Correction to: BMC Rheumatol 4, 4 (2020)https://doi.org/10.1186/s41927-019-0105-4Following publication of the original article , the autThe original article has been updated.AbstractResults: In those without DM GC use had a 4.4-fold increased all-cause mortality RR : 3.77 to 5.07) compared to non-use, whilst those with DM had a lower RR for GC use ). However, those with DM had a higher RD associated with GC use because of their higher baseline risk. In those with DM, GC use was associated with an additional 44.9 deaths/1000 person-years (pyrs) (95% CI: 32.9 to 56.8) compared to non-use, while in those without DM GC use was associated with an additional 34.4 deaths/1000 pyrs (95% CI: 30.1 to 38.7). A similar pattern was seen for CV mortality. The adjusted Cox proportional hazards model showed no evidence of multiplicative interaction, but additive interaction indicated a non-significant increased risk. For CV mortality there was no interaction on either scale.In the “All-cause mortality” sub-section it now correctly reads “During follow-up there were 1005 deaths” rather than 1002 deaths.


Background: Cancer treatments induce symptoms/signs superimposing on individual patient's clinical status, determining heterogenous toxicity syndromes (TS). We reviewed intensive first line triplet chemotherapy-based regimens in metastatic gastro-intestinal cancers (mGI), based on FIr/FOx schedule, fluorouracil and weekly alternating irinotecan/oxaliplatin, to point out limiting TS (LTS) relevance.Methods: Metastatic colo-rectal (mCRC), pancreatic ductal adenocarcinoma (mPDAC), gastric carcinoma (mGC) patients were enrolled by careful decision-making including age, performance status (PS), and comorbidity status in real life phase II studies: FIr-B/FOx adding bevacizumab (B) overall, FIr-C/FOx-C adding cetuximab (C) in KRAS/NRAS wild-type mCRC; FIr/FOx in mPDAC; FD/FOx adding docetaxel (D) in mGC. Toxicity, individual LTS, LT alone  or associated to other limiting/G2 toxicities  were evaluated, compared by chi-square test. In FIr-C/FOx-C, 5-fluorouracil/irinotecan pharmacogenomic biomarkers, 5-fluorouracil degradation rate (5-FUDR), SNPs ABCB1, CYP3A4, DYPD, UGT1A1 were evaluated, related with LTS.Results: FIr-B/FOx, FIr-C/FOx-C in mCRC, FIr/FOx in mPDAC, FD/FOx in mGC, showed activity, efficacy, toxicities similar to reported triplet regimens. LTS: mCRC FIr-B/FOx 44%, LTS-ms 24%, LTS-ss 20%, in young-elderly 46%, LTS-ms significantly increased vs. LTS-ss; FIr-C/FOx-C 65.5%, significantly increased LTS-ms vs. LTS-ss, in young-elderly 83%; mPDAC FIr/FOx 27.5%, mostly LTS-ms, in young-elderly 38.4% all LTS-ms; mGC FD/FOx 30%, all LTS-ms, in young-elderly 25%. Reduced FUDR, SNPs CYP3A4, UGT1A1, >1 positive pharmacogenomic biomarkers were prevalent in patients with gastrointestinal LTS.Conclusions: LTS is an innovative clinical parameter of toxicity burden, differential treatment-related TS in individual patient. LTS can evaluate pharmacogenomic biomarkers predictive relevance to select mGI patients fit for intensive treatments, at risk of limiting gastrointestinal toxicity.Trial Registrations: The trials were registered at Osservatorio Nazionale sulla Sperimentazione Clinica dei Medicinali (OsSC) Agenzia Italiana del Farmaco (AIFA) Numero EudraCT 2007-004946-34, and Osservatorio Nazionale sulla Sperimentazione Clinica dei Medicinali (OsSC) Agenzia Italiana del Farmaco (AIFA) Numero EudraCT 2009- 016793-32. Addition of more drugs in a chemotherapy combination requires the design of proper schedule and doses, to provide the balance between projected/received (>80%) dose intensity (DI) and treatment-related toxicity . This clThe conventional evaluation of toxicity of cancer treatments depends upon grading of the clinical relevance of each toxicity symptom and sign, by evaluation of the type, prevalence and intensity of toxicity, more specifically of limiting G3-4) toxicity, related to the administered treatment regimen, and determined by National Cancer Institute Common Toxicity Criteria . Thus, safety profile and toxicities induced by cancer treatments are conventionally defined, according to the number of administered cycles and treated patients, as severe (G3-4), moderate G2), mild (G1), or absent. This evaluation does not individually describe the toxicity burden experienced in a single patient, defining a spectrum of toxicity syndromes (TS). To better evaluate individual toxicity of intensive treatments, we introduced the concept of Limiting Toxicity Syndromes (LTS), consisting of at least a limiting toxicity alone  or associated to other limiting or G2-3 non-limiting toxicities   66.7%, progression-free survival (PFS) 12 months, and overall survival (OS) 20 months as first line mCRC treatment. Thus, we developed intensive first line regimens, based on FIr/FOx triplet chemotherapy schedule: FIr-B/FOx (We previously developed doublet chemotherapy schedule of 12 h (10 p.m. to 10 a.m.) timed-flat infusion (TFI) 5-FU , associaIr-B/FOx , respectIr-B/FOx ; FD/FOx Ir-B/FOx .Patients treated with triplet chemotherapy-based regimens were enrolled by decision-making process including age, performance status (PS), and comorbidity status evaluated by Cumulative Illness Rating Scale (CIRS) . CIRS stin label for treatment in Italian public hospitals, and published in Gazzetta Ufficiale Repubblica Italiana . MCRC clinical trials were approved by Local Ethical Committee , and conducted in accordance with Declaration of Helsinki. PDAC study was approved by the Regional Review Board . All patients provided written, informed consent concerning the proposed treatment, and biological evaluations.Proposed regimens were approved by Agenzia Italiana del Farmaco for administration 2, days 1–2, 8–9, 15–16, 22–23; CPT-11 160 mg/m2 days 1,15; OXP 80 mg/m2, days 8, 22; every 4 weeks; B 5 mg/kg, days 1,15. In KRAS/NRAS wild-type mCRC, FIr-C/FOx-C . In mGC, FD/FOx  week week2, dschedule  11): TF: TF2, da, FD/FOx  14): TF: TF2, da6 cells/min, and Single Nucleotide Polimorphisms (SNPs) ABCB1 , CYP3A4 , DYPD1 , UGT1A1 (28) were preliminarily related with the occurrence of LTS . To better evaluate toxicity of intensive treatments in the individual patient, LTS, consisting of at least a limiting toxicity alone (LTS-ss) or associated to other limiting or G2-3 non-limiting toxicities (LTS-ms) were evaluated , 14, 20.e of LTS , 25.2, respectively  per cycle for all the drugs at the recommended doses were ≥80% (2/w); CPT-11 76% (45.5 mg/m2/w); OXP 66% (26.5 mg/m2/w); C 71% (178.5 mg/m2/w); yE patients showed significantly worse OS compared to non-elderly (P 0.045) . . 2, respP = 0.022). In mPDAC patients treated with FIr/FOx, median rDI per cycle were: 5-FU 70.4% (1268.5 mg/m2/w); CPT-11 70% (56 mg/m2/w); OXP 72.5% (29 mg/m2/w). In yE, 5-FU 83.3%, CPT-11 80%, OXP 85%. Overall, 17% discontinued FIr/FOx treatment due to LT. Limiting cumulative G3-5 toxicities were: diarrhea 17%, asthenia 14%, neutropenia 17%, mucositis 6%, hypokaliemia 7%, hypertransaminasemia 7%, nausea/vomiting, hypoalbuminemia, anemia, thrombocytopenia 3%, respectively. One case of toxic death (3%) was observed.In mPDAC patients, FIr/FOx showed ORR 53%, PFS 4 months, OS 11 months; among yE/old-elderly patients, median PFS 4 months, and median OS 5 months ; D 91% (22.75 mg/m2/week); OXP 83.8% (33.5 mg/m2/week). Cumulative G3-4 toxicities were represented by: asthenia 20%, neutropenia 50%, leucopenia 20%, mucositis and hypoalbuminemia 10%.In mGC patients, FD/FOx at recommended 5-FU 1000 mg/m7 months . Median KRAS/NRAS wild-type mCRC patients treated with FIr-B/FOx, overall LTS were observed in 22 patients (44%) (P 0.05). LTS were prevalently characterized by G2-3 diarrhea, 9 patients (69.2%), 8 LTS-ms and 1 LTS-ss. The 2 LTS-ms with double LT were observed in yE patients. The 10 LTS-ms, defined by LT added to other, at least G2, non-limiting toxicities, were prevalently determined by G2-3 diarrhea (90%), plus G2-3 nausea/vomiting (70%). The 10 LTS-ss were prevalently characterized by G3 diarrhea (50%), G3 asthenia, hypertension, hypertransaminasemy, neutropenia, thrombocytopenia 10%, respectively.In ts (44%) : LTS-ms ts (44%) . LTS-ms KRAS/NRAS wild-type mCRC patients treated with FIr-C/FOx-C, LTS were observed in 19 patients (65.5%), 5 out of 6 yE (83%): LTS-ms 17 (59%), LTS-ss 2 (7%) (P 0.006). LTS-ms characterized by ≥2 LT 7 (24%); LT associated to other toxicities 10 (34%). LTS were prevalently characterized by G3-4 diarrhea and G3 asthenia associated to other toxicities.In s 2 (7%) . LTS werIn mPDAC patients treated with FIr/FOx, overall LTS were 8 (27.5%); 5 out of 13 yE/old-elderly (38.4%); LTS-ms 7 (24.1%), LTS-ss 1 (3.4%) . LTS-ms CYP3A4, UGT1A1, and >1 positive pharmacogenomic biomarkers. Specifically, the exploratory analysis of 5-FUDR reduction and ABCB1, CYP3A4, DYPD, UGT1A1 SNPs evaluated in 14 KRAS/NRAS wild-type mCRC patients (48.3%) treated with FIr-C/FOx-C .Furthermore, the analysis of SNPs represented an exploratory analysis in colorectal cancer patients treated with intensive triplet chemotherapy plus cetuximab, according to FIr-C/FOx-C