Bacillus thuringiensis SS2 consists of 426 contigs assembled at the scaffold level, totaling 5,030,306 bp, and contains 5,288 putative PATRIC protein-coding genes, including genes responsible for total benzoate consumption, degradation of halogenated compounds, heavy metal tolerance/resistance, biosynthesis of secondary metabolites, and microcin C7 self-immunity protein.The draft genome sequence of strain  Bacillus thuringiensis is a Gram-positive, aerobic, rod-shaped, mesophilic bacterium that has been reported to metabolize persistent organic pollutants (POPs), pesticides, and heavy metals (B. thuringiensis SS2 was identified during a screen for dioxin-degrading bacteria from dibenzofuran (DF)-polluted waste dump soil obtained with a site with a history of incineration in Cele, Isolo, Lagos, Nigeria , and 2,7-dichlorodibenzo-p-dioxin  (MH714859) and biochemical characterization (7-diCDD) . It was rization .B. thuringiensis SS2 was extracted using the Quick-DNA fungal/bacterial miniprep kit , from a Luria-Bertani culture that had been grown overnight at 28°C. The DNA was quantified using a QuantiFluor double-stranded DNA (dsDNA) system with a Qubit 3.0 fluorometer. Whole-genome sequencing (WGS) was performed through Illumina NextSeq paired-end sequencing. The library preparation and 2 × 150-bp paired-end sequencing were performed using the Nextera XT Index kit v2 (FC-131-2001 to FC-131-2004) and a NextSeq 500 system  at Quadram Institute Bioscience  , forming 426 contigs . The longest contig was 120,678 bp, with an N50 value of 22,410 bp. RASTtk identified 5,288 PATRIC protein-coding sequences and 1,174 hypothetical proteins, 66 tRNA genes, and 4 rRNA genes; the closest relative for B. thuringiensis SS2 is B. thuringiensis 97-27 (serovar konkukian).The genome sequence of B. thuringiensis SS2 revealed putative genes linked to the degradation of dioxin and other POPs similar to those of Serratia marcescens SSA1, which was isolated from the same burnt dump site. These genes include putative genes for total consumption of benzoate via hydroxylation and halogenated aromatic compound  degradation, among others  and copper-translocating P-type ATPase (EC 3.6.3.4) genes, suggesting heavy metal tolerance and resistance. Other putative genes, including those for microcin C7 self-immunity protein (mccf), macrocin O-methyltransferase, and CAAX amino-terminal proteins, could indicate this strain’s diverse metabolic adaptations to stress in its environment.WGS of g others . B. thurB. thuringiensis SS2 at PATRICbrc (BV-BRC) can be viewed at https://www.bv-brc.org/view/Genome/1428.1921. The draft genome sequence was deposited in GenBank under the accession number JAOWLZ000000000; project data are available under BioProject accession number PRJNA888024, with BioSample accession number SAMN31205655 and SRA accession number SRR21870348. The 16S rRNA gene sequence is available under GenBank accession number MH714859.The genome annotation of


Microsphaeropsis, taxonomically classified within the kingdom fungi, phylum Ascomycota, subphylum Deuteromycotina, class Coelomycetes, order Sphaeropsidales, and family Sphaeropsidaceae, exhibit a ubiquitous distribution across various geographical regions. These fungi are known for their production of secondary metabolites, characterized by both structural novelty and potent biological activity. Consequently, they represent a significant reservoir for the advancement of novel pharmaceuticals. In this paper, a systematic review was present, marking the analysis of secondary metabolites synthesized by Microsphaeropsis reported between 1980 and 2023. A total of 112 compounds, comprising polyketones, macrolides, terpenoids, and nitrogen-containing compounds, were reported from Microsphaeropsis. Remarkably, among these compounds, 49 are novel discoveries, marking a significant contribution to the field. A concise summary of their diverse biological activities was provided, including antibacterial, antitumor, and antiviral properties and other bioactivities. This analysis stands as a valuable reference, poised to guide further investigations into the active natural products derived from Microsphaeropsis and their potential contributions to the development of medicinal resources. Microsphaeropsis, taxonomically classified within the phylum Ascomycetes, subphylum Deuteromycotina, class Coelenterata, order Sphaeropsidales, and family Sphaeropsidaceae in the fungal taxonomy [Microsphaeropsis was not accurately recognized, leading to the misplacement of many fungi within the genus Coniothyrium. In 1980, Sutton meticulously elucidated the characteristics of the Microsphaeropsis genus. He subsequently reclassified species previously assigned to the genus Coniothyrium under Microsphaeropsis and renamed the genus [Microsphaeropsis have been unearthed and taxonomically elucidated. As of now, the repository of the Species Fungorum database (https://speciesfungorum.org/Names/Names.asp (accessed on 2 November 2023)) registers a total of 54 species within the genus Microsphaeropsis. It is evident that fungi within the genus Microsphaeropsis exhibit remarkable biodiversity, with a high likelihood of containing structurally novel and biologically active secondary metabolites.taxonomy , are comtaxonomy . For a lhe genus ,4. SubseMicrosphaeropsis genus by Sutton in 1980, various structural categories of compounds, including polyketides, terpenoids, nitrogen-containing compounds, macrolides, and others, were isolated from 2 known and 17 unknown species of Microsphaeropsis, and these demonstrated excellent antibacterial, antifungal, cytotoxic, and other activities  and three known compounds: (4S)-4,8-dihydroxy -3,4-dihydro-1(2H)-naphthalen-1-one (2), (4S)-4,6,8-trihydroxy-3,4-dihydro-1(2H)- naphthalen-1-one (3), and chrysogeside D (94). Notably, compounds 3 and 94 were discovered for the first time in Microsphaeropsis. The research team conducted evaluations of the antifungal activity of these compounds. Compound 1 and compound 94 exhibited moderate antifungal activity against A. tenuissima with minimum inhibitory concentrations (MICs) at 64 μg/mL [Qin et al. 2017) successfully isolated four compounds from the endophyte fungus  successf77), along with the known compounds (R)-mellein (8), -hydroxymellein (9), -hydroxymellein (10), and 4,8-dihydroxy-3,4-dihydro-2H-naphthalen-1-one (11), were successfully isolated from the fungus Microsphaeropsis sp. H5-50, which inhabits the marine sponges M. incrustans. Notably, both compounds displayed significant antifungal activity against E. repens and U. violacea in agar diffusion assays [The novel compound microsphaeropsisin M. arundinis PSU-G18, found within the plant Garcinia hombroniana [110), and seven new phthalides, microsphaerophthalides A-G (29–35), were identified, alongside eleven known compounds . Compound 44 exhibited remarkable antifungal activity, with an IC50 value of 8 μg/mL. Additionally, microsphaerophthalides A (29) and microsphaerophthalides E (33) displayed moderate antifungal activity against M. gypseum SH-MU-4 and C. neoformans, both with MIC values of 64 μg/mL. Decaspirone (57), isolated from the endophyte fungus Microsphaeropsis sp. 7291, demonstrated significant antifungal activity against M. violaceum, evidenced by inhibition zone diameters measuring 18 mm [Sommart et al. (2012) isolated nineteen compounds from the endophyte fungus broniana . Among t112), was successfully isolated from the endophyte fungus Microsphaeropsis sp. 7820, found inhabiting the plant Salsola oppositifolia. Remarkably, compound 112 exhibited moderate antifungal activity against M. violaceum, resulting in inhibition zone diameters measuring 9 mm [A novel polychlorinated triphenyl diether, microsphaerol (ing 9 mm .74) and microketides B (75), from the fungus Microsphaeropsis sp. RA10-14, and both compounds exhibited significant antifungal activity against Candida albicans, Colletotrichum truncatum, Gloeosporium musarum, and Pestalotia calabae [74) exhibited IC50 values of 1.56 μg/mL, 1.56 μg/mL, 3.13 μg/mL, and 1.56 μg/mL, respectively. Microketides B (75) displayed equal IC50 values of 3.13 μg/mL.Liu et al. (2020) isolated two novel metabolites, microketides A , were isolated from Microsphaeropsis sp. F2076 and Microsphaeropsis sp. F2078. Notably, the four microsphaerins, A, B, C, and D, exhibited significant antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA), with IC50 values of 3 μM, 3 μM, 5 μM, and 1 μM, respectively. To delve deeper into their antibacterial activity, microsphaerins D (48) was selected for further investigation, encompassing Gram-positive and Gram-negative bacteria. Results revealed that it demonstrated moderate antibacterial activity against various Gram-positive bacteria; however, it displayed inactivity against Gram-negative bacteria, except for K. pneumoniae [Four metabolites, namely microsphaerins A–D (eumoniae .1 (56), palmarumycin M2 (58), papyracillic acid C (61), microsphaeropsins A (78), and microsphaeropsins B (62), were successfully isolated, alongside three known compounds, decaspirone (57), papyracillic acids A (59), and papyracillic acids B (60), from the endophyte fungus Microsphaeropsis sp. 7291, which resides within the plant Pilgerodendron uviferum. Of significance, compounds 56, 57, and 58 exhibited moderate antibacterial activity against B. megaterium, resulting in inhibition zone diameters of 6 mm, 6 mm, and 7 mm, respectively [Five new metabolites, namely palmarumycin Mectively .Microsphaeropsis sp. 8875 residing in the plant Lycium intricatum [64–66), in addition to two known compounds, citreorosein (67) and emodin (68). Moreover, from a Microsphaeropsis sp. 7177 strain sourced from the plant Zygophyllum fortanesii, they identified two new metabolites, 3,4-dihydrofusidienol A (70) and microsxanthone (73), along with three known compounds, fusidienol A (69), 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylic acid methyl ester (71), and methyl 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (72). Remarkably, all these compounds exhibited significant antibacterial activity, particularly against the Gram-negative bacterium E. coli. Microsphaerol (112), derived from the endophyte fungus Microsphaeropsis sp. 7820, displayed moderate antibacterial activity against B. megaterium and E. coli, evidenced by inhibition zone diameters measuring 9 mm and 8 mm, respectively [Krohn et al. 2009) conducted a comprehensive study resulting in the isolation of several metabolites from the endophyte fungus 09 conducectively . 