been examined. Herein, we show for the first time that FHL2 acts as an important regulator in colon cancer cell migration and invasion under specific conditions. Furthermore, we found that miR-340-5p has a functional binding site on FHL2 mRNA and that targeting of FHL2 with miR-340-5p mimic reduces cancer cell migration and invasion. Thus, targeting of FHL2 by miR-340-5p could be a useful strategy to inhibit colon cancer metastasis.et al (FHL2 has been demonstrated to play important roles in the cancer process including proliferation, differentiation and migration . Howeveret al  reportinAccumulating data suggest that miRNAs have characteristic expression patterns in different types of cancers and can regulate cancer progression and metastasis ,38–41. FTo investigate the transient effect of serum on the stressed cancer cells, we cultured transfected cells in low serum conditions for 24 h and then exposed the cells to serum for 30 min. We observed that this short serum exposure increased FHL2 expression in colon cancer cells and that transfection of cells with miR-340-5p mimic significantly reduced serum-induced FHL2 expression both in mRNA and protein levels. Moreover, we found that transfection of HT-29 cells with miR-340-5p reduced cancer cell proliferation in terms of ki67 positive cells. Considering the fact that FHL2 regulates cancer cell migration and proliferation , these fet al reporting that irregular distribution of E-cad is associated with EMT (et al (Knowing that down-regulation of E-cad is an important step in cancer cell progression and metastasis ,47; we nwith EMT . In addiT (et al  showed tT (et al , breast T (et al  and prosT (et al . NotablyTaken together, we conclude that expression of FHL2 in stressed cancer cells play an important role in colon cancer cell migration and invasion. This study shows that inhibition of FHL2 expression by using miR-340-5p mimic reduces colon cancer cells migration and invasion. Moreover, inhibition of FHL2 attenuates cancer cell proliferation and increases E-cad expression in colon cancer cells, suggesting that targeting FHL2 by miR-340-5p might be a useful approach to antagonize colon cancer cell metastasis.


Expression of the phosphatase of regenerating liver-3 (PRL-3) is known to promote tumor growth in gastrointestinal adenocarcinomas, and the incidence of tumor formation upon inflammatory events correlates with PRL-3 levels in mouse models. These carcinomas and their onset are associated with the impairment of intestinal cell homeostasis, which is regulated by a balanced number of Paneth cells and Lgr5 expressing intestinal stem cells (Lgr5+ ISCs). Nevertheless, the consequences of PRL-3 overexpression on cellular homeostasis and ISC fitness in vivo are unexplored. Here, we employ a doxycycline-inducible PRL-3 mouse strain to show that aberrant PRL-3 expression within a non-cancerous background leads to the death of Lgr5+ ISCs and to Paneth cell expansion. A higher dose of PRL-3, resulting from homozygous expression, led to mice dying early. A primary 3D intestinal culture model obtained from these mice confirmed the loss of Lgr5+ ISCs upon PRL-3 expression. The impaired intestinal organoid formation was rescued by a PRL inhibitor, providing a functional link to the observed phenotypes. These results demonstrate that elevated PRL-3 phosphatase activity in healthy intestinal epithelium impairs intestinal cell homeostasis, which correlates this cellular mechanism of tumor onset with PRL-3-mediated higher susceptibility to tumor formation upon inflammatory or mutational events.Key messages• Transgenic mice homozygous for PRL-3 overexpression die early.• PRL-3 heterozygous mice display disrupted intestinal self-renewal capacity.• PRL-3 overexpression alone does not induce tumorigenesis in the mouse intestine.• PRL-3 activity leads to the death of Lgr5+ ISCs and Paneth cell expansion.• Impairment of cell homeostasis correlates PRL-3 action with tumor onset mechanisms.The online version contains supplementary material available at 10.1007/s00109-021-02097-9. PTP4A3 knockout mouse model was shown to be grossly normal, supporting non-essential roles for PRL-3 in healthy tissue  × 30 cycles; 72°C × 5min.For protein extraction and immunoblots, mouse tissues or organoids were lysed on ice in RIPA lysis buffer  containing complete protease inhibitors (Roche). For organoids beforehand, lysis Matrigel was removed using cell recovery solution (Corning) as described in manufacturer’s protocol. Samples were then boiled for 10 min and cleared by centrifugation. Equal amounts of lysate protein were separated performing SDS-PAGE. Western blotting was performed with anti HA mouse primary antibody (Roche) and anti actin rabbit primary antibody (SIGMA) and then with secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase . Visualization was achieved with the Western Lightning Plus-ECL solution (PerkinElmer).https://qupath.github.io/.For immunohistochemical staining, formalin-fixed paraffin embedded 5-μm sections derived from different mouse tissues were used. Following deparaffinization with xylene and rehydration with graded ethanol, antigen retrieval was performed using 0.09% (v/v) unmasking solution (Vector Labs) for 30 min in a steamer. Inactivation of endogenous peroxidases was carried out using 3% hydrogen peroxide (Sigma) for 10 min. Primary antibodies used, anti HA mouse , anti ki67 pre-diluted (MEDAC 275R-18) mouse, and/or anti cleaved caspase-3 rabbit  were diluted in 10% goat serum (Jackson Immuno) in TBS-Tween 0.1%. Secondary antibody staining and biotin-streptavidin incubation were performed using species-specific VECTASTAIN Elite ABC kits (Vector Labs). DAB Peroxidase Substrate kit (Vector Labs) was utilized for antibody detection. Eosin Y and hematoxylin were from Bio-Optica and Vector Labs. Tissue pictures were obtained using Tissue tek. Pictures were then analyzed using the QuPath software from For the immunofluorescence staining of 3D cell cultures, first the samples were rinsed with phosphate-buffered saline (PBS) twice and fixed with 4% paraformaldehyde (PFA) for 20 min. PFA was then quenched with 0.1 M glycine in PBS for 10 min, and then cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and blocked with 10% FBS in PBS with 0.5% Triton X-100 (blocking solution) for 60 min. Then, samples were incubated with primary antibodies anti HA rat high affinity (Roche 11867423001), and cleaved caspase-3 rabbit , or anti KI67 pre-diluted (MEDAC 275R-18) mouse in blocking solution at 4°C overnight, followed by washing and incubation with the corresponding secondary Alexa-fluorophore-conjugated antibody  for 1 h. Rhodamine-conjugated phalloidin 1:1000  and Hoechst 33342 (10 μg/ml) were also used for membrane and nuclei labeling, respectively. Cell images were acquired at room