Synechocystis sp. PCC 6803’ by Sharon L. Smolinski et al., RSC Adv., 2022, 12, 14655–14664, https://doi.org/10.1039/d2ra01295b.Correction for ‘The influence of electron utilization pathways on photosystem I photochemistry in  The authors regret that in the Experimental section on Page 14657, there are six instances where the abbreviations of micromolar (μM), microliter (μl) and microgram (μg) are incorrectly noted as “mM”, “ml” and “mg”. The corrected units are given below:Section 2.6. Fluorescence emission analysis700. Fractions containing PSI monomers and trimers that were isolated using sucrose gradients were normalized to 16 μg chl per ml and were measured at room temperature and 77 K to determine spectral properties.Fractions containing PSI trimers and monomers that were isolated using anion-exchange chromatography were normalized to 3.0 μg chl per ml and were measured at 77 K to quantify P700 spectroscopic analysisSection 2.7. P700 spectroscopic analysis on isolated fractions containing PSI monomers or trimers were normalized to equivalent amounts of P700 (0.84 μmol) and measured using 720 nm actinic light. Samples (2 ml) were placed in a quartz cuvette containing P700, 10 mM sodium ascorbate, and 10 μM 2,6-dichlorophenolindophenol (DCPIP) in 20 mM HEPES–NaOH, pH 7.5, with 10 mM CaCl2, 10 mM MgCl2, 10 mM NaCl, and 0.04% DDM.P2.8. Flavodoxin photoreduction assays700 (44 nmol) were added to a quartz cuvette containing 10 mM sodium ascorbate, 30 μM phenazine methosulfate, and 100 μM flavodoxin (Fld), in 20 mM HEPES–NaOH, pH 7.5, with 10 mM CaCl2, 10 mM MgCl2, 10 mM NaCl, and 0.04% DDM, at a final volume of 350 μl, similar to ref. 26.In order to determine the capacity of PSI monomers and trimers to transfer electrons out of PSI, equivalent amounts of PThe Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Callinectes sapidus puberty molting. However, it is unclear that whether the mode of CFSH action involves a vertebrate-type sex steroid hormone in crustaceans. To this end, E2 levels were first measured using a competitive ELISA in the hemolymph and the potential CFSH target tissues from both prepuberty and adult females; the presence of E2 was further confirmed with a liquid chromatography tandem mass spectrometry method. Then, the cDNAs of the following genes known to be associated with vertebrate steroidogenic pathways were isolated: StAR-related lipid transfer protein 3 (StAR3); 3β-hydroxysteroid dehydrogenase (3βHSD); two isoforms of 17β-hydroxysteroid dehydrogenase 8 (17βHSD8); and, estradiol-related receptor (ERR). RT-PCR analysis revealed that these genes were widely distributed in the eyestalk ganglia, hepatopancreas, brain, ovary, spermathecae, ovigerous and plumose setae tissues of adult females. The 17βHSD8 transcripts were localized in the follicle cells, the periphery of the nuclear membrane of primary oocytes, and yolk granules of the vitellogenic oocytes using in situ hybridization, and the corresponding protein was detected in the follicle cells and ooplasm of primary oocytes using immunohistochemistry. Furthermore, the adult females injected with CFSH-dsRNA (n = 30 times) had E2 and StAR3 transcripts levels lower in the ovigerous and plumose setae, spermathecae than controls. These results suggested that the mode of CFSH action in C. sapidus might involve E2 in these adult-female-specific tissues.17β-estradiol (E2) has been proved to control reproduction, sexual differentiation, and the development of the secondary sexual characteristics of vertebrate females. In decapod crustacean species, crustacean female sex hormone (CFSH), a protein hormone, is required for developing adult-specific ovigerous setae for embryo brooding and gonophores for mating at the blue crab  Nephrops norvegicus  is a female-dominant sex steroid hormone in vertebrates that exerts critical functions in reproduction and sexual differentiation -related lipid transfer protein and estradiol receptor (ER)  was detected in the ovary, and 3βHSD and 17α-hydroxylase activities were identified in the hepatopancreas  proteins, hydroxysteroid dehydrogenase (HSD) enzymes, and sex steroid receptors known to be involved in the vertebrate steroidogenic pathway , 15 wererculatus  and the pancreas . These fCallinectes sapidus, regulates