74) and microketide B (75), were successfully isolated from the fungus Microsphaeropsis sp. RA10-14, which inhabits the gorgonian Anthogorgia ochracea from the South China Sea. Both compounds exhibited significant antibacterial and antifungal activities, with a notable emphasis on their antibacterial effects. Specifically, compound 74 demonstrated significant antibacterial activity against B. subtilis, B. megaterium, E. aerogenes, K. rhizophila, and P. aeruginosa, all with equal MIC values of 0.19 μg/mL, mirroring the potency of ciprofloxacin [Two novel metabolites, microketide A , was isolated alongside seven known compounds from the fungus Microsphaeropsis sp. BCC 3050. This fungal strain was collected from the lichen Dirinaria applanata. The known compounds in this collection include preussomerins E (18), F (19), G (20), H (21), I (17), deoxypreussomerins A (22), and bipendensin (23). Remarkably, all compounds, except compound 23, exhibited activities against M. tuberculosis, and compounds 16, 17, and 18 showed moderate antibacterial activity, with IC50 values of 25 μg/mL, 12.5 μg/mL, and 25 μg/mL, respectively. Compounds 19, 20, 21, and 22 showed significant antibacterial activity, with IC50 values of 3.12 μg/mL, 3.12 μg/mL, 6.25 μg/mL, and 1.56 μg/mL, respectively [94), sourced from the endophyte fungus Microsphaeropsis arundinis PH 30472, showed moderate antibacterial activity against E. coli [The new isomer, 3′-O-demethylpreussomerin I ((21), I 1, deoxypr79), B (80), and C (81), were isolated alongside three known compounds from the endophyte fungus M. arundinis E12-2112, which resides within the plant Ulmus macrocarpa. Among the known compounds, one isochroman-1-one, arundinone A (27), a polyoxygenated benzofuran-3(2H)-one dimer, arundinone B (28), and 1β-hydroxy-α-cyperone (82) were identified. Significantly, compound 82 displayed moderate antibacterial activity against S. aureus with an value of 11.4 μg/mL [Three novel ent-eudesmane sesquiterpenoids, namely arundinols A (.4 μg/mL .R)-1′--1′-oxobutan-3′-ylacetate (4), was isolated, alongside six known compounds, (R)-1--3-hydroxybut -anone (5), 1--2-buten-1-one (6), (R)-6-hydroxy-2-methyl-4-chrom -anone (7), modiolide D (84), modiolide E (85), and modiolide A (86). These compounds were obtained from the endophyte fungus M. arundinis, residing within the plant Paepalanthus planifolius [5 exhibited moderate cytotoxic activity against murine breast adenocarcinoma (LM3), with IC50 values of 36.83 ± 4.86 μg/mL, while compound 6 displayed moderate cytotoxic activity against human breast cancer (MCF-7), with IC50 values of 33.95 ± 3.62 μg/mL [A new metabolite, , through alterations of the culture medium for the fungus M. olivacea F010 [98 demonstrated significant cytotoxic activity against murine leukemic cells (L1210), achieving an inhibition ratio of 90% [Keusgen et al. (1996) successfully isolated a cerebroside, namely cea F010 . Remarkao of 90% .99), C (101), and E (103), in conjunction with two known compounds, TAN-1496 B (100) and D (102), were successfully isolated from the fungus Microsphaeropsis sp. FL-16144. Extensive research has revealed that these compounds act as specific inhibitors of calf thymus Topo I [Three novel metabolites, TAN-1496 A (s Topo I . Notablys Topo I . Additios Topo I .87) and macrosphelide B (88), from the fungus Microsphaeropsis sp. FO-5050 [50 values of 3.5 μM and 36 μM, respectively. Notably, in vitro cell growth assays showed that these compounds had no discernible effect on the growth of cells, including P388 leukemia, human prostate tumor cells, and L929 fibroblast cells, even when administered at a high dose of 200 mg/kg for 5 days [89) and macrosphelide D (90) from the fungus Microsphaeropsis sp. FO-5050 [89) and macrosphelide D (90) exhibited moderate cytotoxic activity, with IC50 values of 67.5 μM and 25 μM, respectively. Fukami et al. (1998) continued their exploration of the fungus Microsphaeropsis sp. FO-5050 and made further discoveries [91), macrosphelide K (92), and 6-epi-5’-hydroxymycosporulone (12). Although these compounds displayed cytotoxic activity, their IC50 values exceeded 100 μg/mL. Collectively, these findings underscore the exceptional potential of the fungus Microsphaeropsis sp. FO-5050 as a source of novel macrolides with cytotoxic properties. These substances hold high promise for future development in the treatment of leukemia.It has been widely reported that cell adhesion molecules play pivotal roles in numerous physiological and biochemical processes, ranging from cancer ,42,43 to FO-5050 . To explr 5 days . Takamat FO-5050 . Macrospcoveries . They is18, 19, and 20 possess the capability to inhibit Ras farnesyl-protein transferase [16–21 have demonstrated significant cytotoxicity against a spectrum of cell lines, including human epidermoid carcinoma (KB cells), human breast cancer (BC-1 cells), and African green monkey kidney fibroblast (vero cells). Compound 28, sourced from the endophyte fungus M. arundinis E12-2112, has exhibited moderate cytotoxicity against human bladder carcinoma cells (T24) and human lung carcinoma cells (A549), characterized by IC50 values of 35.4 μg/mL and 81.6 μg/mL, respectively [Singh et al. (1994) previously reported that compounds nsferase . Seephonnsferase . Furtherectively .49), graphislactone A (50), ulocladol (51), botrallin (52), 2,5-diacetylphenol (53), 7-hydroxy-2,4-dimethyl-3(2H)- benzofuranone (54), and enalin (55), were isolated from the endophyte fungus M. olivacea, which resides within the plant Pilgerodendron uviferum. Remarkably, graphislactone A (50) and botrallin (52) exhibited significant activity against acetylcholinesterase (AChE), characterized by IC50 values of 8.1 μg/mL and 6.1 μg/mL, respectively. However, all these compounds displayed no discernible antibacterial or antifungal activities [Seven known compounds, namely butyrolactone I (tivities .76), from the fungi Microsphaeropsis sp. FO-5050 [76 holds great promise as a novel class of anti-influenza virus drugs, representing a potential breakthrough in the realm of influenza treatment.Fukami et al. (2000) isolated a novel anti-influenza virus antiviral, known as 10-norparvulenone ( FO-5050 . Interes95), was isolated from the endophyte fungus Microsphaeropsis sp. MF6057, found within the plant Prosopis glandulosa. Compound 95 demonstrated an IC50 value of 71 μM in binding to a cloned human B2 receptor with 3H-bradykinin, marking a noteworthy development in the pursuit of potential treatments [Bradykinin, an endogenous peptide, exerts its effects through specific cell receptors, giving rise to a plethora of physiological reactions, including pain, allergic responses, and muscle contractions. Bradykinin antagonists hold the potential to inhibit these physiological reactions, offering prospects for treating conditions such as inflammatory edema, rhinitis, and asthma. In a significant discovery, a novel non-peptide bradykinin-binding inhibitor, L-755,807  and microsphaerones B (97), were successfully isolated from the fungus Microsphaeropsis sp. KMPB W-22. Interestingly, neither of these compounds exhibited significant cytotoxicity. Additionally, they displayed either no activity or only moderate activity against S. littoralis and A. salina [Two novel . salina .83 and 104) and three fresh 1,3,6,8-tetrahydroxyanthraquinone congeners (13–15) were successfully extracted from the fungus Microsphaeropsis sp. KMPB W-22. Remarkably, all these compounds, with the exception of compound 104, exhibited moderate inhibitory activity against protein kinases such as PKC, CDK4, and EGF-R, encompassing an IC50 range of 18.5–54.0 μM [Two novel betaenone derivatives  first reported on the in vitro activity of preussomerins against lciparum . Compoun44, sourced from the endophyte fungus M. arundinis PSU-G18, has exhibited significant antiplasmodial activity, boasting an IC50 value of 9.63 μg/mL. Notably, it also demonstrates significant radical scavenging capabilities, with an IC50 value of 0.018 mg/mL [1 (56), decaspirone (57), and papyracillic acids A (59), all originating from the endophyte fungus Microsphaeropsis sp. 7291, have demonstrated anti-algal activity against C. fusca. Their inhibitory effects are reflected in inhibition zone diameters of 6 mm, 13 mm, and 7 mm, respectively [112, derived from the endophyte fungus Microsphaeropsis sp. 7820, has displayed anti-algal activity against C. fusca, characterized by an inhibition zone diameter of 8.5 mm [64–73, apart from 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylic acid methyl ester (71), have demonstrated anti-algal activity against C. fusca [74) and microketides B (75), isolated from Microsphaeropsis sp. RA10-14, have exhibited moderate antiphytoplankton activity against P. helgolandica, showcasing IC50 values of 12.3 μg/mL and 16.8 μg/mL, respectively [Compound 18 mg/mL . In anotf 8.5 mm . Additioectively .Microsphaeropsis genus, and their activities have yet to be determined. For instance, Liu et