temperature on a spinning disc confocal microscope PerkinElmer UltraView ERS FIJI software . Contrast or brightness adjustment was applied to the whole image. In FIJI software, a 50-pixel rolling ball radius was used to subtract background, and a 1-pixel-radius median filter was applied.For immunofluorescence in intestinal frozen tissue samples OCT (Tissue Tek) embedded 10-μm tissue sections were warmed for 30 min and then washed with PBS-Tween 1% for 15 min. The sections were then blocked with 2% BSA (SIGMA) and 8% goat serum (Jackson Immuno-research) in PBS-Tween 0.1% for 1h. Primary antibodies in the blocking buffer were added to the slides and incubated overnight at 4°C (HA rat Roche 11867423001 (1:1000) and rabbit anti-GFP Abcam 6556 (1:1000)). Samples were washed with PBS. The secondary antibodies were also dissolved in blocking buffer (Goat anti-Rat IgG (H+L) Alexa Fluor 647 A-21247; Goat anti-rabbit IgG (H+L) Alexa Fluor 594 A11037; Hoechst 33342 H3570  Samples were washed with PBS and mounted with Fluoromount-G (eBioScience). Immunofluorescence images were acquired at room temperature on a Zeiss LSM880 laser scanning confocal microscope using a Plan-Apochromat 25×0.8 NA glycerin objective. Images were processed using FIJI software. Contrast or brightness adjustment was applied. For visualization reasons, the brightness of the red HA-PRL-3 signal in the HA-PRL-3 homo/Lgr5-GFP sample was slightly increased compared to the HA-PRL-3 het/Lgr5-GFP sample   as (a) ISCs as Lgr5- EGFPOrdinary two-tailed one- or two-way ANOVA and Tukey’s or Dunnett’s multiple comparison tests were performed to study statistical significance: *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001. Statistical analysis and graphs were generated with Prism (GraphPad).Control samples for in vivo experiments were obtained from animals containing the transgenes but kept on a normal diet or HA-PRL-3 wt/R26-rtTA het mice on dox.Non-induced organoid cultures (without dox) derived from HA-PRL-3 wt/R26-rtTA het mouse intestine on a normal diet were considered as controls in the in vitro assays.Fig s1.Design and characterization of PRL-3 TG mice. A: Schematic representation of the plasmid construct encoding the HA-Ptp4a3 gene used for ES cell electroporation and of the strategy for insertion in the ColA1 locus. B: Western blot analysis of ES clones electroporated with HA-PRL-3 vector using HA antibody. Cells were treated in vitro with 1μg/mL of dox for 48h before the protein extraction. C: PRL-3 mRNA extracted from the denoted organs from HA-PRL-3 het / R26-rtTA het TG mice (FA1) after 15 days on dox was assayed using one-step RT-PCR using mRNA encoding β-actin as a loading control. D: Western blots using tissue samples derived from HA-PRL-3 wt / R26-rtTA het (control) fifteen days on dox and HA-PRL-3 het / R26-rtTA het (HA-PRL-3 het) mice fed with dox impregnated pellets for 0 (not treated), 2 or 15 days. HA and actin (loading control) signals were detected using specific antibodies. E: spleen weight or F: liver weight relative to body weight (a.u. = arbitrary units), in control and HA-PRL-3 het mice from the FA1 colony treated 12 months with dox. N=10. G: Body weight by gender of males (N=4) and females (N=8) from the FB1 mouse colony fed with dox food for 12 months showed no significant (ns) differences in body weight (g=grams) by Oneway Anova with Turkey’ s multiple comparison test compared to control mice. H, I same representations as in E, F but for FB1 mouse colony. N=11. D, E, G, H: Unpaired T test, non-significant differences. K: Kaplan Meier survival curve of HA-PRL-3 het / R26-rtTA het (PRL-3 het), HA-PRL-3 homo / R26-rtTA het (PRL-3 homo) and HA-PRL-3 wt / R26-rtTA het (control) mice without doxycycline. N=10 for each genotype. All genotypes presented the same life span when PRL-3 expression was not induced with doxycycline. (PNG 1038 kb)High Resolution Image (TIF 1259 kb)Fig S2.B: Quantification of the Western Blot shown in A. PRL-3 HA expression is presented in relation to β actin loading control (HA-PRL-3 / β actin ratio). HA-PRL-3 het and HA-PRL-3 homo demonstrate the average of sample 1 and 2, respectively. C: Representative images of KI67 staining of colon biopsies from mice treated 3 months with dox upper panel, or 12 months middle panel. The lower panel shows the HA staining for PRL-3 detection in mice after 12 months on dox food. D: Percentage of KI67 positive cells  in the colonic crypts in the different conditions represented in C. 1000 cells were analyzed per tissue sample. Values represent mean ± s.d. of three biological replicates. Statistics: P≤0.1 t-test, compared to control mice. (PNG 2868 kb)A: Western Blot analysis of small intestine derived tissue samples from HA-PRL-3 wt / R26-rtTA het (control), HA-PRL-3 het/R26-rtTA het (HA-PRL-3 het) and HA-PRL-3 homo / R26-rtTA het (HA-PRL-3 homo) mice treated two days with dox. HA signal was detected using specific antibodies. β actin was used as a loading control. High Resolution Image (TIF 3616 kb)Fig S3.Phenotype and HA-PRL-3 expression of colon  and SI  organoids. 3D cell cultures derived from HA-PRL-3 het / R26-rtTA het mice and HA-PRL-3 homo / R26-rtTA het mice were grown for 2 days after passaging. Subsequently, HA-PRL-3 expression was induced in vitro with 1μg/mL of dox for 2 days (late induction). A, B: Representative bright field inverted microscopy images. Pictures were taken after four days in culture for all conditions. All dox-induced organoids showed the death phenotype compared to non-induced cultures. Scale bar = 50 μm. C, D: Western blot analysis of 3D cell culture after four days. HA signal was detected using specific antibodies. β-actin was used as a loading control. E, F: Quantification of the Western blot shown in C, D. PRL-3 HA expression is presented in relation to β actin loading control (HA-PRL-3 / β actin ratio) and normalized to HA-PRL-3 het induced with dox signal. HA expression in homozygous cultures were increased compared to heterozygous. (PNG 1652 kb)High Resolution Image (TIF 2102 kb)Fig S4.A: PI assay to detect cell death in organoids never induced or late induced. B: Percentage of KI67 positive organoids observed in Fig 4a. The quantification corresponds to the percentage of organoids with any KI67 positive cell with no differences observed between control and PRL-3 het organoids. Although, the pictures in A shows higher number of KI67 positive cells in the control compare to HA-PRL-3 het organoids (with only one or two cells stained for KI67), the number of KI67 positive cells in each organoid was not possible to quantify due to the 3D structure of the organoids. C: Representative bright field inverted microscopy images of small intestinal organoids derived from HA-PRL-3 het mice never induced (no dox) or late induced (dox added in the growth media