schedule, to better evaluate the safety profile, particularly gastrointestinal. Patients who reported gastrointestinal LTS, prevalently showed reduced fluorouracil degradation rate (FUDR), Single Nucleotide Polimorphisms (SNP) -C/FOx-C , 25, andOver the past 10 years, we developed intensive triplet chemotherapy-based regimens in fit mGI cancer patients , 11, 14,KRAS/NRAS wild-type mCRC , to obtain the description of cumulative and prevalent limiting (G3-G4), moderate (G2), mild (G1), or absent toxicities directly determined by the cancer treatment, according to the number of administered cycles and treated patients. This way does not really represent the clinical burden of toxicity in the individual patient, nor define prevalent individual TS affecting a patient population equivalently treated and their variability. Thus, in reported intensive first line triplet chemotherapy-based regimens developed in mGI cancer patients, we added the description of individual TS, and specifically of LTS, to describe cumulative and individual toxicity, that include differential spectrum and intensities of TS, depending from medical treatment and patients' individual clinical conditions.The integration of the evaluation of LTS to the conventional treatment-related toxicity, contributed a patient-related clinical indicator of toxicity burden, providing a global evaluation of patient-related limiting toxicity , 14, 20:In yE patients, LTS evaluation showed the differential tolerability of intensive triplet chemotherapy based regimens: in mCRC, FIr-B/FOx LTS 46%, but significantly prevalent LTS-ms , 27; FIrKRAS/NRAS wild-type MCRC patients treated with FIr-C/FOx-C showed that reduced 5-FUDR, and CYP3A4 and UGT1A1 SNPs may predict individual LTS occurrence, particularly at recommended doses, specifically gastrointestinal LTS  and in 9 who showed LTS (47.4%), exploratory data of pharmacogenomic biomarkers compared with LTS occurrence in Thus, LTS meets the need of an innovative clinical parameter of patient-related toxicity burden, to measure personalized safety of intensive first line triplet chemotherapy-based regimens proposed in mGI, also associated to targeted agents (B or C). Its clinical relevance should be prospectively evaluated as a model in clinical practice. The integration of LTS and conventional toxicity evaluations may help proper selection of patients fit for intensive first line medical treatments in mGI cancers, to more properly weigh toxicity analysis with activity and clinical outcome and its contribution to address selection of patients suitable for intensive triplet chemotherapy-based regimens. The equivalent, integrated evaluation and monitoring of individual safety by LTS, can help properly select first line intensive medical treatment, and safely administer and manage an intensive first line regimen, that could guarantee increased clinical outcomes and good safety profile in real life.In the era of precision oncology, integrating molecular characterization of cancer affecting the individual patient to specifically address targeted treatments, such as in mCRC –32, the Analysis of TS could be integrated in the therapeutic pathway of cancer patients in clinical practice and in clinical studies to globally evaluate tolerability of cancer treatments. It could be, also, particularly useful for a more proper evaluation of tolerability in the therapeutic pathway of individual cancer patients unfit for standard treatments, due to elderly status and/or their clinical status, or unfit for intensive regimens , 34, to More, in intensive regimens such as FIr-C/FOx-C, characterized by high discontinuation rate of treatment, highly prevalent (>50%) LTS and heterogeneity of LT, LTS may help directly relate individual patient- and drugs-related toxicity with pharmacogenomics biomarkers referred to the individual genetic identity of the cancer patient.TS, specifically LTS, represents an innovative clinical parameter of cumulative and individual patient-related toxicity burden, defining differential spectrum and intensities of treatment-related TS, depending from the clinical status of the individual cancer patient, particularly according to elderly, and PS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Proposed regimens were approved by Agenzia Italiana del Farmaco for administration in label for treatment in Italian public hospitals, and published in Gazzetta Ufficiale Repubblica Italiana . MCRC clinical trials were approved by Local Ethical Committee , and conducted in accordance with Declaration of Helsinki. PDAC study was approved by the Regional Review Board . All patients provided written, informed consent concerning the proposed treatment, and biological evaluations.GB contributed in conceptualization, data curation, formal analysis, investigation, methodology, project administration, validation, and writing original draft. ER contributed in conceptualization, data curation, formal analysis, investigation, methodology, project administration, supervision, validation, and writing—review original draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Populus trichocarpa) upon infection with the generalist root pathogen Phytophthora cactorum (Oomycetes). P. cactorum infection led to an induced accumulation of terpenes, aromatic compounds, and fatty acids in poplar roots. Transcriptome analysis of uninfected and P. cactorum-infected roots revealed a terpene synthase gene PtTPS5 that was significantly induced upon pathogen infection. PtTPS5 had been previously reported as a sesquiterpene synthase producing two unidentified sesquiterpene alcohols as major products and hedycaryol as a minor product. Using heterologous expression in Escherichia coli, enzyme assays with deuterium-labeled substrates, and NMR analysis of reaction products, we could identify the major PtTPS5 products as -guaia-4(15)-en-11-ol and -guaia-4-en-11-ol, with the former being a novel compound. The transcript accumulation of PtTPS5 in uninfected and P. cactorum-infected poplar roots matched the accumulation of -guaia-4(15)-en-11-ol, -guaia-4-en-11-ol, and hedycaryol in this tissue, suggesting that PtTPS5 likely contributes to the pathogen-induced formation of these compounds in planta.Pathogen infection often leads to the enhanced formation of specialized plant metabolites that act as defensive barriers against microbial attackers. In this study, we investigated the formation of potential defense compounds in roots of the Western balsam poplar ( Plants are constantly under attack from a multitude of pests, including pathogens and herbivores. Such biotic stresses often induce the formation of specialized plant metabolites that play major roles in plant defense. Terpenoids represent the largest class of natural compounds, and to date, more than 200,000 terpenoids are known, of which ~40,000 can be produced by plants . Beside 10), -farnesyl diphosphate , and -geranylgeranyl diphosphate . The prenyl diphosphates are substrates for terpene synthases (TPS), which catalyze the formation of the basic mono-(C10), sesqui-(C15), and diterpene (C20) skeletons D20 = +47.8 . HRMS (EI): m/z = 222.1978 . GC (HP5-MS): I = 1660. MS : m/z (%) = 222 (0.3), 204 (29), 189 (24), 175 (2), 161 (24), 149 (22), 133 (11), 121 (19), 107 (31), 91 (41), 81 (54), 67 (25), 59 (100), 53(13), 41 (35). IR (diamond ATR): /cm–1 = 2953 (m), 2923 (s), 2854 (m), 1714 (w), 1650 (w), 1456 (m), 1376 (m), 1260 (m), 1094 (s), 1020 (s), 873 (m), 800 (s).-Guaia-4-en-11-ol (2). Yield: 0.6 mg . TLC (pentane/ether = 4:1): Rf = 0.21. Optical rotation: [α]D20 = +21.7 . HRMS (EI): m/z = 222.1975 . GC (HP5-MS): I = 1661. MS : m/z (%) = 222 (4), 204 (81), 189 (69), 175 (7), 161 (75), 147 (27), 133 (29), 119 (37), 105 (61), 91 (81), 79 (68), 67 (30), 59 (100), 51(5), 41 (55). IR (diamond ATR): /cm–1 = 2954 (s), 2923 (s), 2854 (s), 1723 (w), 1670 (w), 1459 (m), 1376 (m), 1260 (w), 1096 (w), 1025 (w), 800 (w).S,5S,7R,10R)-guaia-4(15)-en-11-ol and -guaia-4-en-11-ol. For the reactions with DMAPP (1 mg in 1 mL water) and (Z)-IPP or (E)-IPP (1 mg in 1 mL water), protein preparations of PtTPS5 (1 mL) and FPPS  and incubation buffer (6 mL) were added. For the reactions with (R)-IPP or (S)-IPP (1 mg in 1 mL water), protein preparations of PtTPS5 (1 mL), FPPS  and incubation buffer (6 mL) were added. After incubation with shaking at 28 °C overnight, the reaction mixtures were extracted with C6D6, the extracts were dried with MgSO4 and analyzed by NMR and GC-MS.Isotopic labelling experiments were performed to determine the absolute configurations of , MgCl2 S , IDI , MgCl2 Throughout the manuscript, data are presented as means ± SE. Statistical analysis was performed with SigmaPlot 11.0 for Windows  and is described in the figure and table legends for the respective experiments. Whenever necessary, the data were log transformed to meet statistical assumptions such as normality and homogeneity of variances.Raw reads of the RNAseq experiment were deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession PRJNA660564 ‘Oomycete-induced changes in the root transcriptome of poplar’.