al. (2018) isolated six known metabolites, including butyrolactones I (24), butyrolactones IV (25), aspernolide D (26), fumiquinazolines L (105), fumiquinazolines N (106), and notoamide D (107), from the fungus Microsphaeropsis sp. CF09-11, sourced from marine sediment [24–26 and 105–107) were obtained from Microsphaeropsis for the first time. In a unique discovery, uncommon methyl-branched unsaturated fatty acids, 10-methyl-9Z-octadecenoic acid (108) and glyceride (109), were isolated from the endophyte fungus M. olivacea, which inhabits the marine sponge Agelus sp. This marks the first report of fungal fatty acids with methyl branches on a cis-double bond [63), papyracillic acids A (59), and papyracillic acids B (60), were isolated from the endophyte fungus Microsphaeropsis sp., originating from the plant Arbutus unedo. Importantly, this research led to the establishment of the absolute configuration of massarigenin A (63) [Furthermore, several compounds were isolated from fungi within the sediment . It is nble bond . Addition A (63) .Microsphaeropsis in 1980, fifty-four different species have been included. Currently, this genus has yielded an impressive array of secondary metabolites, with 112 secondary metabolites reported, of which 49 (41.96%) were identified for the first time. These compounds are distributed across various structural classes, including 76 polyketones (67.86%), 14 nitrogenous compounds (12.5%), 10 macrolides (8.9%), 7 terpenoids (6.25%), and 5 other compounds (4.46%), indicating polyketones are the predominant structural type  Employ non-directional activation strategies to activate the biosynthesis of secondary metabolites, such as the OSMAC (one strain many compounds) strategy [Microsphaeropsis has not been reported in the literature so far. Thus, whole gene sequencing and analysis will be the focus of further work. (iii) Expand the scope of activity screening of existing compounds and delve into their mechanism of action to verify their potential for pharmaceutical applications. For instance, microbetides A (74), a novel polyketone compound, demonstrates remarkable antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, with an IC50 of 0.19 μg/mL, comparable to ciprofloxacin. Polyketone 10-norparvulone (76) exhibits the potential to effectively inhibit virus replications, positioning it as a promising candidate for a new anti-influenza drug. Macrolide compounds like macrosphelide A (87) and macrosphelide B (88), characterized by their unique chemical structures and cytotoxicity against leukemia, hold promise as novel anticancer drugs.Fungi occupy an exceptionally pivotal role in drug discovery . Many restrategy , co-cultstrategy , and epistrategy . (ii) Hastrategy , transcrstrategy , the hetstrategy , DNA-assstrategy , and ribstrategy . However


Escherichia coli (ECOL) and Enterobacter cloacae complex (ENTCC) demonstrate a range of MEM MICs. The objective of this study was to assess the in vitro killing activity of dose-optimized MEM against CRE with or without KPC that demonstrate variable MEM MICs and to determine if vaborbactam potentiates the killing activity of MEM against non-CP CRE.KPC producing CRE are common in the US; however non-carbapenemase producing (CP) CRE are as common. KPC- and non-CP Table); isolates demonstrated MEM MICs ranging from 1 to 8 mg/L. MEM standard (1g q8h over 30 minutes) and optimized (2g q8h over 3 hours) regimens were tested in 1-compartment and hollow fiber infection models (HFIM); targeted exposures were confirmed by LC-MS/MS. In HFIM, MEV (4g q 8h over 3 hours) was also tested. Resistant subpopulations were selected by agar plates containing MEM at 4x baseline MIC.8 ECOL and 8 ENTCC were characterized by whole-genome sequencing  or ENTCC . Bacterial regrowth occurred more rapidly against KPC compared to non-CP isolates. For MEM optimized regimens, 72-hour log-kills decreased as MEM MICs increased against non-CP ECOL; optimized MEM regimens were not bactericidal against KPC ECOL with MICs ≥2 mg/L. For ENTCC, optimized MEM resulted in greater log-kills against non-CP than KPC isolates at each MIC level. Across all non-CP and KPC isolates with MEM MICs ≤2 mg/L (n=4 each), mean log-kills at 72 hours with optimized MEM were -4.43 and -0.26 logs, respectively (P=0.04). Colonies selected on MEM containing agar demonstrated a median (range) MEM MIC of 64 mg/L (16 – >256 mg/L). Next, we compared optimized MEM and MEV in 10 day HFIMs against isolates with MEM MICs of 4mg/L. MEV demonstrated potent bactericidal killing against KPC isolates. Unexpectedly, MEV was also bactericidal against both non-CP isolates, and potentiated MEM killing against ECOL0174 .Standard MEM regimens were largely not bactericidal against ECOL . Optimized dosing MEM 2g q8h, 3 hour infusion (green). Limit of detection is 0.7 log colony forming units (CFU)/mL, denoted by dashed line. BMD = broth microdilution. KPC = Klebsiella pneumoniae carbapenemase.Standard dosing MEM 1g q8h, 30 minute infusion (red). Optimized dosing MEM 2g q8h, 3 hour infusion (green). Lower limit of detection is 0.7 log CFU/mL, denoted by dashed line. BMD = broth microdilution. KPC = Klebsiella pneumoniae carbapenemaseStandard dosing meropenem 1g q8h, 30 minute infusion (red). Optimized dosing meropenem 2g q8h, 3 hour infusion (green), or meropenem-vaborbactam 4g q8h, 3 hour infusion (purple). Lower limit of detection is 0.7 log CFU/mL. MIC = minimum inhibitory concentration.in vitro killing of CR ECOL and ENTCC is dependent on MEM MICs and presence of KPC. Even at low MICs, optimized MEM exposures were ineffective against KPC isolates. Against non-CP CRE, killing decreases as a function of increased MEM MIC. MEV may play an additive role in killing against non-CP isolates that are not killed by MEM alone.Our data show Ryan K. Shields, PharmD, MS, Allergan: Advisor/Consultant|Cidara: Advisor/Consultant|Entasis: Advisor/Consultant|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Menarini: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Utility: Advisor/Consultant|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support


In the original publication , there wThere was an error in the original publication. We examined correlations among biomarkers, which should have been analyzed by Spearman’s correlation method instead of Pearson’s correlation.A correction has been made to Section 2, “Results”; Section 2.4, “Correlations between Major CSF Proteins in CSF of CN, MCI, and AD”; and Section 2.5, “Correlations between Major CSF Proteins and AD Biomarkers”.rs = 0.86) and L-PGDS (rs = 0.47) levels (Table 1), while GlcNAc-Tf also correlated with L-PGDS (rs = 0.57). In contrast, Man-Tf levels did not correlate with Sia-Tf or TTR levels. High correlations were thus found among Man-Tf, GlcNAc-Tf, and L-PGDS in the CN group. In MCI, Man-Tf levels correlated well with GlcNAc-Tf (rs = 0.73) and L-PGDS (rs = 0.66) levels, while GlcNAc-Tf correlated well with L-PGDS (rs = 0.69). Man-Tf levels were negatively, but weakly, correlated with TTR (rs = −0.36) levels but not with Sia-Tf. Again, high correlations were observed among brain-derived proteins in the MCI group. In AD, Man-Tf levels correlated well with GlcNAc-Tf (rs = 0.79) and L-PGDS (rs = 0.62) levels but not with Sia-Tf or TTR. On the other hand, GlcNAc-Tf levels correlated well with L-PGDS (rs = 0.61) levels. These results suggest that the brain-derived proteins showed significant correlations with each other across all clinical groups.Correlations of the CSF proteins were also analyzed for the CN, MCI, and AD clinical groups. In CN, Man-Tf levels correlated with GlcNAc-Tf (rs = 0.56), tau (rs = 0.56), Aβ40 (rs = 0.73), and Aβ42 (rs = 0.52); GlcNAc-Tf vs. p-tau (rs = 0.76), tau (rs = 0.79), Aβ40 (rs = 0.76), and Aβ42 (rs = 0.49); L-PGDS vs. p-tau (rs = 0.84), tau (rs = 0.79), Aβ40 (rs = 0.79), and Aβ42 (rs = 0.52). The Aβ42/Aβ40 ratio showed negative correlations with Man-Tf (rs = −0.15), GlcNAc-Tf (rs = −0.34), and L-PGDS (rs = −0.44); however, none of these were statistically significant. In contrast, neither Sia-Tf nor TTR levels correlated with any AD markers. Taken together, the brain-derived CSF proteins correlated significantly with p-tau, tau, Aβ40, and Aβ42 levels in the CN group but the blood-derived proteins did not. In MCI, CSF protein levels also correlated with AD markers as follows: Man-Tf vs. p-tau (rs = 0.71), tau (rs = 0.53), Aβ40 (rs = 0.45); GlcNAc-Tf vs. p-tau (rs = 0.65), tau (rs = 0.54), and Aβ40 (rs = 0.43); L-PGDS vs. p-tau (rs = 0.49) and tau (rs = 0.41). The Aβ42/Aβ40 ratio showed negative correlations with Man-Tf (rs = −0.24), GlcNAc-Tf (rs = −0.45), and L-PGDS (rs = −0.23) but none were statistically significant. Overall, in the MCI group, the brain-derived proteins correlated moderately with AD markers. In AD, p-tau showed a moderate, though significant, correlation with the CSF proteins (rs = 0.51~0.55) but not with other AD markers. TTR negatively correlated with p-tau (rs = −0.42) and tau (rs = −0.72), while Sia-Tf correlated with tau (rs = −0.41). These results suggest that AD markers tend to correlate with the brain-derived CSF proteins in the AD and MCI groups; however, with the exception of p-tau, their correlation declined concomitantly with AD progression.Our previous study demonstrated that Man-Tf and p-tau were co-expressed in hippocampal neurons in the AD brain and that CSF Man-Tf levels correlated well with p-tau levels in MCI and AD. Other CSF proteins, such as Man-Tf, GlcNAc-Tf, L-PGDS, TTR, and Sia-Tf, were therefore examined for their correlation with AD markers (Table 2). In CN subjects, the CSF proteins showed significant correlations with AD markers as follows: Man-Tf vs. p-tau (The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.