developing adult-female-specific reproductive phenotypes of ovigerous setae for embryo brooding and gonopores for mating at the puberty-terminal molting , identified from the eyestalk of the female blue crab  molting . Like P.elagicus  and P. sinimanus , C. sapidulthood  and re-uStAR3 (StAR-related lipid transfer protein 3), 3βHSD, two isoforms of 17βHSD8, and ERR (estradiol-related receptor) that were initially identified from the blue crab C. sapidus transcriptomes, were analyzed for the tissue distributions by RT-PCR and localized 17βHSD8 mRNAs and its protein in the ovary by in situ hybridization and immunohistochemistry, respectively. Moreover, the effects of CFSH on E2 and steroidogenesis gene transcript levels were determined using CFSH-dsRNA injection.Herein, E2 levels were measured in the hemolymph and potential/putative CFSH target tissues using a competitive ELISA, and the presence of E2 was further confirmed with a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Subsequently, several steroidogenesis genes, including 0 stage (n = 10) were kept in five tanks in a closed circulating aquaculture system (91 cm W × 112 cm H × 58 cm D). Each tank held 300 liters of ASW under the following conditions: 16 hours dark, 8 hours light at 18-20°C , Baltimore, MD]. After reaching prepuberty, the females were monitored for their molt stages, early premolt and late premolt . For the0 stage  was plac 18-20°C . The wati.e., 450 mM NaCl, 10 mM KCl, 10 mM NaHCO3, 1 mM EDTA, 10 mM HEPES, pH 7.3) in a 1:1 ratio (v:v). The animals were then placed on ice for 10 min before dissection. The eyestalk ganglia, brain, and ~100 mg of the hepatopancreas, ovary, spermatheca, ovigerous setae, and plumose setae tissues were dissected in ice-cold DEPC-treated crustacean saline . A hemolymph sample (200 µl) was withdrawn directly into an insulin syringe containing crab anticoagulant solution . Data were presented as follows: mean ± SE pg/tissue (n = 5) for the eyestalk ganglia and brain; mean ± SE pg/10 mg wet weight (n = 5) of the hepatopancreas, ovary, spermathecae, ovigerous setae, and plumose setae tissues; and, mean ± SE pg/10 µl (n = 5) of hemolymph.E2 levels in the tissues were estimated using an ELISA kit by following the manufacturer’s protocol (Enzo Biochem). The standard curve was generated using E2 solutions with known concentrations of 29.3 – 30,000 pg/ml. The standards and experimental samples were assayed in triplicate, and the plate was read at an absorbance of 405 nm using a SpectraMax M5 reader (Molecular Devices). The EC® C18 solid-phase extraction (SPE) cartridge (Waters) to remove interfering substances  was confirmed by an LC-MS/MS method modified from previous protocols –28. Firsbstances . The SPE4OH (pH 10.5) and (B) methanol with 0.1% NH4OH. Isocratic elution was conducted with 30% A at a flow rate of 200 µl/min. The column compartment was maintained at 40°C, and the overall method run time was 8 min. Negative electrospray ionization mode was employed for LC-MS/MS analysis, and two characteristic product ions were used to quantify and confirm each analyte. The surrogate standard recovery was 89% in the brain sample. The detailed analytical parameters are available in E2, E2-d3, and EE2-d4 were measured using an UltiMate 3000 LC coupled to a Thermo TSQ Quantum Access Max MS/MS (Thermo). Briefly, 50 µl of the reconstituted SPE extracts were injected onto a Waters XBridge C18 guard column  connected to an analytical column  with the same material. The mobile phase was comprised of (A) LC-MS grade water with 0.1% NHE. coli competent cells. Clones containing the inserts were isolated and cultured at 37°C overnight for subsequent DNA sequencing (ABI).The total RNA (~1-1.5 μg) from the eyestalk ganglia was subjected to 5’ and 3’ rapid amplification of cDNA ends (RACE) cDNA synthesis using the SMART cDNA amplification kit (BD Bioscience) according to the manufacturer’s protocol. Gene-specific primers for 5’ and 3’ RACE (https://web.expasy.org). The conserved domain was searched via SMART (http://smart.embl-heidelberg.de/). ClustalW (www.genome.ad.jp) was used to align the amino acid sequences, which were then modified by ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The phylogenetic tree was