after 2 days in culture). After 2 days of induction the dox was removed from the media and the organoids were grown in dox free media for two more days. Scale bars: 100 μm. D: Percentage of organoids developed with branches, relative to total organoids, before and after DOX withdrawal. Data are mean ± s.d. N=3, ns: not significant, ****P≤0.0001 ordinary two-tailed one-way ANOVA with Tukey's multiple comparison test. (PNG 2313 kb)High Resolution Image (TIF 2832 kb)ESM 5Entire Western blot membranes. A: Entire western blot membranes for the ES clones electroporated with HA-PRL-3 vector using HA antibody and showed in Fig S1B. B: Entire western blot membranes showed in Fig 1a and Fig S1D using tissue samples derived from HA-PRL-3 wt / R26-rtTA het (control) 15 days on dox and HA-PRL-3 het / R26-rtTA het (HA-PRL-3 het) mice fed with dox impregnated pellets for 0 (not treated), 2 or 15 days. Upper membranes correspond to anti HA antibody for HA-PRL-3 detection and lower membranes anti β actin antibody as a loading control. C: Entire western blot membranes showed in Fig S2A using small intestine tissue samples derived from HA-PRL-3 wt / R26-rtTA het (control), HA-PRL-3 het/R26-rtTA het (HA-PRL-3 het) and HA-PRL-3 homo / R26-rtTA het (HA-PRL-3 homo) 2 days on dox. Upper membranes correspond to anti HA antibody for HA-PRL-3 detection and lower membranes correspond to β actin used as a loading control. D: Entire western blot membranes showed in Fig S3C,D. Upper membranes correspond to anti HA antibody for HA-PRL-3 detection and lower membranes anti β actin antibody as a loading control. For further information see Fig S3 caption. (PDF 2433 kb)


Acrostalagmus, Arthrinium, Chaetomium, Cladosporium, Coniothyrium, Curvularia, Dichotomomyces, Eurotium, Eutypella, Exophiala, Fusarium, Hypocrea, Microsphaeropsis, Microsporum, Neosartorya, Nigrospora, Paecilomyces, Penicillium, Pleosporales, Pseudallescheria, Scedosporium, Scopulariopsis, Stagonosporopsis, Thielavia, Westerdykella, and Xylariaceae. Around 347 alkaloid metabolites were isolated and identified via chromatographic and spectroscopic techniques comprising 1D and 2D NMR  which were further confirmed using HR-MS (high resolution mass spectrometry) and Mosher reactions for additional ascertaining of the stereochemistry. About 150 alkaloids showed considerable effect with respect to the tested activities. Most of the reported bioactive alkaloids showed considerable biological activities mainly cytotoxic followed by antibacterial, antifungal, antiviral, antioxidant; however, a few showed anti-inflammatory and antifouling activities. However, the rest of the compounds showed weak or no activity toward the tested biological activities and required further investigations for additional biological activities. Thus, alkaloids isolated from marine-associated fungi can afford an endless source of new drug entities that could serve as leads for drug discovery combating many human ailments.Marine-associated fungal strains act as a valuable reservoir of bioactive diverse secondary metabolites including alkaloids which are highly popular by their biological activities. This review highlighted the chemistry and biology of alkaloids isolated from twenty-six fungal genera associated with marine organisms and marine sea sediments. The selected fungi are from different marine sources without focusing on mangroves. The studied fungal genera comprises  Nowadays fungi isolated from marine resources serve as promising tools for the alleviation of a large number of hazardous diseases that adversely affect human health such as bacterial and viral infections as well as cancers ,2. TheseIn addition, these fungal metabolites showed an acceptable oral-bioavailability and physico-chemical manner offering a safer biomedical alternative relative to synthetic entities that constitute a crucial importance in the process of formulation of various dosage forms ,9. BesidAcrostalagmus, Arthrinium, Aspergillus, Chaetomium, Cladosporium, Coniothyrium, Curvularia, Eurotium, Eutypella, Exophiala, Fusarium, Hypocrea, Microsphaeropsis, Microsporum, Neosartorya, Nigrospora, Paecilomyces, Penicillium, Pleosporales, Pseudallescheria, Scedosporium, Scopulariopsis, Stagonosporopsis, Thielavia, Westerdykella, and Xylariaceae. The fungal genera were classified in an alphabetical order where 347 alkaloid compounds were reported. The majority of the reported alkaloids revealed antioxidant, cytotoxic, antiviral, antifungal, antibacterial, and anti-inflammatory as well as antifouling activities. Relevant data of the mentioned fungal strains were collected till December 2020 from different databases including Scifinder  for studies about chemical constituents; however, for biology-related researches, data were gathered from both PubMed  as well as Web of Knowledge .Thus, this review aimed to comprehensively explore the diverse alkaloids isolated and identified from nearly most of the fungal strains belonging to diverse genera associated with different marine organisms and sediments regarding their chemistry and biology. The selected fungi are from different marine sources without focusing on mangroves. These include Acrostalagmus luteoalbus obtained from deep-sea marine sediments existing in the South China Sea in addition to eight previously isolated alkaloids (3–10) as illustrated in 50 ranging between 0.23 and 1.29 μM ..Ircinia Chaetomium globosum derived from a deep- sea marine sediment from the Indian oceans led to the isolation of a series of cytoglobosins comprising chaetoglobosin B (23), chaetoglobosin C (24), isochaetoglobosin D (25), chaetoglobosin E (26), chaetoglobosin F (27), chaetoglobosinFex (28) in addition to the new alkaloids, cytoglobosins H (29) and I (30). In the evaluation of the cytotoxicity of the previously mentioned compounds on MDA-MB-231, B16F10, and LNCaP, only chaetoglobosin E (26) exhibited a pronounced antiproliferative activity via induction of apoptosis on LNCaP as well as B16F10 cancer cells displaying IC50 of 0.62 and 2.78 µM, respectively ..EurotiumHibiscus tiliaceus-derived Eurotium rubrum which are variecolorin G (59), 12-demethyl-12-oxoeurotechinulin B (81), variecolorin J (82), variecolorin H (83), cryptoechinuline G (84), alkaloid E-7 (85), isoechinulin B (86) eurotechinulin B (87), 7-isopentenylcryptoechinuline D (88). These compounds showed no antibacterial activity versus Escherichia coli and Staphylococcus aureus and weak antifungal activity against the examined fungi which are Alternaria brassicae, Fusarium oxysporium, and Physalospora piricola. The compounds were evaluated for their cytotoxic activity versus a number of cell lines namely, Hela, Du145, SMMC7721, MCF-7, SW1990, NCI-H460. 