Serum UA , NT-proBNP levels (1090.4 (675.2–2664.9) vs. 759.0 (260.3–1474.8), p = 0.034), and MLHFQ scores (21.0 (14.0–47.0) vs. 14.5 (4.5–25.5), p = 0.012) were significantly higher, whereas 25(OH)D concentrations (17.6 (15.1–28.2) vs. 22.7 (19.5–33.8), p = 0.010) were lower in subjects with severely reduced LVEF. Also, 25(OH)D was independently associated with LVEF in univariate and multiple regression analysis, maintaining its significance even after adjusting for confounders such as age, NT-proBNP, the presence of chronic coronary syndrome, hypertension, and anemia. According to our current findings, 25(OH)D is closely associated with LVEF, further supporting the need to establish correct vitamin D supplementation schemes and dietary interventions in HF. The changes in LVEF, 25(OH)D, serum UA, and albumin levels in HFrEF and HFmrEF indicate a similar pathophysiological background.Vitamin D emerged as an important prognostic biomarker in heart failure (HF), with currently highly debated therapeutic implications. Several trials on vitamin D supplementation in HF showed improvements in left ventricular (LV) remodeling and function and health-related quality of life (HRQoL), which did not translate into mid- to long-term beneficial effects regarding physical performance and mortality. We addressed total 25-hydroxyvitamin D (25(OH)D), serum albumin, and uric acid (UA) levels, focusing mainly on vitamin D deficiency, as potential markers of LV systolic dysfunction in HF with reduced and mildly reduced ejection fraction . Seventy patients with LVEF < 50% were comprehensively evaluated using ECG, echocardiography, lung ultrasound (LUS), blood sampling, and the six-minute walk test (6MWT). HRQoL was also assessed using the Minnesota Living with Heart Failure Questionnaire (MLHFQ). Statistically significant positive correlations were found between LVEF, 25(OH)D, serum UA, and albumin, respectively ( Vitamin D is a pleiotropic hormone showing substantial cardiovascular involvement beyond its regulatory effects on calcium and phosphate homeostasis. Vitamin D deficiency is frequently associated with cardiovascular disease  and is i3 2D3), the biologically active form of vitamin D, exerts its genomic and rapid, non-genomic effects via vitamin D receptors (VDRs) . R. R2 usinEchocardiography was performed using a Philips Epiq7 ultrasound machine  and a Philips X5-1 xMATRIX array transducer . Clinical recommendations for multimodality cardiovascular imaging were applied ,28,29. LThe lung ultrasound (LUS) protocol consisted of an eight-zone evaluation, four on each hemithorax. Semi-recumbent positioning of patients facilitated the assessment of B-lines. The same cardiac probe was used for lung imaging as well, and depth was adjusted individually. The following threshold for abnormality (in terms of lung congestion) was applied: ≥3 B-lines per zone .Blood samples were collected after twelve-hour fasting , and centrifuged for 10 min at 3000 rpm. Laboratory tests were performed, including total-, LDL-, and HDL-cholesterol, serum triglycerides, UA, creatinine, albumin, serum iron, ferritin, and CRP using an Architect C4000 ; plasma fibrinogen was assessed with Sysmex CA-1500 . N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations were measured using an electrochemiluminescent method on an Elecsys 2010 . Complete blood count was assessed using tripotassium ethylenediaminetetraacetic acid precoated tubes on a Mindray BC6000 .Quantitative determination ofthe total 25(OH)D in patients with HF was conducted using a competitive electrochemiluminescence protein binding assay on a Mindray CL-900i Chemiluminescence Immunoassay Analyzer . Patients were divided into tertiles based on serum 25(OH)D levels . None of the participants were taking vitamin D supplements in the two months prior to the evaluation. Dietary vitamin D intake from food sources and sun exposure were not assessed. The reference range for this assay was 30–100 ng/mL.2 test. 25(OH)D concentrations were log-transformed for analysis to improve normal distribution. p-values < 0.05 were regarded as statistically significant. Microsoft Excel 2016  and GraphPad Prism 9.5.0  were used for data curation and processing.The Kolmogorov–Smirnov and/or Shapiro–Wilk normality tests were used for the assessment of data distribution. Paired t-test and Pearson’s correlation were performed for variables showing normal distribution. Data following non-Gaussian distribution were processed using the Mann–Whitney U test and Spearman’s rank correlation analysis. Categorical variables were quantified for absolute and relative frequency, and 2 × 2 contingency tables were analyzed with the χp = 0.019). Patients with HFrEF had higher body mass index (BMI) (p = 0.025), LA volume index (LAVI) (p = 0.032), LVEDV index (LVEDVI) (p < 0.0001), LV end-systolic volume index (LVESVI) (p < 0.0001), lower stroke volume index (SVI) (p = 0.0002), longer-lasting heart disease (p = 0.041), compared to patients with HFmrEF. Also, study participants with HFmrEF showed better quality of life (p = 0.012). Serum UAd and NT-proBNP concentrations were significantly increased , whereas 25(OH)D levels were significantly decreased (p = 0.010) in HFrEF. Loop diuretic therapy was more frequently prescribed to patients with HFrEF. None of the participants took serum UA-lowering medication at the time of evaluation.Seventy patients  with a mean age of 66 ± 11 years were included in the study. p < 0.01) (Both log 25(OH)D and serum UA showed significant differences and an inverse relationship in the two study groups ( < 0.01) .p = 0.023), LVEF , platelet count , serum albumin , and HDL-cholesterol concentrations . Statistically significant negative associations were reported between serum 25(OH)D and UA levels , LVESVI , and LVGLS . No significant relationship was found connecting 25(OH)D to CRP , 6MWT distance , or NYHA class . Patients with 25(OH)D levels in the first tertile vs. the third tertile associated type II diabetes mellitus (DM) more frequently, but it did not reach statistical significance .Also, when considering 25(OH)D levels in all patients, significant positive correlations were found with diastolic blood pressure values , BMI (p = 0.006), and E/e’ ratio (p = 0.037). LVEF also showed strong negative associations with NT-proBNP (p = 0.029), serum UA levels (p = 0.009), GGT activity (p = 0.006), and thyroid-stimulating hormone (TSH) concentrations (p = 0.040). Interestingly, LVEF was not linked to 6MWT distance, age, duration of heart disease, or duration of HF. Significant positive correlations were found between LVEF and both components of blood pressure—systolic (p = 0.003) and diastolic blood pressure (p = 0.006), and also serum 25(OH)D levels (p = 0.008).p = 0.036 and p = 0.030, respectively). In the second model, log 25(OH)D maintained its predictive value, even after adjusting for the presence of chronic coronary syndrome (CCS).Multivariate linear regression models (A and B) were constructed in the overall study population for the prediction of LVEF in HF . Log 25(p = 0.027) (Model C). Furthermore, log 25(OH)D predicted HFrEF, maintaining its significance when corrected for age, NT-proBNP tertiles, GGT tertiles, the presence of hypertension, CCS, anemia, and hypothyroidism (p = 0.036) (Model D).Nonlinear logistic regression models (C and D) were also set up to predict LV systolic dysfunction . In the 2D is released into the bloodstream, circulating in both protein-bound  and free forms [2D deficient mice developed glomerular (podocyte) injury, albuminuria, and subsequent kidney dysfunction, which were reversible upon exposure to 1,25(OH)2D [Over the last few decades, especially during the COVID-19 pandemic, vitamin D received widespread attention, mainly because of its complex biological activity, regulating gene transcription, and modulating signaling pathways . Vitaminee forms . The remee forms . Moreove25(OH)2D . Zhang e25(OH)2D . Our pat25(OH)2D ,37, vita25(OH)2D .+ currents proved to be an important cause of electrophysiological remodeling in cardiac hypertrophy, which was reversed upon exposure to 10 nM of calcitriol [2D. Calcitriol, at concentrations above 30 nM, reduced the proliferative capacity, caused mild hypertrophy, and altered cardiomyocyte morphology [p = 0.010). Currently, there is no guideline recommendation regarding screening for vitamin D deficiency and correct supplementation in HF. Both experimental and clinical trials linked vitamin D deficiency to the excessive activation of the RAAS with increased expression of the renin gene, higher plasma renin activity, and elevated plasma aldosterone and angiotensin II levels [2D increases intracellular calcium concentrations and decreases intracardiac filling pressures, thus enhancing systolic and diastolic ventricular function [p = 0.036 and p = 0.030; p = 0.027 and p = 0.031). Contrary to the results published by Khan et al., we found no significant relationship between 25(OH)D levels and the functional status of patients with HF, reflected by the 6MWT distance and NYHA class [p = 0.061). Vitamin D deficiency was previously linked to DM by regulating inflammation and inducing insulin resistance [A multiple inhibitory role regarding vitamin D in HF has been proposed: it reduces myocardial fibrosis, pathological remodeling, and atherosclerosis, by down-regulating low-grade vascular inflammation . The suplcitriol . Vitaminlcitriol . VDRs werphology . The sizrphology . VDR knorphology . There irphology . Other arphology . Our patI levels . Vitaminsistance . Cassanosistance .3 administered weekly and 400 mg of calcium citrate given BID for 6 months, failed to demonstrate the beneficial effects of cholecalciferol supplementation on physical performance in HF patients with a mean LVEF of 39.2 ± 13.2%, and already on guideline-directed medical therapy [3 daily during a 3-year follow-up. Long-term intake of moderately high doses of vitamin D had no impact on mortality but was associated with a significantly greater need for mechanical circulatory support device implants in patients with baseline circulating 25(OH)D levels ≥ 30 nmol/L, who showed increased in-study concentrations > 100 nmol/L [Several trials addressed the potential benefits and pitfalls of vitamin D supplementation in HF but showed inconsistent results. The VINDICATE study enrolled vitamin D-deficient patients with HF and LVEF ≤ 45% already on optimal medical treatment. After 12 months of well-tolerated high-dose cholecalciferol supplementation (4000 IU daily), LV structure and function showed statistically significant improvements, reflected by the EF, end-diastolic, and end-systolic diameters, and volumes. Unexpectedly, the reversal of LV remodeling was not associated with improved 6MWT distance at 1 year, and further clinical outcomes were not assessed . Another therapy . The mor0 nmol/L . In a ca0 nmol/L . High pl0 nmol/L . A recen0 nmol/L ,54. Vita0 nmol/L .As seen, the trials investigating the effects of vitamin D in HF used different supplementation schemes  and study outcomes, which might explain the inconclusive results.p = 0.005). We also found that patients with 25(OH)D deficiency were prone to develop hyperuricemia. The association might be explained by the severity of LV systolic dysfunction, which is associated with lower levels of vitamin D [Serum UA also sparked controversy regarding its status as a potential cardiovascular risk factor, considering its pro-oxidant–antioxidant activity. Elevated levels were linked to increased risk for cardiovascular disease, including HF . Moreoveitamin D ; both viitamin D ,24. Alsoitamin D . Treatmeitamin D ,64. The itamin D . In patiitamin D . The URRitamin D . It shouWe hypothesize that the associated changes in LVEF, 25(OH)D, serum UA, and albumin levels might indicate similar pathophysiological backgrounds in HF with systolic dysfunction. One possible explanation might rely on the pleiotropic biological impact of inflammation. Abundant evidence already linked low-grade systemic inflammation to HF . TherefoVitamin D, serum UA, and albumin are already established prognostic biomarkers in HF. We found 25(OH)D to be an independent marker of LVEF using univariate and multiple regression analysis. Significant correlations were identified between LVEF, 25(OH)D, serum UA, and albumin, suggesting potential molecular interactions. Although there is currently a plethora of information regarding vitamin D deficiency in HF, its precise mechanism, clinical impact, and management still need clarification.