T (Collection de Souches de l’Unité des Rickettsie CSUR P6417 = Colección Española de Cultivos Tipo CECT 9771) is a new Pedobacter species isolated from the planarian Schmidtea mediterranea. Schmidtea mediterranea are flatworms living in freshwater and exhibiting an unusual ability to regenerate amputated parts. To date, the gut microbiota of Schmidtea mediterranea remains poorly studied. Here, via the culturomics strategy that consists in using diversified culture conditions, we isolated a new bacterium, strain EG, that we characterized using the taxono-genomics approach that combines phenotypic assays and genome sequencing and analysis. Strain EG exhibits a 16S rRNA sequence similarity of 98.29% with Pedobacter nyackensis strain NWG-II14T, its closest neighbour with standing in nomenclature. It is an aerobic bacterium belonging to the family Sphingobacteriaceae. Colonies are small, round, smooth and transparent. Bacterial cells are Gram-negative, rod-shaped, motile and non-spore-forming bacilli with positive catalase and oxidase activities. The genome sequence is 6,198,518 bp–long with a G + C content of 41.13%, and the Ortho-ANI and dDDH values when compared to P. nyackensis are 77.34% and 21.50%, respectively. Strain EGT exhibits unique characteristics that classify it as the type strain of new bacterial species for which we propose the name Pedobacter schmidteae sp. nov.Pedobacter schmidteae sp. nov. strain EG Schmidtea mediterranea is an invertebrate living in environmental water. This flatworm is used as a model of development, because of its extraordinary abilty to regenerate after amputation, because of his high contents in stem cells known as neoblasts1. It has been shown that S. mediterranea is an excellent model to investigate host-pathogen relationships3, notably in the context of human pathogens. To date, the gut microbiota of S. mediterranea remains poorly studied5. Using the microbial culturomics approach6, we investigated the S. mediterranea microbiota. Culturomics is a concept in which diversified culture conditions are used to enable isolation of a maximum of bacterial species from the human microbiota10. During this analysis, we isolated a bacterial strain from S. mediterranea that could not be identified using Matrix Assisted Laser Desorption-Ionisation Time of Flight-Mass Spectrometry (MALDI-TOF-MS). We used the taxono-genomics strategy that combines phenotypic assays and genome sequencing to further characterize this bacterium14. This enabled us to describe a new bacterial strain that exhibited enough genetic and phenotypic differences with closely related bacteria. We propose it as a new species named Pedobacter schmidteae sp. nov.Schmidtea mediterranea asexual clonal line ClW42 is a laboratory planarian strain that has been preserved in our laboratory for the past 10 years, by cutting animals in tree fragments each month. S. mediterranea were kept in the dark, in filtered tap water, at 19 °C without antibiotics. The animals were fed once per week with homogenized calf liver and were starved for at least two weeks prior to studying them. Filtered water was obtained using a device consisting of two 0.2 µm filters, one containing charcoal and ceramics , and the other being a 0.20 µm membrane . Filtered water was checked for sterility prior to be used for planarian culture.S. mediterranea were washed in filter-sterilized water and then one ground worm was inoculated in Buffered Charcoal Yeast Extract (BCYE) , Luria Bertani (LB) and 5% sheep blood-enriched Columbia agar . All inoculated media were incubated at 19, 28 and 37 °C. Each individual bacterial colony was harvested and identified by MALDI-TOF-MS 15. The obtained spectra were imported into the MALDI Biotyper 3.0 software  and analysed against the reference spectra of bacteria included in the database (Bruker database constantly updated with the Mediterranee-Infection database (http://www.mediterranee-infection.com/article.php?larub=280&titre=urms-database). The MALDI Biotyper RTC software was used to interpret the results according to the obtained score values: a colony was likely identified at the species level for a score > 2.0, probably identified for a score between 1.99 and 1.7, but not identified for a score <1.7.Following two weeks of starvation, https://www.codoncode.com/). For taxonomic assignation, a BLASTn search was performed against the nr database16. A sequence similarity threshold of 98.65% by comparison with the phylogenetically closest species with standing in nomenclature was used to delineate a putative new species17. Phylogenetic relationships were inferred from the comparison of 16S rRNA sequences using the MEGA7 software18.Bacterial colonies that were not identified at the species level using MALDI-TOF MS were further tested using 16S rRNA sequencing. Genomic DNA was extracted using an EZ1 automate and the DNA tissue kit . The complete 16S rRNA gene amplification and sequencing was performed using eight primers on an ABI Prism 3130xl Genetic Analyzer capillary sequencer . The primers used were Fd1 (5′-AGAGTTTGATCCTGGCTCAG-3′), Rp2 (5′-ACGGCTACCTTGTTACGACTT-3′), F536 (5′-CAGCAGCCGCGGTAATAC-3′), R536 (5′-GTATTACCGCGGCTGCTG-3′), F800 (5′-ATTAGATACCCTGGTAG-3′), R800 (5′-CTACCAGGGTATCTAAT-3′), F1050 (5′-TGTCGTCAGCTCGTG-3′) and R1050 (5′-CACGAGCTGACGACA-3′) . The CodonCode Aligner software was used for sequence alignment, assembly and correction (strain EG and P. nyackensis strain NWG-II14T (DSM19625) was attempted at various growth temperatures  in 5% sheep blood-enriched Columbia agar (bioMérieux) under anaerobic, aerobic and microaerophilic atmospheres using GasPak™ EZ generators . Sporulation was tested by thermal shock, which consists in exposing bacteria to a temperature of 80 °C for 30 minutes and then monitoring their growth for 4 days. Various salinity  and pH  conditions were tested. Gram staining and motility from fresh colonies were observed using a DM1000 photonic microscope  with a 40 × objective lens. Catalase and oxidase activities were tested by using a BBL DrySlide according to the manufacturer’s instructions . The size of bacterial cells was measured using transmission electron microscopy. API strips  were used to study the biochemical characteristics of the strains. Bacterial susceptibility to benzylpenicillin, amoxicillin, ampicillin, ceftriaxone, imipenem, ciprofloxacin, amikacin, gentamicin, streptomycin, daptomycin, doxycycline, metronidazole, rifampicin, and vancomycin was assessed using E-tests and a 0.5 McFarland concentration of strains EG and NWG-II14T. Cellular fatty acid methyl ester (FAME) analysis was performed by GC/MS. Two samples were prepared with approximately 120 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as described by Sasser30 and GC/MS analysis was carried out as previously described31. Briefly, FAMES were separated using an Elite 5-MS column and monitored by mass spectrometry . Spectral database search was performed using MS Search 2.0 operated with the following Standard Reference Database 1 A  and FAMEs mass spectral database .Culture of strain 32. Briefly, sequencing was performed using the Mate-Pair strategy and a Miseq sequencer . A concentration of 1.5 μg of gDNA prepared following the Nextera Mate-Pair Illumina guide was used to prepare the Mate-Pair library. The gDNA was simultaneously fragmented and tagged using a Mate-Pair junction adapter. Then the fragmentation pattern was validated using a DNA 7500 labchip on a BioAnalyzer . The size of the DNA fragments ranged from 1.5 kb to 11 kb. No size selection was performed and 662 ng of labelled fragments were circularized. Next, circularized DNA was mechanically sheared using a Covaris device S2  into small fragments with an optimal size at 1200 bp. The library profile was analysed on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies) and the concentration library was measured at 61.4 nmol/l. Then, the library was loaded on the sequencer after a denaturation step and a dilution at 15 pM. Automated cluster generation and sequencing were performed in a single 39-h run in a 2 × 251-bp and sequencing reads were assembled using the A5 pipeline. Genomic annotation was obtained using the Prokka software. A search for virulence factors was performed by comparaison with the VFDB database (http://www.mgc.ac.cn/VFs/) using BLASTn34.The bacterial genomic DNA (gDNA) of strain EG was extracted using an EZ1 automate and the DNA tissue kit  and then was quantified using a Qubit assay  at 82.6 ng/μL. The bacterial gDNA was prepared and sequenced as previously describedPedobacter africanus strain DSM 12126 T (NZ_FWXT00000000.1), P. antarcticus strain 4BYT (NZ_JNFF00000000.1), P. ginsengisoli strain T01R-27 (NZ_CP024091.1), P. heparinus strain DSM 2366 T (NC_013061.1), P. nutrimenti strain DSM 27372 (NZ_QKLU00000000.1), P. nyackensis strain DSM 19625 (NZ_FWYB00000000.1), P. panaciterrae strain 048 (NZ_LGEL00000000.1) and P. steynii strain DX4 (NZ_CP017141.1). Degrees of genomic similarity between