12-Demethyl-12-oxoeurotechinulin B (81), variecolorin G (59), and alkaloid E-7 (85) displayed a potent activity versus one or two of these cell lines with IC50 ranging between 20 and 30 μg/mL ..ExophialFusarium oxysporum, isolated from the marine mudflat collected at Suncheon Bay, Korea, resulted in the isolation of a new polycyclic quinazoline alkaloid, oxysporizoline (153) that revealed an antibacterial activity against MRSA and MDRSA with MIC equal to 6.25 µg/mL in addition to notable antioxidant potential manifested by its observable radical scavenging effect versus DPPH with IC50 equals to 10 µM -trione (154) (Fractionation and purification of the different fractions obtained from the extract obtained from marine-derived fungus ne (154)  39]..HypocreaMicrosphaeropsis, which was collected from the Huanghua in the Bohai Sea, resulted in the isolation of three alkaloids which are fumiquinazolines L (155) and N (156) and notoamide D (157)   40]..MicrosphLomentaria catenata, collected at Guryongpo, NamGu, PoHang in Republic of Korea. The alkaloid revealed a potent cytotoxic effect on HeLa cells inducing apoptosis manifested by the p21, p53, Bax, Bcl-2, caspase 3, and caspase 9 expressions. Neoechinulin A (99) effectively enhances cell apoptosis via the downregulation of Bcl-2 expression with concomitant upregulation of Bax expression and enhancement of caspase-3 as evidenced by the Western blot (Neoechinulin A (99), a prenylated indole alkaloid, was isolated from the extract of the culture broth of marine-derived Microsporum sp., isolated from the surface of a marine red alga ern blot  41]..LomentarNeosartorya fischeri, isolated from marine mud in the intertidal zone of Hainan Province of China, resulted in the characterization of three alkaloids, two of which are new namely, tryptoquivaline T (158), tryptoquivaline U (159) in addition to fiscalin B (160). All the tested compounds showed notable cytotoxic potential evidenced by induction of HL-60 cells apoptosis displaying IC50 values of 82.3, 90.0, and 8.88 μM respectively ..NeosartoNigrospora oryzae, a marine-derived fungus isolated from a marine gorgonian Verrucella umbraculum collected in the South China Sea near Sanya City. The former is a pyrrolidinone alkaloid meanwhile the latter is indole type alkaloid. The absolute configuration of both compounds were determined employing Mosher reaction  and nigrospin A (167) were isolated from reaction  45]..NigrospoPaecilomyces variotii, isolated from Grateloupia turuturu, a marine red alga collected from the coast of Qingdao, China, was comprehensively investigated using various chromatographic and spectral techniques and led to the isolation of dihydrocarneamide A (168), iso-notoamide B (169) which were two new prenylated indole alkaloids in addition to varioxepine A (170), a new 3H-oxepine-containing alkaloid. Dihydrocarneamide A (168) and iso-notoamide B (169) displayed weak cytotoxic potential versus NCI-H460 cell lines (human large cell lung carcinoma) with IC50 values equal to 69.3 and 55.9 μmol/L, respectively . Ci. CiPeniccitrinum . ScalusaP. citrinum, isolated from marine sediments collected from Langqi Island, Fujian, China. It revealed a potent anti-proliferative activity in A-375  cells where it significantly enhanced the cell apoptosis via reducing Bcl-2 expression and enhancing Bax expression. Besides, it reduced the metastatic potential of A-375 cells by virtue of controlling MMP-9 and TIMP-1 expression -1,3-oxazine (226), and (E) -2-(-hept-5-enyl)-8-(hydroxyimino)- 3-methyl-4-oxo-6,7,8,8a-tetrahydro-4H-pyrrolo -1,3-oxazine (227) were isolated which were not detected without the addition of ScCl3. These compounds showed no cytotoxicity versus SKMEL-2 (human skin cancer), HepG2 (human liver cancer), XF-498 (human CNS cancer), HCT115 (human colon cancer), and MCF-7 (human breast cancer) ..P. citriP. commune associated with deep sea obtained sediments collected from the South China Sea, Sansha City is a rich source of oxindole alkaloids where nine new compounds, cyclopiamides B–J (232–240) were isolated and structurally elucidated using different spectroscopic techniques in addition their absolute configurations were determined using single crystal X-ray diffraction and ECD techniques . In. InP. cof 7.0 µM .P. griseofulvum derived from the sediment of a deep ocean represents a rich source of bioactive indole alkaloids from which three new alkaloids, variecolorins M–O (253–255), and other known ones were isolated. These known alkaloids are tardioxopiperazine A (256), didehydroechinulin (257), neoechinulin A (99), isoechinulin B (60), neoechinulin (61), variecolorin H (71), echinuline (95), preechinulin (97). Variecolorins M–O (253–255) revealed a weak antioxidant potential manifested by their IC50 values in DPPH radical scavenging capacity assay which are 135, 120, and 91 µM, respectively meanwhile none of them exhibited cytotoxic effect versus BEL-7402, HL-60, and A-549 cell lines as examined using both MTT and SRB assays  and 3-methoxy-4-hydroxy-4-(4′methoxyphenyl)-3,4-dihydro-2 (1H)-quinolinone. Compound (259) only showed cytotoxic effect on SKOV-3 cells -3,4-dihydro-2(1H)-quinolinone (258) and 3R*,4R*dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (259) were isolated from -3 cells  70]..P. janczP. janthinellum, marine rhizosphere soil-derived fungus, acts as a rich source of prenylated indole alkaloids, two of which are new compounds, paraherquamide J–K (261–262) whereas paraherquamides A, E, and SB200437 (263–265) were previously isolated. Unfortunately none of the isolated compounds revealed any antibacterial, topoisomerase I inhibitory effects or lethal effects versus brine shrimp Artemia salina . In. InP. jaompounds .β-carbolines in addition to penipaline C (278), indole carbaldehyde derivative, were isolated from P. paneum derived from deep-sea-sediment. Additional two known alkaloids, dimethyl-1H-β-carboline-3-carboxylic acid (279) and 1,7-dihydro-7,7-dimethylpy-rano indole-3-carbaldehyde (280) were also isolated. Upon testing the cytotoxic effect of the new compounds versus A-549 and HCT-116 cell lines, only penipaline B (277) and C (278) showed notable effect versus both cell lines with IC50 of 20.4 and 21.5 μM, respectively against A-549 and 14.8 and 18.5 for them, respectively versus HCT-116 ..PseudallS. apiospermum fed by different amino acids led to the isolation of various new alkaloids comprising scedapins A–E (320–324) in addition to scequinadoline D (325). Both scedapin C (322) and scequinadoline D (325) showed promising antiviral potential versus hepatitis C  in addition to two known alkaloids, lumichrome (327) and WIN 64,821 (328). They showed weak cytotoxic effect versus e L5178Y mouse lymphoma cell line ..StagonosThielavia, a marine-derived fungus isolated from the marine mudflat collected at Gomso Bay, Korea, displayed antimicrobial effect against MRSA (methicillin-resistant Staphylococcus