Electroconvulsive therapy (ECT) is one of the few non-pharmacological stimulation treatment which is cost effective, effecious and lifesaving in various psychiatric disorders. Although myths and misconceptions prevailed in a society undermine the usefulness of such treatment.Attitude towards Electroconvulsive therapy (ECT) among psychiatric patients.It was a descriptive cross-sectional study conducted at the Department of Psychiatry and Behavioural sciences, Jinnah Postgraduate Medical Centre (JPMC), Karachi from 22-Oct-2019 to 21-April-2020 and a total of 250 psychiatric patients were enrolled.Methode; Attitudes toward ECT were assessed using ECT attitude questionnaire6 (Annexure III). A 15 items questionnaire, each item has three alternatives based on which responses were categorized into positive, negative, or ambivalent attitudes. Mean score was calculated for each.Patients who were given 8 positive answers out of 15 were labeled as having a positive attitude. Patients who were given 8 negative answers out of 15 were labeled as having a negative attitude. Patients who were given 8 I don’t know answers out of 15 were labeled as having ambivalent attitude.Age 18-65 yearsEither genderPsychiatric patients, having awareness regarding their nature of illness and could give consent for study.Patients with duration of illness >3 months.Psychiatry patients who have no awareness regarding their illness.Patients with impaired cognitiveForty-four (45.83%) patients had positive attitude, 36 (37.50%) had negative attitude and 16 (16.67%) had ambient attitude.Further stratification was also performed on the basis of educational status, occupational status, duration of illness, psychiatric diagnosis, and previous experience of ECT. There was no significant association was found of these variables with attitude regarding ECT.Mean age was 39.58±12.48 years included in this study. There were 55 (57.29%) female and 41 (41.71%) male patients. There were 72 (75.00%) patients were household workers, 04 (4.17%) students, 06 (5.25%) unskilled labour, 3 (3.13%) skilled labour, 10 (10.42%) professionals and just 01 (1.04%) were law enforcement worker. 19 (19.79%) patients were diagnosed with schizophrenia, 62 (64.58%) were diagnosed with unipolar depression and 15 (15.63%) were diagnosed with bipolar disorder. Source of ECT information was 11 (11.46%) electronic media, 09 (9.38%) print media, 19 (19.79%) social media and 57 (59.38%) was from health professionals. Forty-four (45.83%) patients had positive attitude, 36 (37.50%) had negative attitude and 16 (16.67%) had ambient attitude.Knowledge regarding electroconvulsive therapy (ECT) was low in psychiatric patients in Pakistan. Only 45.83% patients showed positive attitude towards ECT.Carney S, Geddes J. Electroconvulsive therapy. Br Med J. 2003;326:1343-4Gangadhar BN., Kapur RL., Kalyanasundaram S. Comparison of electroconvulsive therapy with imipramine in endogenous depression: a double blind study. Br J Psychiatry.1982;141:367–71.Kellner CH., Fink M., Knapp R., Petrides G., Husain M., Rummans T., et al. Relief of expressed suicidal intent by ECT: a consortium for research in ECT study. Am J Psychiatry. 2005;162:977–82.Baghai T C, Moller HJ. Electroconvulsive therapy and its different indications. Dialogues Clin Neurosci. 2008 Mar; 10(1): 105–17.Weiner RD., Coffey CE., Folk J., Fochtmann LJ., Greenberg RM., et al. American psychiatric association committee on electroconvulsive therapy, The practice of electroconvulsive therapy. 2nd ed. Washington, DC: American Psychiatric Association; 2001None Declared


Although the survival rate of patients who undergo surgery for gastric cancer has greatly improved, still many patients have a poor prognosis. This retrospective study aimed to investigate the predictive ability of the PNI-IgM score, a combined prognostic nutritional index (PNI), and immunoglobulin M (IgM), on the prognosis of patients undergoing surgery for gastric cancer.340 patients with gastric cancer who underwent surgery from January 2016 to December 2017 were selected. The PNI-IgM score ranged from 1 to 3: score of 1, low PNI (< 48.45) and low IgM (< 0.87); score of 2, low PNI and high IgM, or high PNI and low IgM; score of 3, high PNI and high IgM. We compared the differences in disease-free survival (DFS) and overall survival (OS) among the three groups, while univariate and multivariate analyses calculated prognostic factors for DFS and OS. In addition, the nomograms were constructed based on the results of multivariate analysis to estimate the 1-, 3- and 5-year survival probability.P = 0.053) and the PNI-IgM score group 3 . In stratified analysis, PNI-IgM score 1 had a worse prognosis in the age < 60 years group and CA724 < 2.11 U/m group.There were 67 cases in the PNI-IgM score 1 group, 160 cases in the PNI-IgM score 2 group, and 113 cases in the PNI-IgM score 3 group. The median survival times of DFS in the PNI-IgM score group 1, the PNI-IgM score group 2, and the PNI-IgM score group 3 were 62.20 months, not reached, and not reached, and 67.57 months vs. not reached vs. not reached in three groups for OS. Patients in the PNI-IgM score group 1 had a lower DFS than the PNI-IgM score group 2 (HR = 0.648, 95% CI: 0.418-1.006, PNI-IgM score is a novel combination of nutritional and immunological markers that can be used as a sensitive biological marker for patients with gastric cancer who undergo surgery. The lower the PNI-IgM score, the worse the prognosis. According to the World Health Organization, gastric cancer was the fifth leading cancer in the world, with nearly 1.09 million new cases of gastric cancer worldwide in 2020. Gastric cancer is the fourth leading cause of cancer deaths, with almost 770,000 deaths worldwide in 2020 –5. In reThe nutritional status of patients with gastric cancer, which could predict the progression of the treated cancer, has been identified an important factor –11. It wA large number of previous researches have pointed out that composite indicators of nutrition and immunity, such as Glasgow Prognostic Score (GPS), platelet-to-lymphocyte (PLR), and Controlling Nutritional Status (CONUT) score could predict the prognosis of gastric cancer patients , 27. HowIn this study, we evaluated the predictive effect of the PNI-IgM score on efficacy and prognosis in 340 patients with gastric cancer who underwent surgery. To further validate the PNI-IgM score, we performed a subgroup analysis and created nomograms.This is a retrospective study, so the Ethics Committee of Harbin Medical University Cancer Hospital waived informed consent. In total, we collected 340 consecutive patients with gastric cancer who received surgery at Harbin Medical University Cancer Hospital between January 2016 and December 2017 and were tested for lymphatic subsets and specific proteins. Statistical analysis of 340 patients and their clinical information was implemented according to the Helsinki Declaration and its amendments. All patients were included according to the following criteria: (1) all patients underwent surgical treatment; (2) all patients had no chronic disease; (3) all patients were tested for lymphatic subsets and specific proteins; (4) all patients did not display inflammatory response. (5) Patients with gastric cancer combined with other primary malignant tumors were excluded. Patients without complete clinical information and regular review after surgery were exclusion criteria. The flow chart of clinical case selection is shown in Patients were followed up by telephone or outpatient visit, every 3-6 months during the first 2 years, and every 6-12 months from the 3rd to 5th year, and annually thereafter. Disease-free survival (DFS) was comprehended as the period from the first day of surgery date to the date of disease progression. The evidence of progression was obtained by chest and abdomen X-ray or computed tomography. DFS was also defined as the date of death, death from any cause, or the date of withdrawal from the follow-up. Overall survival (OS) was described as the period from the first day of surgery date to the date of death, the date of withdrawal from the follow-up, or the time of the last follow-up. Electronic medical record system was used to acquire patients’ clinical and pathological information.9/L). The cut-off point was obtained by the receiver operating characteristic (ROC), which was based on OS for the prediction of patients’ death. The area under the ROC Curve (AUC) was used to evaluate the predictive ability of PNI and IgM. The optimal cut-off values of PNI and IgM with the highest Youden index were obtained.The peripheral venous blood was collected in fasting state after admission in all patients. The counts of peripheral lymphocytes (L) were measured and analyzed by an automatic blood analyzer (BACKMAN COULTER LH750), the levels of peripheral albumin were measured and analyzed by an automatic blood analyzer (ADVIA-2400), and the levels of peripheral IgM were measured and analyzed by a specific protein analyzer (IMMAGE800). PNI was calculated as follows: PNI = albumin (g/L) + 5 × total lymphocyte counts  and 95% confidence interval (CI). The Cox proportional hazards regression model was constructed to analyze independent prognostic factors for DFS and OS. The nomograms were also constructed to predict the 1-, 3-, and 5-year survival probability for DFS and OS. The calibration curve analysis was used to assess the prognostic predictive ability of nomogram. All