strain EG and compared genome were evaluated using the GGDC (http://ggdc.dsmz.de/ggdc.php#)16 and Orthologous Average Nucleotide Identity (Ortho-ANI) (https://www.ezbiocloud.net/tools/orthoani)35 softwares.The genome from strain EG was compared to those of Pedobacter nyackensis strain NWG-II14T with which it exhibited a sequence similarity of 98.29%. When compared to other Pedobacter species, strain EG exhibited 16S rRNA similarity values of 97.84%, 97.69%, 97.63%, 97.93%, 97.43%, 97.28%, 97.33%, 97.62%, 97.93%, 97.32%, 97.32% and 97.0% with P. heparinus strain DSM 2366 T, P. steynii strain WB2.3–45 T, P. metabolipauper strain WB2.3–71 T, P. nutrimenti strain J22T, P. duraquae strain WB2.1–25 T, P. africanus strain NBRC 100065 T, P. panaciterrae strain Gsoil 042 T, P. ginsengisoli strain Gsoil 104 T, P. seoulensis strain THG-G12T, P. trunci strain THG-DN3.19 T, P. humi strain THG S15–2T and P. antarcticus strain 4BYT 40, respectively .Strain EG was isolated on 5% sheep blood-enriched Columbia agar (bioMérieux) after 2 days at 28 °C in aerobic atmosphere at pH 7. The 16S rRNA-based phylogenetic tree demonstrated that strain EG was most closely related to S. mediterranea revealed the presence of strain EG in 11 S. mediterranea sp. nov. Strain EG is a member of the family Sphingobacteriaceae within the phylum Bacteroidetes(Table 1).The analysis of 13 , rod-shaped, motile, and non-spore-forming bacilli. Using the Image J software, their mean length and width were 1.98 µm and 0.69 µm, respectively , without any flagellum. For the two strains EG and P. nyackensis NWG-II14T, no growth was obtained in anaerobic or microaerophilic conditions. Both strains grew at temperatures ranging from 6 to 30 °C in aerobic atmosphere at pH values ranging from 5.5 to 9; the strains also grew at salinity concentrations lower than 25 g/L. Catalase and oxidase activities were positive for both strains.After 4 days of culture on blood-enriched Columbia agar, colonies from strain EG were small (0.4 mm of diameter), transparent, round with a convex shape and smooth. Bacterial cells were Gram-negative Fig. , rod-shaely Fig. , withoutP. nyackensis strain NWG-II14T were susceptible to benzylpenicillin, ampicillin, ceftriaxone, imipenem, gentamicin, doxycycline and rifampicin (Table ). Both strains were resistant to amoxicillin, amikacin, daptomycin, metronidazole and vancomycin. Pedobacter nyackensis, but not P. schmidteae, was susceptible to ciprofloxacin and streptomycin.Strain EG and in Table . Both stP. nyackensis NWG-II14T, strain EG differed by exhibing of Esterase (C4), Esterase lipase (C8), Valine arylamidase, β-galactosidase and β-glucosidase activities (Table ). By comparaison with all other tested species, strain EG differed in production of esterase (C4) and β-glucosidase.Positive and negative reactions obtained using API 50CH, API 20NE, API Zym, and API 20E strips are presented in Table es Table . By comp(Table ). 15-methyl-Hexadecenoic acid, 14-Methylpentadec-9-enoic acid is detected only in strain EGT. In contrast to P. nyackensis NWG-II14T, the strain EG have in its membrane composition the 3-hydroxy-Hexadecanoic acid, 13-methyl-Tetradecanoic acid, 3-hydroxy-13-methyl-Tetradecanoic acid, 3-hydroxy-15-methyl-Hexadecanoic acid, 15-methyl-Hexadecenoic acid and 14-Methylpentadec-9-enoic acid; and for the absence of Dodecanoic acid, 3-hydroxy-8-methyl-nonanoic acid, 9-Heptadecenoic acid, 9-Octadecenoic acid.The analysis of the composition in fatty acid of the strain EG revealed that the major fatty acids were 13-methyl-tetradecanoic acid 49.8%), 9-Hexadecenoic acid (27.7%) and 3-hydroxy-15-methyl-Hexadecanoic acid (7.5%). Other fatty acids included 15-methyl-Hexadecenoic acid (3.6%), 14-Methylpentadec-9-enoic acid (3.4%), Hexadecanoic acid (3.3%), 3-hydroxy-13-methyl-Tetradecanoic acid (1.9%), Tetradecanoic acid (1.4%) and 3-hydroxy-Hexadecanoic acid (1.0%). Minor amounts (<1%) of the fatty acids were also detected as Pentadecanoic acid and 9,12-Octadecadienoic acid 9.8%, 9-H−3) and a score of 52 with the GacS [GacS/GacA two-component system]42 sensor histidine kinase/response regulator in Pseudomonas putida strain KT2440 according to the VFDB database. It has been reported that the two-component system signaling pathways are the major signaling mechanisms in bacteria and as well in Archaea43 to monitor external and internal stimuli 44. This pathway is also found in simple eukaryota and higher plants45. In opportunistic bacterial pathogens, the use of two-components systems is required to regulate the expression of genes necessary for the transition from the environmental reservoir to the host46. Digital DNA-DNA hybridization values (dDDH) obtained using the GGDC software are reported in Table P. nutrimenti to 21.50% with P. nyackensis. Such values were lower than the 70% threshold recognized to delineate distinct species. Similarly, Ortho-ANI values  ranged from 70.98% with P. nutrimenti to 74.65% with P. nyackensis, which is lower than the 95% threshold used to discriminate bacterial species. Thus, we could confirm that strain EG belonged to a separate Pedobacter species for which we propose the name Pedobacter schmidteae sp. nov.The genome sequence from strain EG was assembled into one contig of 6,198,518 bp with a G + C content of 41.13%. We identified a total of 5,012 predicted protein-coding genes, in addition to 3 complete rRNA operons, 52 tRNAs and 1 tmRNA. Comparison of these genomic data with those from of closely related species is presented in Table P. schmidteae sp. nov. Interestingly, P. schmidteae sp. nov. is present in 84.6% of S. mediterranea worms tested. To best of our knowledge, P. schmidteae sp. nov has never been identified anywhere else than in S. mediterranea. Indeed, no nucleotide sequence linked to this new strain has been found in the nr database (BLASTn https://blast.ncbi.nlm.nih.gov). To date, we assume that P. schmidteae sp. nov is unique to S. mediterranea, but we cannot exclude formally that it might be present in other living organisms of the environment.Using the taxono-genomic approach, we concluded that strain EG is the representative strain of the new species 47. Pedobacter schmidteae . The bacterium belongs to the family Sphingobacteriaceae within the phylum Bacteroidetes. The type strain EGT (CSUR P6417 = CECT9771) was isolated on 5% sheep blood-enriched Columbia agar after 2 days at 28 °C in aerobic atmosphere at pH 7 from the microbiota of the planarian S. mediterranea. Colonies are small, round, smooth, transparent and convex. Bacterial cells are Gram-negative, rod-shaped, motile and non-spore-forming bacilli with positive catalase and oxidase activities. The main cellular fatty acids detected are 13-methyl-tetradecanoic acid (49.8%), 9-Hexadecenoic acid (27.7%), 3-hydroxy-15-methyl-Hexadecanoic acid (7.5%), 15-methyl-Hexadecenoic acid (3.6%), 14-Methylpentadec-9-enoic acid (3.4%), Hexadecanoic acid (3.3%), 3-hydroxy-13-methyl-Tetradecanoic acid (1.9%), Tetradecanoic acid (1.4%) and 3-hydroxy-Hexadecanoic acid (1.0%). We found traces (<1%) of Pentadecanoic acid and 9,12-Octadecadienoic acid.The protologue is to standardize the format of descriptions of new taxa, supported by the Judicial Commission of the International Committee on Systematic BacteriologyT exhibits positive reactions for catalase, oxidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, Naphtol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, esculin ferric citrate, glucose, D-mannose, N-Acetyl-glucosamine and D-maltose, but negative reaction for lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-mannosidase, α-fucosidase.Using an APIZYM strip, strain EGT is unable to metabolize glycerol, starch, amygdalin, arbutin, D-adonitol, D-arabinose, D-arabitol, D-cellobiose, D-fructose, D-fucose, D-galactose, D-glucose, D-lactose, D-melezitose, D-melibiose, D-raffinose, D-ribose, D-saccharose, D-sorbitol, D-tagalose, D-trehalose, D-turanose, dulcitol, D-xylose, erythritol, gentiobiose, glycogen, inositol, inulin, L-arabinose, L-arabitol, L-fucose, L-rhamnose, L-sorbose, L-xylose, methyl-αD-Glucopyranoside, methyl-αD-Mannopyranoside, methyl-βD-xylopyranoside, potassium 2-CetoGluconate, potassium 5-Cetogluconate, potassium gluconate, salicin, xylitol.Using a API 50CH strip, strain EGT use potassium nitrate, L-tryptophane, L-arginine, urea, gelatin, D-mannitol, malic acid, phenylacetic acid, L-lysin, trinatriumcitrate, L-ormithin, Esculin ferric citrate, Glucose, D-mannose, N-Acetyl-glucosamine and D-maltose.In addition, strain EGT is 6,198,518 bp–long with a G + C content of 41.13%.The genome of strain EGThe 16S rRNA gene and genome sequence are deposited in GenBank under accession numbers LS453293 and LS999839, respectively.Pedobacter schmidteae strain EGT is available at http://www.mediterranee-infection.com/article.php?laref=936The MALDI-TOF MS spectrum of T is TA00631 in the http://imedea.uib-csic.es/dprotologue/ website.The 16S rRNA gene sequence and genome sequence were deposited in GenBank under accession numbers LS453293 and LS999839, respectively. The Digital Protologue database Taxonumber for strain EGT  was deposited in the CSUR (Collection de Souches de l’Unité des Rickettsies WDCM 875) and CSCT (Colección Española de Cultivos Tipo) strain collections under numbers CSUR P6417 and CECT9771, respectively.Strain EG