aureus) and MDRSA (multidrug resistant Staphylococcus aureus) with MIC values equal to 6.25 and 12.5 µg/mL, respectively. In addition, it showed an effective radical-scavenging activity against 2,2-dipheny1–1-picrylhydrazyl (DPPH) displaying 11 µM as an IC50 (Thielaviazoline (338) isolated from  an IC50  88]..ThielaviWesterdykella dispersa, obtained from marine sediments, which were collected at South China Sea, Guangzhou, Guangdong province, China. It showed substantial effect against B. subtilis with MIC equals 12.5 µg/mL. In addition, it showed certain inhibitory potential versus MCF-7, HepG2, A549, HT-29, and SGC-7901 cell lines with IC50 values ranging between 25.6 and 83.7 µM. (Gymnastatin Z (339), a new tyrosine-derived alkaloid was isolated from 83.7 µM.  89]..WesterdyXylariaceae species, isolated from the South China Sea gorgonian coral Melitodes squamata, is a rich source of alkaloids, from which 5-(2′-hydroxypropyl) yridine-3-ol (340), a new alkaloid in addition to seven known compounds termed 3-hydroxy-5-methyl-5,6-dihydro7H-cyclopenta[b]yridine-7-one (341), penicillenol A1 and A2 (342–343), quinolactacin A1, A2, C1, and C2 (344–347) was isolated  revealed strong antifouling potential versus Bugula neritina larval settlement with an EC50 value of 6.21 μg/mL A marine-derived isolated . CompounA pie chart representing the percentages of the biological activities exerted by the different bioactive alkaloids is represented in Thus, it can be concluded that marine-derived fungal strains are a very rich source of alkaloids. About 347 alkaloid metabolites were isolated from about twenty-six genera of fungi. About 150 alkaloids showed considerable effect with respect to the tested activities. Most of the reported bioactive alkaloids showed considerable biological activities mainly cytotoxic followed by antibacterial, antifungal, antiviral, antioxidant; however, a few showed anti-inflammatory and antifouling activities. However, the rest of the compounds showed weak or no activity toward the tested biological activities. Thus, alkaloids isolated from marine-associated fungi can afford an endless source of new drug entities that could serve as leads for drug discovery combating many human ailments. However, further investigations for additional biological activities for alkaloids that revealed no activity should be performed.


One week after second vaccinations were administered, an outbreak of B.1.1.7 lineage severe acute respiratory syndrome coronavirus 2 infections occurred in a long-term care facility in Berlin, Germany, affecting 16/20 vaccinated and 4/4 unvaccinated residents. Despite considerable viral loads, vaccinated residents experienced mild symptoms and faster time to negative test results. Outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in long-term care facilities (LTCF) are of great concern and have been reported to have high case-fatality rates (https://www.pfizer.com) has demonstrated high efficacy against COVID-19  mRNA vaccine BNT162b2  B.1.1.7 rapidly became the predominant lineage in Europe in 2021. Analyses estimated that B.1.1.7 has increased transmissibility and a https://www.ema.europa.eu/en/documents/scientific-guideline/ich-e-6-r2-guideline-good-clinical-practice-step-5_en.pdf).We investigated a SARS-CoV-2 B.1.1.7 outbreak in a LTCF, which involved 20 BNT162b2-vaccinated residents and 4 unvaccinated residents. We report on clinical outcomes, viral kinetics, and control measures applied for outbreak containment. The study was approved by the ethics committee of Charité–Universitätsmedizin Berlin (EA2/066/20) and conducted in accordance with the Declaration of Helsinki and guidelines of Good Clinical Practice  result in 1 caregiver in a LTCF in Berlin, Germany. Among 24 residents of the unit under their responsibility, 20 (83%) residents had received the second dose of BNT162b2 on January 29 or 30, 2021 . Four reThe next week, testing detected 7 additional infections, resulting in 4/4 unvaccinated infected residents and 16/20 vaccinated infected residents. The remaining 4 vaccinated residents tested negative throughout the 30-day observation period .In addition to residents, 11/33 (33%) staff members from the unit tested positive for SARS-Cov-2 by February 18; of those, none were twice-vaccinated staff members, 2/8 (25%) had received 1 dose of BNT162b, and 9/22 (40.9%) had not been vaccinated. No infected staff required hospital treatment.Respiratory symptoms, including cough and shortness of breath, occurred in 5/16 (31.3%) vaccinated patients and all 4 unvaccinated patients Table. Al>3 weeks, and residents in the adjacent unit underwent AgPOCT every 2–3 days. The outbreak was contained within the unit; no further cases were detected.Containment measures in place included mandatory use of FFP2 or N95 masks and daily AgPOCT screening for anyone entering the facility. Immediately after detection, the facility was closed to visitors and additional containment measures were put in place, including designated staff and separate entrance, elevator, and changing rooms. Staff were required to change personal protective equipment before entering each room. Residents of all 7 units of the LTCF underwent weekly AgPOCT for All SARS-CoV-2 RNA-positive samples were tested for presence of SARS-CoV-2 VOCs by RT-PCR and complete genome sequencing . RT-PCR 10 copies/mL; p = 0.10). Furthermore, duration of SARS-CoV-2 RNA shedding was considerably shorter in vaccinated patients than in unvaccinated patients  (6 copies per mL, below which virus isolation in cell culture is usually not successful, were detected in all 4 unvaccinated patients but only in 7/16 vaccinated patients (We performed serial RT-PCR testing of nasopharyngeal swab specimens from 22 patients. SARS-CoV-2 RNA concentrations peaked within 5 days  Figure 210 SARS-CoV-2 RNA copies/mL, yielded a positive isolation outcome. Isolation attempts from samples of the same patient taken in the next 4 days and from 21 samples taken from 13 other patients were unsuccessful.We further assessed the level of infectiousness in 22 samples from 14 patients by virus cell culture . One samFive weeks after initial testing, 8/8 vaccinated and infected residents and 2/2 unvaccinated and infected residents showed robust antibody responses against SARS-CoV-2 spike antigens, virus neutralization capacity, and interferon-γ release of SARS-CoV-2–specific T cells  Figure 3We performed a longitudinal study of SARS-CoV-2 infections in a LTCF unit. Nearly all infected residents were symptomatic, including most residents that had received a second BNT162b2 dose the week before. The outbreak was caused by SARS-CoV-2 VOC lineage B.1.1.7, which might partly explain the high attack rate and lack of protection in vaccinated residents. Nevertheless, we reported a lower attack rate, a shorter duration of SARS-CoV-2 RNA shedding, and a lower proportion of symptomatic COVID-19 requiring hospitalization and oxygen support for vaccinated patients. However, despite the limited sample size and the short interval between second vaccination and infection, this outbreak raises questions about the effectiveness of the vaccination regimen in the elderly (<2 weeks after second BNT162b2 vaccination. Therefore, single-dose regimens and extended dosing intervals might be insufficient for fully protecting this population (This outbreak highlights that older adults have reduced protection Additional information about outbreak of SARS-CoV-2 B.1.1.7 lineage after vaccination in long-term care facility, Germany, February–March 2021