statistical analyses were completed through the R 4.1.3 , IBM SPSS Statistics 25 . Finally, we considered two-sided P values < 0.05 as statistical differences.Continuous variables are presented as means with standard deviations or as medians with interquartile ranges. Categorical variables were expressed as percentages. The comparison between continuous variables used the t-tests, one-way ANOVA, Kruskal-Wails rank sum test. We used the Chi-square test or Fisher’s exact test to compare the discrepancies between categorical variables. The Kaplan-Meier survival curve was used to compute the survival rate and the Log-rank test to compare the survival time difference. Univariate and multivariate analyses were performed using the Cox proportional hazards model. Variables that achieved statistical significance at P = 0.006), weight loss (P = 0.012), fatigue (P < 0.001), pTNM stage (P < 0.001) and tumor size (P = 0.003). The one-way ANOVA Kruskal-Wails rank sum test showed that PNI-IgM score was related to age (P < 0.001), BMI (P = 0.012), bleeding volume (P = 0.012). The detailed clinical characteristics of all 340 cases grouped by PNI-IgM score are displayed in The median age of patients was 60 years, and there were 105 women (30.9%) and 235 men (69.1%) in all two groups’ cases. The optimal cut-off value of PNI was 48.45. The optimal cut-off value of IgM was 0.87 g/L. According to the optimal cut-off values of PNI and IgM, all patients were divided into three groups: PNI-IgM score of 3 (n = 113): high IgM (≥ 0.87) and high PNI (≥ 48.45); PNI-IgM score of 2 (n = 160): high IgM (≥ 0.87) and low PNI (< 48.45), or low IgM (< 0.87) and high PNI (≥ 48.45); PNI-IgM score of 1(n = 67): low IgM (< 0.87) and low PNI (< 48.45). The Chi-square test or Fisher’s exact test showed that PNI-IgM score was related to melaena (P = 0.017), γ- GT (P < 0.001), TBIL (P < 0.001), TP (P < 0.001), ALB (P < 0.001), GLOB (P < 0.001), UA (P = 0.002), ALP (P = 0.027), WBC (P < 0.001), RBC (P < 0.001), IgA (P < 0.001), IgG (P < 0.001), KAP (P < 0.001), LAM (P < 0.001) and CD3 +/CD4 + CD8 + cells (P = 0.004).In this study, we also collected patients’ nutritional and hematological parameters before surgery, including total protein (TP), PALB, alanine aminotransferase (ALT), aspartate transaminase (AST), glutamyl transpeptidase (γ- GT), lactate dehydrogenase (LDH), total bilirubin (TBIL), globulin (GLOB), urea, creatinine (CREA), urate (UA), alkaline phosphatase (ALP), monocyte (Mono), eosinophils (Eosi), basophil (Baso), red blood cell (RBC), platelet (P), carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), carbohydrate antigen 724 (CA724), carbohydrate antigen 125II (CA125II). We also collected lymphatic subsets and specific proteins, including immunoglobulin A (IgA), immunoglobulin G (IgG), transferrin (TRF), light-chain immunoglobulin (KAP), heavy-chain immunoglobulin (LAM), KAP/LAM, CD3 + cells (T cells), CD3 +/CD4 + cells (Th cells), CD3 +/CD8 + cells (CTL cells), CD4 +/CD8+ cells, CD3 +/CD4 + CD8 + cells, CD19 + cells (B cells), CD3 -/CD16 + CD56 + cells (NK cells), CD3 +/CD16 + CD56 + cells (NKT cells). We analyzed their relationship to the PNI-IgM score by Kruskal-Wails rank sum test (P = 0.001), BMI (P = 0.043), tumor size (P < 0.001), pTNM stage (P < 0.001), radical resection (P < 0.001), TBIL (P = 0.020), Eosi (P = 0.032), RBC (P = 0.032), CA724 (P < 0.001), CA125II (P = 0.003), IgG (P = 0.028), PNI-IgM score (P < 0.05). And the prognosis factors of patients in this study for OS were age (P = 0.001), melaena (P = 0.048), tumor size (P < 0.001), pTNM stage (P < 0.001), radical resection (P < 0.001), TBIL (P = 0.016), Eosi (P = 0.030), RBC (P = 0.031), CA724 (P = 0.004), CA125II (P = 0.004), IgG (P = 0.036), PNI-IgM score (P < 0.05). The multivariate analysis indicated that age (P = 0.036 vs. P = 0.035), pTNM stage (P < 0.001 vs. P < 0.001), radical resection (P = 0.006 vs. P = 0.001), and CA724 (P = 0.027 vs. P = 0.015) were both independent prognostic factors for DFS and OS. In addition, melaena (P = 0.003) was the independent prognostic factor for OS  and in CA724 < 2.11U/m group   or the PNI-IgM score 3 . Patients with the PNI-IgM score 1 have a shorter OS than patients with the PNI-IgM score 2  or the PNI-IgM score 3  , 65.4% (95% CI: 54.7%-78.3%), 53.1% (95% CI: 41.9%-67.3%). The PNI-IgM score 1 group’s median survival time for OS was 67.57 months, the 1-, 3-, and 5-year OS probability was 89.5% (95% CI: 82.4%-97.2%), 69.0% (95% CI: 58.6%-81.3%), 55.6% (95% CI: 44.5%-69.4%). The PNI-IgM score 2 group’s median survival time for DFS and OS were both not achieved. The 1-, 3-, and 5-year DFS and OS probability were 89.3% (95% CI: 84.6%-94.2%), 74.0% (95% CI: 67.4%-81.3%), 68.2% (95% CI: 61.1%-76.1%); 89.9% (95% CI: 85.4%-94.7%), 77.8% (95% CI: 71.6%-84.6%), 69.7% (95% CI: 62.8%-77.3%), respectively. The PNI-IgM score 3 group’s median survival time for DFS and OS were both not achieved. The 1-, 3-, and 5-year DFS and OS probability were 93.8% (95% CI: 89.4%-98.4%), 80.5% (95% CI: 73.4%-88.4%), 80.5% (95% CI: 73.4%-88.4%); 95.6% (95% CI: 91.9%-99.4%), 82.0% (95% CI: 75.1%-89.4%), 81.0% (95% CI: 74.0%-88.7%), respectively. To further determine whether the PNI-IgM score could predict the prognosis of gastric cancer patients. The ROC curves were based on OS for the prediction of patients’ death. For the traditional clinicopathologic factors, including ALB, L, PNI, and IgM, each feature and the combined PNI-IgM score were plotted, and the point with the highest AUC was illustrated on the ROC curve. The PNI-IgM score exhibited a higher prognostic accuracy for DFS and OS than PNI and other clinicopathological risk factors (P = 0.626) or the PNI-IgM score 3 . Patients with the PNI-IgM score 1 also have a shorter OS than patients with the PNI-IgM score 2 group  or the PNI-IgM score 3 group . Patients in the advanced pTNM stage had lower DFS  and OS  than those early pTNM stage patients  group (221 patients) and advanced pTNM stage (III + IV) group (119 patients). The median survival time for DFS and OS in the early pTNM stage group were both not reached. The 1-, 3-, and 5-year DFS and OS probability were 98.2% (95% CI: 96.4%-100.0%) vs. 98.2% (95% CI: 96.4%-100.0%), 89.2% (95% CI: 85.2%-93.5%) vs. 89.9% (95% CI: 86.0%-94.0%), 86.7% (95% CI: 82.2%-91.4%) vs. 87.4% (95% CI: 83.1%-92.0%). The median survival time for DFS and OS in the advanced pTNM stage group were 30.90 months and 40.27 months. The 1-, 3-, and 5-year DFS and OS probability were 76.9% (95% CI: 69.6%-84.9%) vs. 79.6% (95% CI: 72.7%-87.2%); 45.7% (95% CI: 37.2%-56.2%) vs. 54.1% (95% CI: 45.7%-64.0%); 34.7% (95% CI: 26.6%-45.4%) vs. 39.0% (95% CI: 30.9%-49.3%). Patients with the PNI-IgM score 1 have a shorter DFS than patients with the PNI-IgM score 2 (HR = 0.814, 95% CI: 0.356-1.860, This study found that age, radical resection, CA724, and pTNM stage were the independent prognostic factors for DFS. By constructing Cox proportional hazard regression model, age, melaena, radical resection, CA724, and pTNM stage were the independent prognostic factors for OS. Based on the results of multivariate analysis, the nomograms to predict the 1-, 3-, and 5-year survival probability for DFS and OS were established (Gastric cancer is a common malignant tumor in China, with the third highest incidence and mortality rate . AlthougOur study is the first to assess the association between the PNI-IgM score, which a composite indicator of immunity and nutrition, clinicopathological factors and survival. It also demonstrated that the PNI-IgM score predicted prognosis among gastric cancer patients who underwent resection. We found that low PNI and IgM were associated with poor patient prognosis. This study found that age, pTNM stage, radical resection, and CA724 were independent prognostic factors for DFS and OS. In addition, an independent prognostic factor for OS was melaena. In the Stratified analyses, we found age <60 years and CA724 < 2.11 U/m had shorter OS in PNI-IgM score 1.Although many studies have confirmed the prognostic association of PNI and IgM with gastric cancer –35 and oThis study also has some limitations. First, this was a single-region, single-center retrospective study with limited sample size and potential selection bias. Second, we only selected patients with gastric cancer who underwent surgery. Third, the cut-off value of PNI and IgM is usually derived from the ROC curve, and the optimal cut-off value is uncertain. Therefore, multi-regional, multi-center, and larger sample size studies are needed to validate our findings.In conclusion, our study found that the PNI-IgM score is a valid scoring tool for patients with gastric cancer who received surgery. Patients in the PNI-IgM score 1 group had a worse prognosis than those in the PNI-IgM score 2 group, and the PNI-IgM score 3 group. Therefore, the PNI-IgM score could be used as a biomarker to develop better treatment strategies for patients undergoing surgery for gastric cancer.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the ethics committee of Harbin Medical University Cancer Hospital. All patients provided written informed consent before the study.Writing-original draft and Writing-review & editing: ZD, HaS; Data curation and Investigation: RZ, GD, and HP; Methodology and Supervision: YZ, RH; Resources, Funding acquisition and Project administration: YX, and HoS. All authors contributed to the article and approved the submitted version.