Escherichia coli strain Tj, isolated from the Varzob River in Tajikistan, is presented. This strain possesses four prophage elements related to Shigella phage SfV, E. coli O157:H7-specific phage ϕV10, lambdoid phage HK225, and coliphage Ayreon. It contains a gene encoding a hemolysin E toxin.The 4.6-Mbp draft genome sequence of  Escherichia coli strain Tj, isolated from the Varzob River in Tajikistan, is presented. This strain possesses four prophage elements related to Shigella phage SfV, E. coli O157:H7-specific phage ϕV10, lambdoid phage HK225, and coliphage Ayreon. It contains a gene encoding a hemolysin E toxin.The 4.6-Mbp draft genome sequence of  Escherichia coli (Migula 1895) Castellani and Chalmers 1919 is a Gram-negative, facultatively anaerobic, enteric bacterium that inhabits the intestines of warm-blooded animals and humans and frequently contaminates environmental waters and food. E. coli strain Tj was isolated from the Varzob River in Tajikistan, during a laboratory course at the Center of Biotechnology of the Tajik National University, on Endo LES agar plates at 44°C as a dark-red colony and confirmed as E. coli by its API 20E profile (5-0-4-4-5-5-2). The 16S rRNA gene sequence, obtained by PCR amplification and Sanger sequencing as described elsewhere . The genome was sequenced at Eurofins Genomics using a NEBNext Ultra II DNA library preparation kit and Illumina HiSeq 2500 paired-end sequencing technology with a read length of 2 × 150 bp, yielding 6,416,190 reads and 1,924,857,000 sequenced bases. Reads with a maximum of 7 bases with a Phred score below 28 were initially discarded, and additional quality control procedures were performed using the Trim Reads tool in the CLC Genomics Workbench v. 8.5.1. Unless otherwise stated, all software was used with default values. Assembly was performed using the CLC de novo assembly tool, resulting in 4,627,784 bp of unique sequence data distributed into 96 contigs with an N50 value of 111,402 bp, coverage of 417×, and GC content of 50.8%. The draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok). Genome completeness was estimated as 99.96% by CheckM v. 1.0.18  values of ≥97.0% (http://www.mgc.ac.cn/VFs) (E. coli phage Ayreon (P-1) (Shigella flexneri (P-2) (NC_019717), and phage ϕV10, which specifically infects E. coli O157:H7 (P-4)   using IsE. coli strain Tj have been deposited in DDBJ/ENA/GenBank under accession numbers MT920313 and GCA_007049865.1, respectively. The associated BioProject, SRA, and BioSample accession numbers are PRJNA506592, SRR12336902, and SAMN10464432, respectively.The partial 16S rRNA gene and whole-genome shotgun sequences of


In Russia, cancer is the second leading cause of death following cardiovascular diseases. Adoptive transfer of NK cells is a promising approach to fight cancer; however, for their successful use in cancer treatment, it is necessary to ensure their robust accumulation at tumor foci, provide resistance to the immunosuppressive tumor microenvironment, and to engineer them with higher cytotoxic activity. NK lymphocytes are known to kill cancer cells expressing a number of stress ligands; and the balance of signals from inhibitory and activating receptors on the surface of the NK cell determines whether a cytotoxic reaction is triggered. We hypothesized that stronger cytotoxicity of NK cells could be achieved via gene editing aimed at enhancing the activating signaling cascades and/or weakening the inhibitory ones, thereby shifting the balance of signals towards NK cell activation and target cell lysis. Here, we took advantage of the CRISPR/Cas9 system to introduce mutations in the coding sequence of the shp-2 (PTPN11) gene encoding the signaling molecule of inhibitory pathways in NK cells. These shp-2 knock-outNK cells were additionally transduced to express a chimeric antigen receptor (CAR) that selectively recognized the antigen of interest on the target cell surface and generated an activating signal. We demonstrate that the combination of shp-2 gene knockout and CAR expression increases the cytotoxicity of effector NK-like YT cells against human prostate cancer cell line Du-145 with ectopic expression of PSMA protein, which is specifically targeted by the CAR. Natural killer cells are cells whose primary function is toeliminate infected and transformed cells in the body. Cytotoxicactivity of NK cells is regulated by the balance of signalstriggered by the interaction of activating and inhibitory receptorswith appropriate ligands on the surface of target cells.Inhibitory receptors play an central role in NK cell biologysince most of their ligands are represented by Major HistocompatibilityComplex I (MHC-I) molecules, that are presenton the surface of nearly all healthy cells and frequently absenton cancer or infected cells . This ensures that normal cells are spared by NK cells,whereas the cells that have lost MHC-I expression becomeeliminated . Nevertheless,absence of MHC-I expression does not universallyguarantee that an NK cell would kill a target cell, as this activityis also heavily dependent on the presence of ligands toactivating receptors of NK cells . Inhibitory receptor signal is known to be mainlytransduced via SH2-containing inositol 5′ polyphosphatase 1(SHIP-1) and/or tyrosine phosphatases Shp-1 and Shp-2. Thelatter two proteins dephosphorylate tyrosine residues withinthe ITAM motifs of activating receptors thereby aborting thesignaling cascade .NK cells are successfully used in therapy of oncologicaldiseases . Nevertheless, because tumorcells actively withstand elimination by establishing an immunosuppressivetumor microenvironment, the cytotoxicityof NK cells can be strongly affected . There are several waysof enhancing the cytotoxicity of NK cells. These includemodification of signaling pathways, altering the levels andrepertoire of NK cell receptors, or boosting the activity ofNK cells with appropriate cytokines . In this study, we explored whethermodification of the inhibitory signaling pathway through theshp-2 gene knockout in NK cells would result in the increaseof the cytotoxic activity of model NK cells.Cell lines and cell culture. YT, Du-145-PSMA, and HEK293Tcell lines were maintained in Iscove’s Modified Dulbecco’sMedium (IMDM) supplemented with 10 % fetal bovine serum(FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin(Sigma) in a humidified atmosphere of 5 % CO2 at 37 °C.Plasmid construction. Two shp-2-specific sgRNAs wereidentified using publicly available bioinformatics tools . The following target sequences were selected: gtgcagatcctacctctgaaagg andacagtactacaactcaagcagg (PAM site underlined).The lentiCRISPRv2 vector provided by Prof. Feng Zhang  was usedfor co-delivery of sgRNAs and Cas9. Oligonucleotides correspondingto the selected targets were denatured in a boilingwater bath followed by slow renaturation and cloned into thelentiCRISPRv2 vector between BsmBI restriction sites.The plasmid DNA of clones lentiCRISPRv2-Shp2g1 andlentiCRISPRv2-Shp2g2 encoding shp2-specific sgRNA1 andsgRNA2, was mixed with the DNA of packaging plasmidspsPAX2 and pMD2.G (provided by Prof. D. Trono) in thefollowing weight ratio: 10:10:7.5:2.5, in the total amountof 3 μg. Using calcium phosphate transfection , the resulting plasmid DNA mix was delivered toHEK293T cells. Supernatants containing VSV-G pseudotypedlentiviral particles were collected 48 hours after transfection,filtered through 0.45 μm PES filters and used either fresh orstored at –70 °C.YT cell transduction. To improve transduction rate ofYT cells, a spinoculation protocol was used . YT cells were seeded in 24-well plates (1 × 105 cells) inthe presence of polybrene (8 μg/ml) followed by the additionof supernatants containing pseudotyped lentiviral particles.Cells were centrifuged at 500 g for 40 minutes at 32 °C andincubated in a CO2 incubator for 16 hours. The next day,the supernatant was replaced with a fresh culture medium.After 3 days, selection of transduced cells was performed byadding puromycin  to a final concentrationof 5 mg/ ml (1 week), with non-transduced cells serving ascontrols.Western blot analysis. For western blot analysis, transducedYT cells and control non-transduced cell lines YT-wtand HEK293T were lysed in lysis buffer . Cell lysateswere centrifuged, boiled in a water bath for 5 minutes,separated by SDS-PAGE in a 10 % polyacrylamide gel andtransferred onto a nitrocellulose membrane . After blocking in 3 % milk in PBST, the membrane wasincubated with rabbit monoclonal antibodies against Shp-2, followedby an HRP-labeled secondary goat-anti-rabbit antibody. Equal loading was controlled byusing hybridization with control antibodies against β-actin. Signal was detected usingan ECL-prime substrate in accordance with the manufacturer’srecommendations  and AmershamImager 600 with an exposure time of 1 min.Flow cytometry. For phenotyping CAR-YTshp-2–/– celllines, 105 cells were washed in PBS and incubated for 30 minuteswith the biotinylated protein L (3.3 μg/ml)  at a temperature of 4 °C. Then the cells werewashed and incubated with streptavidin-APC  in accordance with the manufacturer’s recommendations.Vital dye 7AAD  was used to excludedead cells from the analysis. The samples were analyzed usingBD FACSCanto® II (Becton Dickinson and Company) andBD FACSDiva software.Real Time Cytotoxicity Assay (RTCA). Adherent targetcells (5 × 104 cells per well) were seeded in 8-well E-plates and left to grow for 16–18 hours.The next day, the medium was removed and replaced witha fresh one containing 105 effector cells. Cell growth wasmonitored for 24 hours using the RTCA iCELLigence system.Cytotoxicity was calculated using the formula: [CI (targetcells without effector cells) – CI (target cells with effectorcells) × 100/CI (target cells without effector cells)], where CIis the normalized impedance value in the wells .Flow cytometry-based cytotoxicity assay. Cells were labeledwith Cell Proliferation Dye eFluor 670 . 