This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bovine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evidence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also emphasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity. Reoviridae family and contains an 11-segmented double-stranded RNA (dsRNA) genome. The genome encodes six structural proteins that form the viral triple-layered particle (TLP), which consists of the outer capsid (VP4 and VP7), the inner capsid (VP6) and the core (VP2 encasing VP1 and VP3) proteins that enclose the nucleic acid material. The viral genome also encodes five or six non-structural proteins, NSP1-6 [Rotavirus is an enteric pathogen that affects the young of many mammalian and avian species . The virhttps://talk.ictvonline.org/taxonomy/ (accessed on 20 September 2021)) based on the nucleotide sequence of the VP6 gene. RVA have been extensively studied and a classification system based on the whole genome is used to characterise these viruses. The classification is defined as genotypes Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, where x indicates a numerical number and represents the encoded proteins: VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 /P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3 genotypes . Four stThe three G6 bovine strains clustered within lineages IV and V. In lineage IV, Bov1 (detected in the FS in 2009) and Bov4 (detected in the NW in 2003) formed a discrete cluster with the previously characterised, contemporary South African bovine strain, RVA/Cow-wt/ZAF/MRC-DPRU3010/2009/G6P[5] which was detected in KZN in 2009 . These sThe four G10 sequences identified in this study formed two different sub-clusters within lineage V . Strain The five P[11] study strains  formed a discrete monophyletic cluster in lineage III and shared 96.93–98.46% nucleotide identity (97.98–98.85% amino acid identity). These strains were moderately distinct to global P[11] strains, most closely related to RVA/Human-wt/SVN/SI-R56/2007/G6P[11] and RVA/Rabbit-tc/NLD/K1130027/2011/G6P[11] sharing 96.35–97.84% nucleotide identity (97.98–98.85% amino acid identity). Similar to the G6 VP7 analysis, the two P[5] sequences of Bov1 and Bov4 clustered together with the same South African bovine strain, RVA/Cow-wt/ZAF/MRC-DPRU3010/2009/G6P[5]. These sequences shared 98.46–99.06% nucleotide identity (98.97–99.36% amino acid identity) and were distinct to global P[5] sequences . Again, In the VP6 phylogenetic tree, the study strains grouped in three different clusters independent of their G and P genotypes. Four study strains  formed a distinct monophyletic cluster with other South African bovine strains, two detected in the Western Cape (RVA/Cow-wt/ZAF/1603/2007/G6P[5] and RVA/Cow-wt/ZAF/1605/2007/G6P[5]) and RVA/Cow-wt/ZAF/MRC-DPRU3010/2009/G6P[5], as well as a South African porcine strain, RVA/Pig-wt/ZAF/MRC-DPRU3878/2008/G5P[X]. MRC-DPRU457 was identical to an unpublished South African strain, RVA/Cow-wt/ZAF/MRC-DPRU456/2009/G6P[11] . These sAll the study strains except MPT93 formed a monophyletic group within recognised lineages for VP1 (XII) and VP2 (XIV). The strains were dispersed throughout lineage X in the VP3 tree, clustering with other South African animal strains, as well as a bovine-like Mozambican strain (RVA/Human-wt/MOZ/0060b/2012/G12P[8]P[14]) previously detected in a diarrhetic infant . MPT93 wThree of the study strains  contained the rarely identified A13 NSP1 genotype. Bov4 clustered with MRC-DPRU457 and RVA/Cow-wt/ZAF/MRC-DPRU456/2009/G6P[11], sharing 98.30% nucleotide identity (98.57% amino acid identity) . As was The majority of NSP2 sequences of the study strains  formed a diverse monophyletic group together with previously characterised South African animal strains in lineage XV. Bov7 and Bov4 clustered distinctly in lineage XIII with other diverse bovine strains. MPT93 clustered in lineage V with two bovine-like strains previously detected in humans from Mozambique, RVA/Human-wt/MOZ/0060b/2012/G12P[8]P[14] and Uganda (RVA/Human-wt/UGA/MUL-13-204/2013/G8P[6]), respectively .The T6 NSP3 genes of the study strains formed two sub-clusters. MRC-DPRU457 clustered with the two Mozambican strains, MPT307 and MPT93, and formed a diverse monophyletic cluster with previously identified South African animal strains that was distinct from global strains. The study strains shared 97.04–98.68% nucleotide identity (98.68–99.34% amino acid identity) , whereasThe E2 NSP4 genes of the study strains formed three sub-clusters; two of which fell within lineage XV. MRC-DPRU457 and MPT307 clustered with previously characterised South African animal strains and a bat strain form Zambia in the southern African region. MPT93, Bov1 and 1162 clustered with a South African buffalo strain  and a French bovine strain (RVA/Cow-xx/FRA/DijonA036/2006/GXP[X]) . These sAll study strains contained a NSP5-encoding gene with a H3 genotype. All strains, except Bov7, clustered together alongside previously characterised South African bovine strains and a Zambian bat strain and shared 98.10–98.92% nucleotide identity (98.36–99.18% amino acid identity) . Bov7, sThe presence of bovine-like rotavirus strains in humans from Africa has frequently been reported ,18,19. HThese study strains contained genotypes typically associated with rotavirus detected in bovine hosts: G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3. It is noteworthy that three of the study strains contained the rarely identified A13 genotype adding to the 14 complete sequences in the NCBI GenBank. The only other published study describing an African strain with this genotype was detected in an African buffalo . The stuThe majority of the study strains almost always clustered with each other or other African animal strains. Of note, study strain RVA/Cow-wt/ZAF/MRCDPRU457/2009/G10P[11] clustered with the contemporary, previously characterised strain RVA/Cow-wt/ZAF/MRC-DPRU456/2009/G6P[11] across all genes analysed, except for the VP7 encoding gene. Bovine , buffalo  and porcine (RVA/Pig-wt/ZAF/MRC-DPRU3878/2008/G5P[X]) strains from South Africa were the most frequent closest relatives to the study strains. These were detected in the Western Cape, Limpopo and North West provinces of South Africa between 2002 and 2009 as well as in Mozambique in 2012. This indicates prolonged and extensive circulation of rotavirus strains throughout the two countries. However, the lack of whole genome data of animal rotavirus strains from South Africa and Mozambique must be noted. Unsampled local diversity is also represented in the long branch lengths in some of the trees and the low nucleotide identity between the study strains and their closest relatives.In some cases, the study strains clustered with rotaviruses detected in humans from African countries such as the NSP5 sequence of Bov7 which grouped with RVA/Human-wt/MOZ/0060b/2012/G12P[8]P[14] in a separate clade. The majority of the strains in this clade was, however, detected in animals and 0060b was also described as a bovine rotavirus strain detected in a child . This inThe VP1 sequence of MPT93 may also be derived from an interspecies reassortment event as it clustered in lineage V with mostly human strains. The closest relative was a human rotavirus strain, RVA/Human-tc/MWI/QEC287/2006/G8P[8] with a shared nucleotide identity of 98.48% . This huThe other Mozambican strain, MPT307, clustered closely to the South African strains for all the segments except that of NSP1-A13, indicating a close relationship between these strains. Additionally, these study strains group closely to strains from other African countries indicating possible transboundary movement of animals and subsequent transmission events. The frequency and extent of transboundary movement in the region is, however, unknown. This observation calls for combined regional efforts between veterinary services to manage infections across international borders.The limited data available due to local subsampling complicate full characterisation of these study strains but at the same time highlight the significant contribution this study makes to current knowledge about bovine rotaviruses in Africa. Apart from the limited knowledge, the data presented in this study represent a diverse genetic pool of bovine rotavirus strains in Africa that are shaped by extensive reassortment events. These strains circulated for more than a decade in various geographical regions, across country borders. Extensive reassortment is seen, resulting in endemic variants, as well as interspecies transmission in the VP1 and NSP2 genes. Results suggest that transboundary movement and interaction of hosts influence the diversity of rotavirus in Africa. This study calls for extensive surveillance of bovine rotavirus in African countries to understand bovine rotavirus diversity and the extent of interspecies transmission.Collection and testing of bovine faecal samples in South Africa were performed with ethical approval by the Medunsa Research Ethics Committee (MREC/P/103/2008:PG). In Mozambique, the study protocol was approved by the Institutional Bioethics Committee for Health from Instituto Nacional de Saúde (CIBS-INS), reference number 378.050/CIBS-INS/2020.Seven rotavirus-positive bovine faecal samples, five from South Africa [(RVA/Cow-wt/ZAF/Bov7/2003/G10P[11] (Bov7), RVA/Cow-wt/ZAF/Bov4/2003/G6P[5] (Bov4), RVA/Cow-wt/ZAF/MRC-DPRU457/2009/G10P[11] (MRC-DPRU457), RVA/Cow-wt/ZAF/Bov1/2009/G6P[5] (Bov1), RVA/Cow-wt/ZAF/1162/2012/G6P[11] (1162)] and two from Mozambique [(RVA/Cow-wt/MOZ/MPT307/2016/G10P[11] (MPT307) and (RVA/Cow-wt/MOZ/MPT93/2016/G10P[11] (MPT93)] were characterised in this study . The SouRNA extraction was performed as previously described . BrieflySequencing was performed at the University of the Free State Next Generation Sequencing (UFS-NGS) Unit using the Nextera XT DNA Library Preparation Kit  and the MiSeq Reagent Kit V3 (600 cycles).G4 and for VP7 (G6), VP4 (P[5]), NSP1 (A3) and NSP5 (H3) Tamura 3 + GG4 + I was used. The Tamura-Nei model + GG4 + I was used for VP4 (P[11]) and VP1 (R2). For VP2 (C2), NSP2 (N2) and NSP3 (T6) the General Time Reversible + GG4 and for VP3 (M2) and NSP1 (A11 and A13) General Time Reversible + GG4 + I was used. Lineages for the DS-1-like genotypes [Sequencing data were assembled as previously described and consensus sequences were analysed using BLASTn . Genotypenotypes  and G- aenotypes  were ass


In this study, sewage treatment plants (STPs) servicing well-defined catchment areas were surveyed for the presence of CR-GNB bearing carbapenemase genes (Klebsiella-Enterobacter (KE) group amounted to 116 isolates (35.7%). CR-KE isolates were recovered from TS, DW (35.7%) and RS samples (44.2%) (p = 0.001); but not from UW samples. KE isolates represented 65.8% of all blaKPC or blaNDM positive strains. The frequency of blaKPC-or-NDM strains was positively associated with the occurrence of district hospitals located near STPs, as well as with the number of hospitalizations and of sewer connections serviced by the STPs. blaKPC-or-NDM strains were recovered from ST samples in 7 out of 14 STPs, including four tertiary-level STPs; and from 6 out of 13 DW spots whose RS samples also had blaKPC-or-NDM strains.A total of 325 CR-GNB were recovered from raw (RS) and treated (TS) sewage samples as well as from water body spots upstream (UW) and downstream (DW) from STPs. blaKPC-or-NDM resist sewage treatments and spread into environmental aquatic matrices mainly from STPs impacted by hospital activities.Clinically relevant GNB bearing The online version contains supplementary material available at 10.1186/s12866-021-02400-1. K. pneumoniae strains harboring resistance gene blaKPC was confirmed ; however, they were recovered at a higher frequency (p = 0.015) from UW samples [66.6% (12/18)]  from TS and DW samples [respectively 31.5% (28/92) and 38.0% (30/84)], showing a higher recovery frequency (p = 0.001) from RS samples [48.0% (58/131)] , 10.7% (n = 34),1.8% (n = 6) and 1.2% (n = 4) of the tested CR-GNB, respectively. Five strains from RS samples were positive for more than one tested gene. Four isolates were positive for 2 carbapenemase genes . Furthermore, one K. pneumoniae strain from RS samples harbored three carbapenemase genes (blaKPC + blaNMD + blaIMP).Carbapenemase genes were detected in 124 (39.2%) out of 316 CR-GNB isolates. The genes blaKPC-or-NDM strains (p < 0.0001) with the predominance of K. pneumoniae (53.8% - 63/117) (p < 0.0001), followed by E. cloacae (11% - 13/117) and E. aerogenes (3.4% - 4/117)  represented 11.1% (13/117) of blaKPC-or-NDM strains  and Pantoea agglomerans (93.3% confidence level). Lastly, the transposon Tn4401 was detected in 97.1% (69/71) of the blaKPC strains, including isolates of Klebsiella spp., Enterobacter spp., Aeromonas spp., Citrobacter spp., P. putida