Kidney function can alter drug clearance. This is pertinent for tenofovir-based pre-exposure prophylaxis (PrEP) regimens where drug concentrations confer protection against HIV. Among transgender (TG) individuals whose kidney function is dynamic due to use of gender affirming therapy (GAT), it is unclear if there is a relationship between tenofovir diphosphate dried blood spots (TFV-DP DBS) and direct measures of glomerular filtration rate (mGFR) The objective of this study was to quantify this relationship and compare if mGFR differs between TG persons with perfect/imperfect adherence to tenofovir alafenamide/emtricitabine (TAF/FTC).A prospective cohort study was performed among TG individuals on TAF/FTC for PrEP. Inclusion criteria were age ≥18 years old, TG identity, HIV negative, use of PrEP for ≥12 weeks, and willing to receive iohexol for direct measurement of GFR. TFV DBS concentrations were evaluated after being on ≥12 weeks of TAF/FTC. Perfect adherence to TAF/FTC was defined as a TFV-DP DBS of >1800fmol/punch . Iohexol clearance was used for mGFR by subcutaneously administering 0.5ml iohexol. Plasma iohexol concentrations were assayed 3h post-injection. Pearson’s correlation coefficients were computed to quantify the relationship between TFV-DP DBS and iohexol clearance. Median iohexol clearances were compared between those with perfect/imperfect adherence.2=0.10, 95% confidence interval, CI: -0.29 – 0.45, p=0.62). TFV-DP DBS >1800fmol/punch (perfect adherence) was observed in 22 participants. Median (95% CI) iohexol clearance between those with and without perfect adherence did not reach significance: 78 (68-96) vs 69 (60-83)mL/min, respectively (p=0.26).Among the 28 individuals included, median  age was 33.0 (26.3 – 36.8) years. Three quarters were on GAT and over two thirds (67.9%) were assigned male at birth (AMAB). Median  iohexol clearance and TFV-DP DBS concentrations were 76 (65 – 95)ml/min and 2611 (2114 – 3240), respectively. There was no significant correlation between TFV-DP DBS and iohexol clearance (rNo significant relationship was observed between iohexol clearance and TFV-DP DBS among TG individuals taking TAF/FTC for PrEP.Sheldon Morris, MD, Gilead: Grant/Research Support


Skin and soft tissue infections (SSTIs) are very common bacterial infections. Numerous clinical trials have compared antibiotics for SSTI treatment. However, trials typically have non-inferiority designs that give little insight as to which antibiotic classes have superior efficacy.We performed a systematic literature review and a network meta-analysis of published SSTI treatment trials. Using standardized key words, we searched PubMed and Embase for SSTI clinical trials published from 1/1/66 to 5/31/22. We excluded trials on diabetic foot infections, non-generalizable populations , and studies with outcomes not involving clinical resolution or cure. Abstracts with relevant clinical trial data were pulled and, if inclusion criteria met, had manuscript data abstracted. Two reviewers independently performed abstract and manuscripts reviews and data abstraction. For the network meta-analyses, the comparator antibiotic class was glycopeptides. Two analyses were performed using intention to treat (ITT) and clinically evaluable (CE) populations. Clinical trial quality was measured using the PEDro score.We reviewed 5,469 abstracts, of which 260 were pulled for data abstraction and 93 contained analyzable trial data . Clinical trial quality varied widely . We identified 26 analyzable antibiotic classes or combinations for comparison. In the ITT model, cure rate for oxazolidinones (OR 1.28 [95% CI 1.10-1.49]), flucloxacillin plus clindamycin (OR 1.61 [95% CI 1.03-2.53]), lipopeptides (OR 1.66 [95% CI 1.04-2.66]), and tetracyclines OR 1.69 [95% CI 1.24-2.30]), significantly differed from glycopeptides.  In the CE model, cure rate for oxazolidinones (OR 1.29 [95% CI 1.01-1.64]) and tetracyclines (OR 2.05 [95% CI 1.37-3.06]) significantly differed.We found that most (22/26) antibiotic classes or combinations used for SSTI treatment had similar efficacy to glycopeptides. However, oxazolidinones, lipopeptides, flucloxacillin plus clindamycin, and tetracyclines may have superior efficacy compared to glycopeptides. Treatment guidelines may wish to favor these latter classes for patients at high risk of treatment failure.Amy Y. Kang, Pharm.D., BCIDP, Paratek: Grant/Research Support Elliot I. Miller, BS, Epic Systems, Verona, WI: Employee Loren G. Miller, MD MPH, ContraFect: Grant/Research Support|GSK: Grant/Research Support|Medline: Grant/Research Support|Merck: Grant/Research Support|Paratek: Grant/Research Support


A safe and effective method to protect against cytomegalovirus (CMV) infection is a public health priority. mRNA-1647 is an investigational mRNA-based vaccine against CMV consisting of 6 mRNA sequences encoding 2 CMV antigens (glycoprotein B and the pentameric glycoprotein complex) in lipid nanoparticles in a lyophilized presentation.This phase 2, randomized, observer-blind, placebo-controlled, dose-finding trial of mRNA-1647 was conducted in CMV-seronegative and -seropositive healthy adults aged 18-40 years (NCT04232280). In Part 1, males and females were randomized 3:1 to receive mRNA-1647  or placebo at Months 0, 2, and 6. In Part 2, females were randomized 3:1 to receive mRNA-1647 100 µg or placebo at Months 0, 2, and 6. Primary endpoints were safety throughout the study, including solicited adverse reactions (ARs) up to 7 days after each dose, and neutralizing antibody (nAb) titers against epithelial cell infection and against fibroblast infection up to 12 months after last dose. Parts 1 and 2 results are combined.Figure). Robust nAb responses against epithelial cell infection were observed after each mRNA-1647 dose and sustained through the end of the study (12 months after last dose); nAb titers against epithelial cell infection in seronegative mRNA-1647 100-µg recipients exceeded the geometric mean titer of all seropositive recipients at baseline.Of 315 adults randomized, solicited ARs following dose 1 were reported in 54.7% (placebo), 84.4% (50 µg), 87.5% (100 µg), and 91.1% (150 µg) of seronegative adults and 59.3%, 94.4%, 94.6%, and 94.4%, respectively, of seropositive adults. The most common local and systemic solicited ARs across serostatus groups after dose 1 were pain  and fatigue , respectively. Similar trends in ARs were observed after doses 2 and 3. nAb titers against epithelial cell infection and against fibroblast infection were observed in all mRNA-1647 groups (Antibody-Mediated Immunogenicity of mRNA-1647 by CMV Serostatus(A) nAb titers against epithelial cell infection and (B) nAb titers against fibroblast infection are presented from day 1 through month 18 for CMV-seronegative (colored solid lines) and CMV-seropositive (colored dashed lines) treatment groups. Data are presented by serostatus, treatment group, and visit as GMT and corresponding 95% CI. Data are from the Per-Protocol Set. The solid black lines represent nAb GMTs against epithelial cell infection (GMT = 4575.7) and against fibroblast infection (GMT = 4215.5) for all CMV-seropositive participants at baseline (n = 87). Doses 1, 2, and 3 were administered on D1, M2, and M6, respectively, as represented by an arrow and syringe.CMV, cytomegalovirus; D, day; GMT, geometric mean titer; M, month; nAb, neutralizing antibody.mRNA-1647 was generally safe and well-tolerated and induced antigen-specific immune responses at all dose levels in both CMV-seronegative and -seropositive participants. These results guided selection of the 100-µg dose for the mRNA-1647 phase 3 trial.Lori Panther, MD, MPH, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Sandeep Basnet, MD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Richard Leggett, DO, Crossroads Clinical Research: Contract employee James Peterson, MD, Moderna, Inc.: Received payment as a study investigator Jiang Lin, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Kai Wu, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Heather Lee, BS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Roxane Hasselbeck, BA, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Andrew Natenshon, MA, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds Jacqueline Miller, MD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds


Asaia bogorensis strain SC1 isolated from a human-blood-fed Aedes aegypti mosquito crop. Metabolic pathway characteristics of aerobic respiration were present in the genome, along with multiple putative antibiotic resistance mechanisms.Understanding microbe-host interactions is key to combating disease transmission by mosquitoes. Here, we report the genome sequence of  Asaia bogorensis strain SC1, isolated from a human-blood-fed Aedes aegypti mosquito crop. Importantly, Asaia sp. is dominant in the crop of Ae. aegypti  Aedes aegypti mosquito crop. Briefly, the crop was dissected from a mosquito using ethanol-flamed forceps, homogenized in 1× PBS pH = 7.4, and inoculated on Luria-Bertani (LB) agar at 30°C. To ensure a pure culture, individual colonies were sequentially streaked on fresh LB plates three times. DNA was extracted from overnight LB broth cultures  using the Qiagen DNeasy PowerSoil Kit  and quantified via Qubit . For long-read sequencing, the Native Barcoding Kit 24 V14 Kit  was used to barcode samples and prepare libraries. DNA was not sheared or size selected prior to library prep. Sequencing was performed with an R10.4.1 flow cell (FLO-MIN114) and minION device under high-accuracy mode (280 bp/s). Basecalling was performed using Guppy 6.4.6, and reads with quality scores <7 were removed. Reads were filtered with Filtlong 0.2.1 (ng 0.2.1  to removcy mode 20 bp/s. Bl FLO-MIN4 and minng 0.2.1 . Potenting 0.2.1 . Taxonomng 0.2.1  and the ng 0.2.1 .Asaia bogorensis (96.36% average nucleotide identity). GhostKOALA identified complete pathways for central carbon metabolism, including glycolysis and pyruvate oxidation, TCA cycle, and the pentose phosphate pathway. A complete oxidative phosphorylation pathway was annotated. Interestingly, pyruvate decarboxylase (pdc) was absent, but other putative fermentation genes, aldehyde dehydrogenases  and alcohol dehydrogenase (adhA), were present. GhostKOALA identified genes potentially conferring resistance to vancomycin, beta-lactams, and cationic antimicrobial peptides. The genome sequence of Asaia bogorensis strain SC1 from the mosquito crop provides a valuable resource for further investigating host-microbe interactions of public health interest.The SC1 genome is 3.4 Mbp with 59.69% GC content . GTDB id