50,000 target cells per well were seeded into a 96-wellplate. Next, an equal number of effectors was added .Cells were incubated for 4 hours in a humidified atmosphereof 5 % CO2 at 37 °C. Then the vital dye 7AAD was added toeach well and the percentage of living target cells was measuredon a BD FACSCantoII flow cytometer.Statistical analysis  was performed using Prism software(GraphPad version 8.0). One-way analysis of variance (onewayANOVA) was used to detect the differences between theactivity of control cell lines that did not carry the shp-2 geneknockout and that of knockout cells. All data are presentedas mean ± standard error of the mean.In this study, we have chosen human immortalized cell lineYT that has an NK-like phenotype. These cells are known todisplay perforin-mediated target cell lysis, lack Fc receptor expressionand do not require conditioning of growth media withIL2 . To knock-out shp-2, CRISPR/Cas9 system was used.Two sgRNAs specific to the 3rd and 5th exons of the genewere cloned into a lentiviral vector lentiCRISPRv2 . The two resulting constructs lentiCRISPRv2-Shp2g1 and lentiCRISPRv2-Shp2g2 were used for producingVSV-G-pseudotyped lentiviral particles, which then wereused for transduction of YT-cells. Given that lentiCRISPRv2vector carries a puromycin resistance gene, transduced cellswere subjected to antibiotic selection and single-cell cloning. A panel of monoclonal derivatives of YT cells was analyzedby Sanger-sequencing the genomic fragments centered atsgRNA-binding sites in the shp-2 gene, in order to identifythe clones carrying biallelic mutations in this gene .Four clones  were obtained that displayedtruncated shp-2 sequence . Knock-outswere verified by western-blot analysis, which confirmed thatthese clones did not express a full-length Shp-2 protein .One way to re-target NK cell cytotoxicity is via expressionof chimeric antigen receptors (CARs). Even if the target celllacks typical stress markers recognized by endogenous NK cellreceptors, binding of a CAR to its cognate target may inducepro-activation signaling in a CAR-NK cell. To test whetherCAR-mediated cytotoxicity is enhanced upon shp2-knock out,we used our previously published second-generation CAR(scFv(J591)-CD8hinge-CD28TM-CD28-CD3z)  that is specific for PSMA, a common surfacemarker of prostate cancer cells . Wild-type and shp2-mutant YT cell lines weretransduced to express this CAR, which was verified by flowcytometry .shp-2–/– cells exerted significantlyhigher cytotoxicity than CAR-YT cells with a functionalshp- 2 gene; no difference between the knock-out subcloneswas observed .Next, we compared the cytotoxic activity of the B1-CAR,C1-CAR, and C4-CAR cells vs CAR-YT cells against Du-145 prostate cancer cells engineered to ectopically expressPSMA. Du-145 cells have been reported to be resistant toNK cell killing , which establishes theman appropriate model for assaying possible enhancement ofNK cell-mediated cytotoxicity. We used iCELLigence platform for the cytotoxicity analysis.It was found that CAR-YTshp-2–/– cells may displayunwanted cytotoxicity against healthy cells, cytotoxicity assaywas conducted with PBMCs isolated from a healthy donor. Weobserved that the percentage of dead normal cells did not differsignificantly between the wells where CAR-YTshp-2–/– cells orno effector cells (control) were added . This was alsotrue for cultures, where normal cells were co-incubated witheither CAR-YT or CAR-YTshp-2–/– cells.To understand whether CAR-YTCytotoxic activity of NK cells is known to be regulated bythe balance of activating and inhibitory receptor signals . Cytotoxic reaction occurswhen two conditions are met. Namely, if an activating signalis present, (i. e. when the target cell expresses the ligand(s) tothe activating receptors) and if the negative signaling is sufficientlyreduced or absent  . Whenever positive signalingis absent or negative signaling outweighing the positive signaling,cytotoxic reaction will not be launched. In the contextof NK cell therapy, this translates to the failure to eliminatethe tumor cells .Absence of activating signal can be effectively solved byengineering CAR expression in NK cells, an approach alreadyactively used in practice . Nonetheless,tumor cells overexpressing the ligands of inhibitoryNK cell receptors may still remain protected from CAR-NKcell mediated lysis .One way to address this problem is to create NK cells withknock-outs of inhibitory receptors. Although technicallyfeasible, this may not be as straightforward, considering thelarge number of these receptors. In our opinion, knockingout Shp-2, a single “hub” of NK cell signaling appears as aviable alternative . Our experiments show that YT cell line derivativeslacking SHP2 expression display stronger CAR-mediatedcytotoxicity towards NK-resistant cell line Du-145-PSMA, unlike CAR-YT cells . Thisis likely attributableto the fact that Du-145-PSMA cellshave a high density of inhibitory ligands, which results in thesuppression of activating signaling in NK cells, even uponCAR engagement. Indeed, it was reported that Du-154 cellsexpress low levels of ligands to the activating NK receptorsNKp30 and NKp46, while MHC-I expression is very high.shp-2–/– cells are safe, testing ona wide panel of different healthy cell types obtained frommultiple donors may be required, which is a challenging taskfrom a technical and ethical points of view. In the currentstudy, we have measured cytotoxicity of CAR-YTshp-2–/– cellstowards the lymphocytes of a healthy donor, and no significantdifference with negative control was observed. This suggeststhat reducing the inhibitory signaling in NK cells likely posesno threat provided that no strong activating signals are available.However, to address this important safety concern a morecomprehensive study is indeed required. We envisage that arepresentative and diverse panel iPS-derived normal humancell types can be obtained to test for the possible off-targetactivity of shp2-mutant YT cells.Safety of such combination is yet to be found, as it is possiblethat this may lead to increased cytotoxicity towardshealthy tissues, as a result of weakening of inhibitory signaling.To ensure that the YTThus, the best option for enhancing cytotoxicity of NK cellsand the YT cell line in particular is to induce activating signalsusing CARs in combination with the suppression of inhibitorysignaling via knocking out its key mediator, the SHP-2protein. In the future it is necessary to comprehensivelystudy the safety of modified lymphocytes, in particular, tostudy their cytotoxic activity against a wider range of healthytissues.The authors declare no conflict of interest.Becker P.S.A., Suck G., Nowakowska P., Ullrich E., Seifried E.,Bader P., Tonn T., Seidl C. Selection and expansion of naturalkiller cells for NK cell-based immunotherapy. Cancer Immunol.Immunother. 2016;65(4):477-484. DOI 10.1007/s00262-016-1792-y.Cerwenka A., Baron J.L., Lanier L.L. Ectopic expression of retinoicacid early inducible-1 gene (RAE-1) permits natural killercell-mediated rejection of a MHC class I-bearing tumor in vivo.Proc. Natl. Acad. Sci. USA. 2001;98(20):11521-11526. DOI10.1073/pnas. 201238598.Chang S.S. Overview of prostate-specific membrane antigen. Rev.Urol. 2004;6(Suppl.10):S13-S18.Chester C., Fritsch K., Kohrt H.E. Natural killer cell immunomodulation:targeting activating, inhibitory, and co-stimulatoryreceptor signaling for cancer immunotherapy. Front. Immunol.2015;6:601. DOI 10.3389/fimmu.2015.00601.Childs R.W., Carlsten M. Therapeutic approaches to enhance naturalkiller cell cytotoxicity against cancer: the force awakens. Nat.Rev. Drug Discov. 2015;14:487. DOI 10.1038/nrd4506.De Charette M., Marabelle A., Houot R. Turning tumour cells intoantigen presenting cells: the next step to improve cancer immunotherapy?Eur. J. Cancer. 