group., P. aeruginosa group, P. agglomerans and K. ascorbate (data not shown).KE isolates were identified in 65.8% (77/117) of the blaKPC-or-NDM were detected at different proportions across analyzed samples (p < 0.001) (supporting information Table blaKPC-or-NDM strains accounted for 50.4% (64/127) of the CR-GNB isolates recovered from RS samples. The proportion of blaKPC-or-NDM strains in TS (26/88–29.5%) and DW (27/83–32.5%) samples was statistically equal (p = 0.742). Conversely, blaKPC-or-NDM strains were not detected in UW samples (supporting information Table Klebsiella-Enterobacter group were predominant among blaKPC-or-NDM strains recovered from RS (70.3% - 45/64), TS (65.3% - 19/27) and DW (70.3% - 17/26) samples  accounted for 75% (48/64) of the isolation of blaKPC-or-NDM strains recovered from RS samples  as a function of the total number of hospitals; the number of hospitalizations; the occurrence in STP vicinities (less than 3 km away) of district hospitals; and the number of sewer connections serviced by the STPs   accomplished by STPs ] and TS [46.4% (13/28)] samples (p = 0.627). In contrast, tertiary-level STPs achieved a 33 percentage-point reduction in the frequency of blaKPC-or-NDM strains in TS samples compared to RS samples. In tertiary-level STPs, blaKPC-or-NDM strains were detected in respectively 55.1% (48/87) and 21.6% (13/60) of the strains recovered from RS and TS samples (p < 0.001).In order to evaluate the effectiveness of sewage treatment in reducing the spread of CR strains, the frequency of TPs Fig. . In secoblaKPC-or-NDM strains were still recovered from 7 out of 14 analyzed STPs, including four tertiary-level STPs  and three secondary-level STPs . Most of blaKPC-or-NDM strains recovered from the TS samples belonged to Klebsiella-Enterobacter group (65.3% - 17/26), followed by mesophilic Aeromonas (19.2% - 5/26)  of all of the blaKPC-or-NDM-positive strains. In contrast, blaKPC-or-NDM-positive strains were not isolated from UW samples. blaKPC-or-NDM-positive strains were recovered from 6 out of 13 (46%) DW sites whose RS samples also had blaKPC-or-NDM strains  and in two secondary-level STPs (STP-2 and STP-12)  of blaKPC-or-NDM-positive strains recovered from DW samples, being followed by mesophilic Aeromonas group (14% - 4/27)  postulates that infectious diseases and drug-related markers, including antibiotic resistance, can be monitored comprehensively through the analysis of population pooled wastewater. Additionally, WBE can provide information on the community and environmental health status as well as on the community exposure to health-threatening issues , 27. Card blaNDM . In thisd blaNDM .P. putida, S. maltophilia and C. violaceum) which were frequently found in soil and water environments. These saprophytic species are not epidemiologically relevant once they rarely harbor mobile carbapenemase genes and rarely produce human infections. In contrast, 142 (43.6%) CR-GNB isolates represented intrinsically carbapenem susceptible, clinically relevant species which are frequently associated with human infections .Our survey for CR-GNB in STPs settings yielded a collection of 325 isolates. As expected, part of these isolates (124–38.2%) represented species expressing intrinsic resistance to carbapenem  over blaNDM strains (34–29.0%). With regard to blaKPC-associated transposon, Tn4401 was detected in 97% of the blaKPC strains including Enterobacterales species, Aeromonas species, non-fermentative bacilli and soil saprophytes species. Therefore, our data evince the role of Tn4401 as the foremost element for spreading blaKPC in different species of GNB, including environmental strains  and directly from treated sewage outlet pipe [Treated Sewage (TS) - n = 35]. Water samples were collected in receiving water bodies on spots located 50 m upstream [Upstream Water (UW) – n = 30] and 50 m downstream [Downstream Water (DW) – n = 38] from the point where sewage outlet pipe discharged the treated sewage. With regard to STP-13 and STP-5, the water body receiving the treated sewage is Lake Paranoá . In order to evaluate the role of STP-4 and STP-10 in spreading antibiotic resistance into receiving water body, their results were then pooled. Samples were collected in sterile conical tubes (50 mL) during the mornings, preserved at room temperature in insulated boxes, and sent for culture at the Central Laboratory for Public Health (LACEN-DF) in the same work shift.Three rounds of sewage and receiving water body sampling were carried out in April, July and August 2017 covering 14 sewage treatment plants (STPs), all located in Brasília. The profile of the regions serviced by the STPs as well as the burden on those plants represented by hospital and farming activities were displayed in Table Five hundred microliters of each sample were cultured in Luria-Bertani broth supplemented with vancomycin [7.5 mg/L] and ertapenem [2.5 mg/L] at 36.5 °C for 24 h. Positive CR cultures were streaked on chromogenic and differential agar (ChromID® ESBL – bioMérieux) and incubated at 36.5 °C for 24 h for isolation and presumptive identification of Gram-negative bacilli (GNB). Three colonies of each presumptive bacterial group were isolated per sample limited to a maximum of 10 colonies per sample. Colonies were preserved into a semisolid nutrient medium (0.8% agar) stored in hermetically sealed tubes (3 mL) at room temperature and were kept safe from direct light exposure.Escherichia coli strain ATCC™ (American Type Culture Collection) 8739 was used as a positive control. The Myla® database was accessed for the identification of bacterial isolates. A confidence level greater than 80% was adopted for genus assignment and greater than 90% for species.The bacterial isolates were recovered and grown on Mueller Hinton agar for 24 h at 37 °C for obtaining isolated colonies. The identification was accomplished using Vitek MS system (Matrix-assisted laser desorption ionization time of flight mass spectrometry - MALDI-TOF MS system - BioMerieux) in accordance with the manufacturer’s instructions. blaKPC, blaNDM, blaIMP, blaVIM and blaOXA-48)   recognizes the blaKPC locus located 250-bp downstream from ISkpn6.Carbapenemase genes were detected by standard polymerase chain reactions (PCR). Supernatants derived from bacterial suspensions in deionized water and treated by boiling (100 °C for 15 min) were used as the source of DNA template. Primers were used to detect multiple alleles of carbapenemase genes . Non-parametric analyses were carried out with Fisher’s Exact Test (2-sided). Linear-by-linear associations of ordered categories were assessed with the Mantel-Haenszel test. With regard to continuous variables, non-parametric Mann-Whitney U-tests were performed to compare differences between independent groups. Results with Additional file 1.