Rf) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1–HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility (rf) haplotype; HT2, a less-functional Rf; HT3–HT5 are semi-functional Rfs; and HT6 and HT7 are functional Rfs. However, the rare haplotypes HT8–HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region.In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-of-fertility ( These s Tanaka) . TherefoRf). The male sterile phenotype is a result of interaction between the CMS-associated gene and a non-functional restorer-of-fertility nuclear gene (rf)  Rf2 being the first  family  . To eluche first . Among t) family . These P) family , with ea) family . In partstigated . Three nstigated ; however1 populations of a cross between different varieties and selected strains harboring the Kishu-cytoplasm (MS-P1). This reduced NPG is linked to the haplotype block derived from kunenbo in the MS-P1 locus (Rf are located in the MS-P1 locus and to determine the influence of a combination of haplotype blocks (including Rf) on the phenotypic variations pertaining to male sterility and fertility in individuals with Kishu-cytoplasm.Male sterility and fertility segregate in the Fytoplasm . Specifiytoplasm . In addiytoplasm . These rytoplasm  and haveP1 locus . TherefoMS-P1 region within the locus using fine mapping, (2) identify candidates for Rf (Rf-MS-P1) through bioinformatic and transcriptional analysis, (3) define the number of allelic haplotypes at the MS-P1 region among the founders; and (4) reveal the association between NPG and the combination of allelic haplotypes (diplotype) at the MS-P1 region. Hybrid varieties, selected strains, and individuals in F1 populations (breeding germplasms) harboring the Kishu-cytoplasm as well as the various diplotypes in each individual were evaluated. Two mitochondrial-targeted PPR family genes at the MS-P1 region showed significant transcriptional differences between sterile and fertile varieties during flower development, indicating that they played an important Rf role in the CMS model. Assessing the combination allelic haplotypes, including the two PPR family genes, would enhance our understanding of the wide phenotypic variations observed in citrus male sterility and fertility associated with a sterile cytoplasm.To elucidate the molecular mechanism underlying male sterility in Kishu-cytoplasm and assess if it fits into the CMS genetic model, the present study aimed to: (1) identify the 1 populations, Okitsu No. 46 × ‘Kara’ (O46-K), ‘Sweet spring’ × Okistu No. 56 (SS-O56) and ‘Harehime’ × Okistu No. 63 (H-O63), and the varieties/selected strains used in this study were maintained in the Division of Citrus Research, Institute of Fruit Tree and Tea Science, NARO   S2. The and 2013 . The NPG1 populations and the 85 varieties/selected strains through a modified protocol using cetyltrimethylammonium bromide and a high-salt precipitation solution . Six DNA markers  in the MS-P1 locus were used in a previous report (MS-P1 locus (C. clementina genome v1.0 (JGI) (https://phytozome-next.jgi.doe.gov/info/Cclementina_v1_0) (https://mikan.dna.affrc.go.jp/) were identified using BLAST (https://mikan.dna.affrc.go.jp/blast/) (https://archive.gramene.org/db/markers/ssrtool) (https://bioinfo.ut.ee/primer3/)  , was obtna_v1_0) . The cor/blast/) . The idessrtool) . The pririmer3/) . Dimericrimer3/) , as descrimer3/) .MS-P1 locus were identified based on the genotype segregation pattern. A graphical genotype of the MS-P1 locus was constructed for the physical map of C. clementina genome v1.0 between 5,153,708 and 19,761,190 bp in scaffold 8. The male sterile genotypes within the MS-P1 locus in recombinant individuals were identified through comparisons with the genotype of male sterile varieties/selected strains  were obtained from Phytozome (https://phytozome-next.jgi.doe.gov/info/Cclementina_v1_0) (Zea mays) Rf2 (U43082), rice (Oryza sativa) Rf2 (AB583697), rice Rf17 (Os04g0475900), and sugar beet (Beta vulgaris) Rf1 (AB646135), all of which belong to the non-PPR family of Rf, were used as the query to perform BLAST analysis against the sequences in the Mikan Genome DB under default settings. The protein sequences of the PPR family at the MS-P1 region were analyzed using TargetP v.2.0  (https://urgi.versailles.inra.fr/predotar/) (https://ihg.helmholtz-muenchen.de/ihg/mitoprot.html) (https://ppr.plantenergy.uwa.edu.au/)  (https://prosite.expasy.org/scanprosite/), and phylogenetic trees were constructed using the neighbor-joining method of MEGA v.10.0.5 (https://www.megasoftware.net) with protein sequences of rice (Oryza sativa) Rf1a (DQ311053), rice Rf1b (DQ311054), rice Rf4 (KJ680249), Sorghum (Sorghum bicolor) Rf1 (Brassica napus) Rfp1 (KX671967), Petunia (Petunia hybrida) Rf-PPR592 (AY10271), Ciclev10030242m, and Ciclev10030361m. Bootstrap values were calculated through a 1,000-permutation test.The gene locus, annotations, and protein sequences at the na_v1_0) . The DNAetP-2.0) , Predotaedotar/) , and Mitot.html)  to prediedu.au/) . Proteinserver/) , while Plor) Rf1 , ChineseC. clementina genome v1.0 (JGI) (https://phytozome-next.jgi.doe.gov/info/Cclementina_v1_0) using HISAT2 v.2.1.0. Transcripts per million (TPM) values were obtained to measure gene expression using StringTie v.2.1.1. TPM values were imported to Subio Platform v.1.24.5853  for normalization. Statistical analysis of the comparisons between ‘Shiranuhi’ and KyOw14 was carried out using the “compare 2 groups” tool of the Subio platform. All RNA-seq data was deposited in the DDBJ Sequence Read Archive under accession number DRA015326.Bulked stamen from a sterile selected strain (KyOw14) and fertile variety (‘Shiranuhi’) were collected seven days before flowering (DBF) and 1 DBF in a field from the Division of Citrus Research, Institute of Fruit Tree and Tea Science, NARO , as two biological replicates. Total RNA was isolated using an RNeasy Plant Mini Kit , while the RNA-seq analysis was carried out by Hokkaido System Science Co., Ltd. . RNA-seq libraries were generated from the total RNA using the NEBNext Ultra RNA Library Prep Kit for Illumina . The 150 bp paired-end sequencing of RNA-seq libraries was performed using the NovaSeq 6000 system , and the sequenced reads were trimmed using fastp v.0.22.0 and mapped to https://phytozome-next.jgi.doe.gov/info/Cclementina_v1_0)  . Quantitative PCR was performed QuantStudio3 Real Time PCR System  using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) under 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. The primers for the gene expression of Ciclev10030242m , Ciclev10030361m , and Ciclev10029947m  were designed at a specific region or 3’UTR referred by Phytozome (na_v1_0) . The expGGATCAT) . SpecifiThe sources of cytoplasm in the 85 varieties/selected strains were determined referring to the pedigree chart .1 populations   Table S1d H-O63) , althougd H-O63)  of genotee chart .https://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html)  .MS-P1 locus between 33.5cM (TSRF161) and 69.1cM (SSR08B66) on linkage group 8 (LG8) in the corresponding linkage map of Okitsu No. 46 × Okitsu No. 56  , 66 functional genes, including 10 PPR family genes, were predicted on the 920 kb genomic region, corresponding to the newly refined MS-P1 region (Rf2 (U43082), rice Rf2 (AB583697), rice Rf17 (Os04g0475900), or sugar beet Rf1 (AB646135), which are non-PPR families of Rf, were not found among the 66 genes here. Bioinformatic analysis using PPRfinder revealed that all PPR family genes at the MS-P1 region belonged to the P-class subfamily  should be expressed more in a male fertile variety/selected strain than in a male sterile variety/selected strain. Transcriptomic analyses of KyOw14 (sterile selected strain) and ‘Shiranuhi’ (fertile variety) stamens were performed at 7 and 1 DBF through RNA-seq analysis. RNA extracted from the stamens (two replicates) were used to build libraries for RNA-seq (MS-P1 region of KyOw14 and ‘Shiranuhi’ was performed (p< 0.05)  than in KyOw14 (sterile strain). In addition, the existence of Ciclev10030242m and Ciclev10030361m at the MS-P1 region provides evidence that molecular mechanisms underlying citrus male sterility fit into the general plant CMS model.The protein sequence analysis showed that Ciclev10030242m and Ciclev10030361m were highly similar proteins, with 86.66% overlap , ChineseRf or rf are located; therefore, we defined allelic haplotypes at the MS-P1 region among the founders of Japanese breeding germplasm. The nearest markers to candidates for Rf-MS-P1 were narrowed down to 00918-1 through fine mapping of the MS-P1 region  were genotyped (1 populations except for ‘Willking’ (see legend in MS-P1 locus (C. tangerina hort. ex Tanaka), Iyo (C. iyo hort. ex Tanaka), Ponkan (C. reticulata Blanco), or Willowleaf mandarin (C. deliciosa Ten.)  were investigated (MS-P1 region (p< 0.05) (MS-P1 region (p < 0.05) (p < 0.05)   corresponding to the MS-P1 region was derived from Willowleaf mandarin, which harbors HT6  to identify Rf-MS-P1 candidates was a reasonable strategy.The results here also suggest that plotypes Table 2.bors HT6 ; therefoMS-P1 (MS-P1 region were not observed (data not shown). ‘Nishinokaori’ and ‘Kara’ exhibited an increased NPG, although ‘Nishinokaori’ harbored HT1/HT3 and ‘Kara’ harbored HT1/HT4, both of which are partial male sterile diplotypes  were detected in our previous study .The original contributions presented in the study are publicly available. This data can be found here: DDBJ, accession DRA015326 (SG acquired funding, designed the study, performed experiments, analyzed data, and wrote the original draft. SG, HF, TE, ToS, and TaS designed the study. HH, SO, and KN produced and managed materials. SG and TaS reviewed and edited this manuscript. All authors contributed to the article and approved the submitted version.