2016;68:134-147. DOI 10.1016/j.ejca.2016.09.010.Deaglio S., Zubiaur M., Gregorini A., Bottarel F., Ausiello C.M.,Dianzani U., Sancho J., Malavasi F. Human CD38 and CD16are functionally dependent and physically associated in naturalkiller cells. Blood. 2002;99(7):2490-2498.Del Zotto G., Marcenaro E., Vacca P., Sivori S., Pende D., DellaChiesa M., Moretta F., Ingegnere T., Mingari M.C., Moretta A.,Moretta L. Markers and function of human NK cells in normaland pathological conditions. Cytometry B. Clin. Cytom. 2017;92(2):100-114. DOI 10.1002/cyto.b.21508.Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W.,Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., VirginH.W., Listgarten J., Root D.E. Optimized sgRNA design tomaximize activity and minimize off-target effects of CRISPRCas9.Nat. Biotechnol. 2016;34(2):184-191. DOI 10.1038/nbt.3437.Edsparr K., Speetjens F.M., Mulder-Stapel A., Goldfarb R.H.,Basse P.H., Lennernäs B., Kuppen P.J.K., Albertsson P. Effects ofIL-2 on MMP expression in freshly isolated human NK cells andthe IL-2-independent NK cell line YT. J. Immunother. 2010;33(5):475-481. DOI 10.1097/CJI.0b013e3181d372a0.Freund-Brown J., Chirino L., Kambayashi T. Strategies to enhanceNK cell function for the treatment of tumors and infections.Crit. Rev. Immunol. 2018;38(2):105-130. DOI 10.1615/CritRevImmunol.2018025248.Golubovskaya V., Berahovich R., Zhou H., Xu S., Harto H., Li L.,Chao C.C., Mao M.M., Wu L. CD47-CAR-T cells effectivelykill target cancer cells and block pancreatic tumor growth. Cancers(Basel). 2017;9(10):139. DOI 10.3390/cancers9100139.Gorchakov A.A., Kulemzin S.V., Kochneva G.V., Taranin A.V.Challenges and prospects of chimeric antigen receptor T-cell therapy for metastatic prostate cancer. Eur. Urol. 2019. DOI10.1016/j.eururo. 2019.08.014.Hanke T., Takizawa H., Mcmahon C.W., Busch D.H., Pamer E.G.,Miller J.D., Altman J.D., Liu Y., Cado D., Lemonnier F.A.,Bjorkman P.J., Raulet D.H. Direct assessment of MHC class Ibinding by seven Ly49 inhibitory NK cell receptors. Immunity.1999;11(1):67-77. DOI 10.1016/S1074-7613(00)80082-5.Hewitt E.W. The MHC class I antigen presentation pathway: strategiesfor viral immune evasion. Immunology. 2003;110(2):163-169. DOI 10.1046/j.1365-2567.2003.01738.x.Hood S.P., Foulds G.A., Imrie H., Reeder S., Mcardle S.E.B.,Khan M., Pockley A.G. Phenotype and function of activatednatural killer cells from patients with prostate cancer: patientdependentresponses to priming and IL-2 activation. Front. Immunol.2019;9:3169. DOI 10.3389/fimmu.2018.03169.Igarashi T., Wynberg J., Srinivasan R., Becknell B., McCoy J.P.,Takahashi Y., Suffredini D.A., Linehan W.M., Caligiuri M.A.,Childs R.W. Enhanced cytotoxicity of allogeneic NK cells withkiller immunoglobulin-like receptor ligand incompatibilityagainst melanoma and renal cell carcinoma cells. Blood. 2004;104(1):170. DOI 10.1182/blood-2003-12-4438.Imai C., Iwamoto S., Campana D. Genetic modification of primarynatural killer cells overcomes inhibitory signals and induces specifickilling of leukemic cells. Blood. 2005;106(1):376-383. DOI10.1182/blood-2004-12-4797.Ingegnere T., Mariotti F.R., Pelosi A., Quintarelli C., De Angelis B.,Tumino N., Besi F., Cantoni C., Locatelli F., Vacca P., Moretta L.Human CAR NK cells: a new non-viral method allowing highefficient transfection and strong tumor cell killing. Front. Immunol.2019;10:957. DOI 10.3389/fimmu.2019.00957.Kärre K., Ljunggren H.G., Piontek G., Kiessling R. Selective rejectionof H–2-deficient lymphoma variants suggests alternativeimmune defence strategy. Nature. 1986;319(6055):675-678.DOI 10.1038/ 319675a0.Kulemzin S.V., Matvienko D.A., Sabirov A.H., Sokratyan A.M.,Chernikova D.S., Belovezhets T.N., Chikaev A.N., Taranin A.V.,Gorchakov A.A. Design and analysis of stably integrated reportersfor inducible transgene expression in human T cells and CARNK-cell lines. BMC Med. Genomics. 2019;12(Suppl.2):44. DOI10.1186/s12920-019-0489-4.Kutner R.H., Zhang X.-Y., Reiser J. Production, concentration andtitration of pseudotyped HIV-1-based lentiviral vectors. Nat.Protoc. 2009;4(4):495-505. DOI 10.1038/nprot.2009.22.Lee S.K., Gasser S. The role of natural killer cells in cancer therapy.Front. Biosci. (Elite Ed). 2010;2:380-391.Malarkannan S. The balancing act: inhibitory Ly49 regulateNKG2D-mediated NK cell functions. Semin. Immunol. 2006;18(3):186-192. DOI 10.1016/j.smim.2006.04.002.Mamessier E., Sylvain A., Thibult M.-L., Houvenaeghel G., JacquemierJ., Castellano R., Gonçalves A., André P., Romagné F.,Thibault G., Viens P., Birnbaum D., Bertucci F., Moretta A.,Olive D. Human breast cancer cells enhance self tolerance bypromoting evasion from NK cell antitumor immunity. J. Clin.Invest. 2011;121(9):3609-3622. DOI 10.1172/JCI45816.Moreno-Mateos M.A., Vejnar C.E., Beaudoin J.D., Fernandez J.P.,Mis E.K., Khokha M.K., Giraldez A.J. CRISPRscan: designinghighly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.Nat. Methods. 2015;12(10):982-988. DOI 10.1038/nmeth.3543.Nayyar G., Chu Y., Cairo M.S. Overcoming resistance to naturalkiller cell based immunotherapies for solid tumors. Front. Oncol.2019;9:51. DOI 10.3389/fonc.2019.00051.O’Doherty U., Swiggard W.J., Malim M.H. Human immunodeficiencyvirus type 1 spinoculation enhances infection throughvirus binding. J. Virol. 2000;74(21):10074-10080. DOI 10.1128/jvi.74.21.10074-10080.2000.Pasero C., Gravis G., Granjeaud S., Guerin M., Thomassin-PianaJ., Rocchi P., Salem N., Walz J., Moretta A., Olive D. Highlyeffective NK cells are associated with good prognosis in patientswith metastatic prostate cancer. Oncotarget. 2015;6(16):14360-14373. DOI 10.18632/oncotarget.3965.Pasero C., Gravis G., Guerin M., Granjeaud S., Thomassin-PianaJ., Rocchi P., Paciencia-Gros M., Poizat F., Bentobji M.,Azario-Cheillan F., Walz J., Salem N., Brunelle S., Moretta A.,Olive D. Inherent and tumor-driven immune tolerance in theprostate microenvironment impairs natural killer cell antitumoractivity. Cancer Res. 2016;76(8):2153. DOI 10.1158/0008-5472.CAN-15-1965.Paul S., Lal G. The molecular mechanism of natural killer cellsfunction and its importance in cancer immunotherapy. Front.Immunol. 2017;8:1124. DOI 10.3389/fimmu.2017.01124.Purdy A.K., Campbell K.S. SHP-2 expression negatively regulatesNK cell function. J. Immunol. 2009;183(11):7234-7243. DOI10.4049/jimmunol.0900088.Quintarelli C., Sivori S., Caruso S., Carlomagno S., Boffa I., OrlandoD., Guercio M., Cembrola B., Pitisci A., Di Cecca S., LiPira G., Vinti L., De Angelis B., Moretta L., Locatelli F. CD19redirected CAR NK cells are equally effective but less toxic thanCAR T cells. Blood. 2018;132(Suppl.1):3491. DOI 10.1182/blood-2018-99-118005.Rehman A.U., Rahman M.U., Khan M.T., Saud S., Liu H., Song D.,Sultana P., Wadood A., Chen H.F. The landscape of proteintyrosine phosphatase (Shp2) and cancer. Curr. Pharm. Des.2018;24(32):3767-3777. DOI 10.2174/1381612824666181106100837.Rezvani K., Rouce R., Liu E., Shpall E. Engineering natural killercells for cancer immunotherapy. Mol. Ther. 2017;25(8):1769-1781. DOI 10.1016/j.ymthe.2017.06.012.Rusakiewicz S., Semeraro M., Sarabi M., Desbois M., Locher C.,Mendez R., Vimond N., Concha A., Garrido F., Isambert N.,Chaigneau L., Le Brun-Ly V., Dubreuil P., Cremer I., CaignardA., Poirier-Colame V., Chaba K., Flament C., Halama N.,Jäger D., Eggermont A., Bonvalot S., Commo F., Terrier P.,Opolon P., Emile J.-F., Coindre J.- M., Kroemer G., Chaput N.,Le Cesne A., Blay J.-Y., Zitvogel L. Immune infiltrates areprognostic factors in localized gastrointestinalstromal tumors. CancerRes. 2013;73(12):3499. DOI 10.1158/0008-5472.CAN-13-0371.Sanjana N.E., Shalem O., Zhang F. Improved vectors and genomewidelibraries for CRISPR screening. Nat. Methods. 2014;11(8):783-784. DOI 10.1038/nmeth.3047.Sivori S., Vacca P., Del Zotto G., Munari E., Mingari M.C., MorettaL. Human NK cells: surface receptors, inhibitory checkpoints,and translational applications. Cell. Mol. Immunol. 2019;16(5):430-441. DOI 10.1038/s41423-019-0206-4.Suen W.C.W., Lee W.Y.W., Leung K.T., Pan X.H., Li G. Naturalkiller cell-based cancer immunotherapy: a review on 10 yearscompleted clinical trials. Cancer Invest. 2018;36(8):431-457.DOI 10.1080/07357907.2018.1515315.Yang L., Shen M., Xu L.J., Yang X., Tsai Y., Keng P.C., Chen Y.,Lee S.O. Enhancing NK cell-mediated cytotoxicity to cisplatin-resistant lung cancer cells via MEK/Erk signaling inhibition.Sci. Rep. 2017;7(1):7958. DOI 10.1038/s41598-017-08483-z.Yodoi J., Teshigawara K., Nikaido T., Fukui K., Noma T., Honjo T.,Takigawa M., Sasaki M., Minato N., Tsudo M., Uchiyama T.,Maeda M. TCGF (IL 2)-receptor inducing factor(s). I. Regulationof IL-2 receptor on a natural-killer-like cell-line (YT-cells).J. Immunol. 1985;134(3):1623-1630.Yusa S.-I., Campbell K.S. Src homology region 2-containingprotein tyrosine phosphatase-2 (SHP-2) can play a direct rolein the inhibitory function of killer cell Ig-like receptors in humanNK cells. J. Immunol. 2003;170(9):4539. DOI 10.4049/jimmunol.170.9.4539.


Egglestonichthysfulmensp. nov. (Teleostei: Gobiidae) is described on the basis of a single specimen (21.7 mm in standard length) collected from 250 m depth off Okinawa Island, Ryukyu Islands, Japan. The new species is characterized by the following combination of characters: anal-fin rays I, 9; pectoral-fin rays 17, lower rays not free from membrane; longitudinal scale series 25; transverse scales 8; pre-dorsal-fin scale rows 8; cheek and opercle naked; pelvic frenum absent; caudal fin lanceolate, its length 32.2% of SL; interorbital width very narrow, 1.2% of HL (much narrower than pupil diameter); no spicules or odontoid processes on outer surface of gill arches; and body whitish, upper half with broken zigzag pattern of bright yellow patches and associated scattered black melanophores in fresh specimens (melanophores retained in preserved specimens). Several characters, including pectoral-fin ray count, interorbital width, and coloration uniquely distinguish the new species from congeners. Egglestonichthys Miller & Wongrat, 1979. Although a generic reassessment of the genus Egglestonichthys is needed , Egglestonichthyspatriciae Miller & Wongrat, 1979, Egglestonichthysrubidus Allen, Erdmann & Brooks, 2020, and Egglestonichthysulbubunitj Larson, 2013]   Fig. ; no spicer) Fig. .SL (% of HL in parentheses): head length 30.3; head depth 16.7 (55.1); head width 18.3 (60.6); body depth 14.9; body width 12.9; caudal-peduncle length 19.1, depth 9.4; snout length 5.9 (19.3); eye diameter 7.3 (24.0); interorbital width 0.4 (1.2); upper-jaw length 11.8 (39.0); pectoral-fin length 31.0; pelvic-fin length 28.1; caudal-fin length 32.2; and longest dorsal-fin spine (2nd) length 12.8.Dorsal-fin rays VI + I, 10; anal-fin rays I, 9; pectoral-fin rays 17; segmented caudal-fin rays 16; caudal-fin ray pattern 9/7; branched caudal-fin rays 7/5; unsegmented (procurrent) caudal-fin rays 6/6; longitudinal series scales 25; transverse scales 8; pre-dorsal-fin scale rows 8 ; circumpeduncular scales 12; gill rakers on outer face of first arch 3 + 11 (counted on right side); vertebrae 10 + 16. The following morphometrics are expressed as percentage of Body slender, compressed, width much less than depth Fig. . Anus siHead lacking sensory canal pores Fig. . Head seBody covered with relatively large cycloid scales. Pre-dorsal- and pelvic-fin regions covered with cycloid scales; anterior margin of pre-dorsal-fin scales reaching to vertical through preopercle margin; pre-pelvic-fin scales reaching to just behind anteroventral point of gill opening. Pectoral-fin base with 2 relatively large cycloid scales. Side of head  naked , last rays well separated from caudal-fin base. Pectoral fin long, pointed, middle rays longest, tips reaching to below origin of 2nd dorsal-fin soft ray; all rays connected by membrane, uppermost and lower 3 rays unbranched. Pelvic fins completely connected by membrane, without frenum; posterior tip reaching below 2nd dorsal-fin origin when appressed; pelvic-fin origin located just below ventral end of pectoral-fin base. Caudal fin lanceolate, its length much greater than head length.First dorsal fin triangular, 2Fresh coloration . Scattered black melanophores throughout bright yellow patches on head and body. Dorsal-fin rays whitish with yellow interspaces (possibly barred in undamaged specimens). Other fins whitish; upper end of pectoral-fin base with yellow spot; caudal-fin base with faint yellow vertical bar and broad rectangular patch of melanophores.ion Fig. . Body whColoration when preserved , neither could be confirmed in the present specimen. A detailed investigation of species currently assigned to Egglestonichthys must precede any redefinition of the genus .The holotype and only known example of the new species lacks head sensory pores, but has a transverse sensory papillae pattern on the cheek and a wide gill opening , thereby matching the diagnostic characters of aracters . Althougenus see : 153. NeEgglestonichthysfulmen is unlikely to be misidentified as one of its congeners, having the following characters: 17 pectoral-fin rays ; interorbit very narrow (width 1.2% of HL), much less than pupil diameter [vs. variously broad: 38.5–47.6% HL in E.bombylios (described as 4.9–5.9 in HL in Larson and Hoese 1996); 17.0–20.4% HL in E.melanoptera (4.9–5.9 in HL in Larson and Hoese 1996); equal to pupil diameter in E.patriciae .E.fulmen and E.melanoptera both lack a pelvic frenum, the former has much lower scale counts than the latter . E.ulbubunitj; all are connected by membrane in E.fulmen. In addition, E.fulmen has fewer anal-fin rays . Egglestonichthysfulmen also has fewer longitudinal series scales than E.bombylios and E.patriciae , a lanceolate caudal fin (length 32.2% of SL), and scales absent on the cheek and opercle .Although


Mycobacterium smegmatis mc2155. While the Awesomesauce genome is 57,054 bp with 94 protein-coding genes, the LastJedi genome is 55,149 bp with 94 protein-coding genes. Nucleotide sequence comparison in Phamerator detected synteny between Awesomesauce gp49 to gp61 and singleton LilSpotty. Whole-genome BLASTn alignments revealed that LastJedi strongly resembles Clifton (99.41% identity).Bacteriophages Awesomesauce and LastJedi infect  Mycobacterium smegmatis mc2155. While the Awesomesauce genome is 57,054 bp with 94 protein-coding genes, the LastJedi genome is 55,149 bp with 94 protein-coding genes. Nucleotide sequence comparison in Phamerator detected synteny between Awesomesauce gp49 to gp61 and singleton LilSpotty. Whole-genome BLASTn alignments revealed that LastJedi strongly resembles Clifton (99.41% identity).Bacteriophages Awesomesauce and LastJedi infect  Mycobacterium smegmatis mc2155, a prominent acid-fast Gram-positive soil bacterium. Awesomesauce inhabited damp, shaded soil at Brown University , while LastJedi was from mud near shrubs at the State University of New York College at Old Westbury . Both were isolated (with enrichment at 23 to 24°C), purified, and amplified in M. smegmatis mc2155, as described in the SEA-PHAGES Discovery Guide (https://seaphagesphagediscoveryguide.helpdocsonline.com/home). The bacteriophages form clear plaques (∼1 mm), exhibit Siphoviridae morphology (images are available at https://phagesdb.org/phages/Awesomesauce and https://phagesdb.org/phages/LastJedi), and are F cluster/F1 subcluster members (Bacteriophage Awesomesauce and LastJedi genomes were examined in collaboration with the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program in efforts to characterize viral diversity. Both bacteriophages infect https://phagesdb.org/DNAMaster). PECAAN resources applied to identify genes included Glimmer v3.0 (https://seaphages.org/software), ARAGORN v1.2.38 (http://www.cbs.dtu.dk/services/TMHMM), TOPCONS v2 (Following DNA extraction (Promega Wizard DNA clean-up system), sequencing libraries were produced (NEBNext Ultra II DNA library prep kit) and sequencing on an Illumina MiSeq system (MiSeq reagent kit v3)  was perfmer v3.0 , GeneMarmer v3.0 , Phameramer v3.0 , Starter v1.2.38 , and tRN v1.2.38 . Functio v1.2.38 , HHpred  v1.2.38 , TMHMM2 PCONS v2 , and thePCONS v2 . All proIn both genomes, key structural and assembly genes are on the left arm and nonstructural coding sequences are on the right arm. On the left arm, both genomes encode small and large terminase subunits, portal protein, capsid maturation protease, scaffolding protein, major capsid protein, head-to-tail adaptor, head-to-tail stopper, tail terminator, major tail protein, tail assembly chaperones, tape measure protein, minor tail proteins, lysin A/lysin B, and DNA polymerase III subunit. On the right arm, both code for genes regulating lysogeny , as well as WhiB family transcription factor, mobile element MPME1, and two glycosyltransferases.MH020243) , Misha28 (MH020242) , and Piper2020 (MK494120) . The Awesomesauce genome also includes Cas4-like exonuclease (gp66), two HNH endonucleases (gp1 and gp74), and two DNA methylases (gp73 and gp76). A rare synteny block (gp49 to gp61) that is highly conserved in 4 of the other 169 F1 mycobacteriophages  was identified. BLASTn comparison revealed remarkable colinearity and sequence similarity (98% identity) between the Awesomesauce synteny block and gp49 to gp61 in the singleton LilSpotty (MK977707), suggesting a common origin for the two. Excise (gp48) and MPME1 (gp62) border the synteny block and may facilitate its transposition. It was also noted that homologues to Awesomesauce gp82 (function unknown) occurred in 13 subcluster A1 bacteriophages but only 2 F1 mycobacteriophages (TootsiePop and Misha28) in addition to LilSpotty, suggesting shared origins for A1 and F1 bacteriophages.Whole-genome BLASTn alignments  revealedMH371115; 99.41% identity, with 95% coverage) and Brocalys . Notable features include a toxin/antitoxin system (gp35 and gp36), two helix-turn-helix DNA binding domains (gp53 and gp54), Ku-like double-stranded DNA break-binding protein (gp57), AAA-ATPase (gp58), HNH endonuclease (gp59), DNA helicase (gp60), and two GIY-YIG endonucleases (gp65 and gp82).LastJedi is highly homologous to F1 bacteriophages Clifton  (The Awesomesauce and LastJedi genomes are available in DDBJ/ENA/GenBank under accession numbers sdb.org) , MargotM


The 2010 Patient Protection and Affordable Care Act includes the Annual Wellness Visit (AWV) for older adult (OA) patients. Medicare pays for an initial AWV per beneficiary and subsequent visits annually. Many Medicare beneficiaries have not taken advantage of the AWV preventive health benefit. The Saint Louis University Geriatrics Workforce Enhancement Program (GWEP) developed an AWV for OA, NH residents. This project describes the NH AWV and reports results. Data include age, gender, comorbidities, medications, hospitalizations, depression, frailty, pain, sarcopenia, sensory impairment, cognition, nutrition, smoking, falls, and advance directives. Two suburban academic for-profit NHs are included in this study (2016-17). OA NH residents (N=247) completed an AWV and 36.8% (n=91) had a 1-year follow-up AWV. OA NH residents were female  and a majority ages 75+ . Most (96.3%) had a documented advance directive. Comorbidities (7.8±2), polypharmacy (92.3%), vision impairment (52.8%), hearing impairment (52.8%), depression (65.2%), frailty (75.7%), sarcopenia (84.4%), risk of weight loss (53.9%), MCI (11.7%), and dementia (75.8%) were prevalent. Among OA NH residents (n=91) with an AWV follow-up, there was modest worsening in total comorbidities and medications as well as frailty, sarcopenia, and cognition scores (ps≤0.05). Pain, depression, and nutrition did not change. To our knowledge, no one has specifically analyzed the Medicare AWV in NHs. Data from the traditional AWV is an extension of the routine clinical care of OAs and therefore could also be useful for healthcare professionals focused on providing care to OA patients in the NH setting.