constructed using the neighbor-joining method (MEGA X).The open reading frames were identified using ExPasy (RT reverse transcriptase reagent kit with gDNA eraser (TaKaRa).Total RNA was extracted from the tissues using QIAzol Reagent (Qiagen) following the manufacturer’s procedures. RNA concentrations were measured using a Nanodrop (Thermo Scientific). Reverse transcription with 1.5 µg total RNA was carried out using a PrimeScripStAR3, 3βHSD, 17βHSD8a, 17βHSD8b, and ERR, was examined in the tissues obtained from an adult female using an endpoint RT-PCR assay with the gene-specific primers reported in AK and eIF4A (eukaryotic translation initiation factor 4A) as reference genes. The PCR conditions were as follows: an initial denaturation step at 94°C for 2.5 min, followed by 35 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s, with a final extension 72°C for 5 min. PCR products were resolved on 1.5% agarose gel and stained with ethidium bromide for visualization.After being diluted with RNase-free water at 12.5 ng/μl, cDNA was amplified for the arginine kinase (AK) gene to examine the quality of each sample . The spaOvaries were dissected and fixed overnight in Bouin’s solution at 4°C. The fixed tissues were progressively dehydrated in ascending ethanol concentrations. The dehydrated tissues were cleared in xylene and embedded in Paraffin wax (60°C). The wax blocks were cut 6 μm in thickness and mounted on APTES (3-aminopropyl triethoxysilane)-coated slides. After being deparaffinized and rehydrated using xylene and a graded series of DEPC-treated ethanol dilutions, respectively, the sections were visualized by staining with hematoxylin and eosin.in situ hybridization was carried out using digoxigenin (DIG)-labeled sense and anti-sense probes synthesized following T7-mediated in vitro transcription using TranscriptAid T7 high yield transcription kit (Roche) from the clone of cDNAs fragment of target genes. With some modifications, ISH procedures followed previously described protocols , 50 μg/ml denatured yeast DNA). Sense or anti-sense probe concentrations were 1 ng/μl, and hybridizations were performed at 58°C overnight. Unbound probes were removed by rinsing with a series of low-salt solutions . The presence of mRNAs was localized with a DIG nucleic acid detection kit (Roche). Specimens were photographed using an Olympus micro/DP70 camera mounted on an Echo Revolve microscope (Echo).An rotocols . Brieflyi.e., 50 mM phosphate buffer, 0.05% NaN3, 0.5% Triton X-100) 3-5 times for 30-60 min/wash at 20°C. The tissues were blocked in 10% normal lamb serum and incubated with anti-antigen serum diluted 1:1000 for 3 days at 4°C (pre-immune and pre-absorption serum for the negative control), followed by incubation with FITC-conjugated goat anti-rabbit IgG (Thermo scientific) at 1:5000 for 2 days. Washing was repeated in PTX 3-5 times, and samples were kept in 50% glycerol and 50% PTX in the dark until photographic analysis with a Leica SP8 confocal microscope.Ovary sections were deparaffinized, rehydrated, and rinsed in DEPC-treated, double-distilled water before being washed in PTX (2) and the average amplification efficiency of samples are listed in AK and eIF4A levels in the same cDNA samples (n = 5) transcripts/μg total RNA.Each cDNA sample containing 25 ng total RNA was assayed in duplicate using Fast SYBR Green Master Mix (Applied Biosystems) and gene-specific primers Table 2  samples . Data wen = 5) or saline-only controls (n = 5). These injection regimes were continued 30 times over four months (Oct. 2019-Feb. 2020). Tissues were sampled (section 2.1) for E2 measurement and expression analysis using RT-qPCR assays.CFSH-dsRNA preparation and injection were carried out as described . The mate.g., different tissues), and a non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test was used for multiple group comparisons. Second, the student’s t-test was used when two groups  were compared. Significance at p < 0.05 was accepted and noted by letters or ‘*’ symbols.All data were analyzed using SPSS 25.0 for statistical significance. The data were analyzed in two ways. First, one-way ANOVA was employed with multiple samples (n = 5) to the highest level in the brain  at early premolt and from 9.6 ± 1.2 pg/10 μl (hemolymph) to 256.4 ± 64.6 pg/10 mg wet weight (brain) at late premolt (n = 5).Tissues of the prepuberty females at early and late premolt were measured for E2 using a competitive ELISA (n = 5), significantly lower (p < 0.01) than early premolt . The E2 content in plumose setae and ovary at late premolt significantly decreased (p < 0.05) by about twofold to 112.3 ± 2.0 pg/10 mg wet weight (n = 5) and 49.1 ± 2.2 pg/10 mg wet weight (n = 5), respectively, compared to those at early premolt. Significantly (p < 0.05) reduced levels of E2 were also observed in the spermathecae . However, no changes were identified for the E2 content of the eyestalk ganglia, brain, hepatopancreas, and hemolymph at early and late premolt.E2 levels differed by molt stage in the following tissues: ovigerous and plumose setae, spermathecae, and ovary. Ovigerous setae at late premolt had E2 levels of 33.1 ± 5.0 pg/10 mg wet weight .According to the ELISA results, the brain contained the highest E2 level. Therefore, this tissue was chosen for further confirmation of E2 StAR3, 3βHSD, 17βHSD8a, 17βHSD8b, and ERR, in C. sapidus involved in the biosynthetic pathway of vertebrate steroid hormones. The full-length StAR3 and 3βHSD sequences were cloned from the eyestalk using PCR with gene-specific primers , and a 630-bp 3’UTR with a poly-A tail (3βHSD cDNA (1604 nt) consisted of a 527 bp 5’UTR, a 912 bp ORF, and a 165 bp 3’UTR, including the poly-A tail (3βHSD/isomerase family (3β_HSD), which illustrated 28-60% homology to vertebrates and invertebrates  and hormone receptor (ligand-binding domain of nuclear hormone)  clade and separated from the vertebrate (zebrafish) clade  with two shrimp species   compared to controls . The ovigerous setae of CFSH-dsRNA injected females had significantly lower E2 of 24.3 ± 1.3 pg/10 mg wet weight (n = 5) than controls, 59.6 ± 3.8 pg/10 mg wet weight (n = 5). Moreover, CFSH-dsRNA injection significantly (p < 0.05) decreased E2 concentrations in the spermathecae  compared to controls .In general, the effect of CFSH-dsRNA injection on E2 levels differed by tissue (n = 5), whereas controls contained 63.8 ± 4.3 pg/tissue (n = 5). E2 levels in the hemolymph were as follows: CFSH-dsRNA injection, 3.8 ± 1.2 pg/10 μl; controls, 5.4 ± 1.4 pg/10 μl, n = 5).CFSH-dsRNA injection slightly reduced E2 levels in the ovary, eyestalk ganglia, brain, hepatopancreas, and hemolymph, but the differences were not statistically different. The brains of the females that received CFSH-dsRNA had E2 levels of 48.5 ± 4.2 pg/tissue  compared to the controls with 2.6 ± 0.8 × 106 transcripts/μg total RNA (n = 5). The animals injected with CFSH-dsRNA had significantly (p < 0.05) reduced StAR3 transcripts in all examined adult-female-specific tissues compared to controls  compared to controls .The effect of CFSH-dsRNA injection on the transcript levels of steroidogenesis genes was determined using a RT-qPCR assay (p < 0.05) lower 17βHSD8a by about 50%, namely 19.2 ± 3.9 × 105 transcripts/μg total RNA (n = 5) and 20.8 ± 4.0 × 105 transcripts/μg total RNA (n = 5) compared to controls with 35.7 ± 2.0 × 105 transcripts/μg total RNA (n = 5) and 32.6 ± 4.9 × 105 transcripts/μg total RNA (n = 5), respectively (p < 0.05) downregulation of 17βHSD8b and ERR expression was only observed in the ovary  changes in 3βHSD were observed  in adult females, the undifferentiated ovaries in prepuberty females had higher E2 levels. This finding appears to be rather different from those reported in other decapods, in which E2 levels in the ovary significantly increased with ovarian development and oocyte maturation , 39, 40.e.g., 70 ± 10 g for prepuberty, 160 ± 20 g for adult, n = 10); therefore, the hemolymph contained the greatest amount of E2 compared to other tissues, suggesting that E2 may act as a hormone in this species.The levels of E2 measured in the tissues were converted and presented as pg/tissue type, with the same 10 mg wet weight wherever possible. In the present study, E2 levels were lowest in the hemolymph of the prepuberty and adult females, but hemolymph accounted for ~15% of body weight , P450c17/cyp17a, and P450arom/cyp19a (P450 aromatase), were not identified in this study. The presence of P450c17 has been documented in echinoderms , molluskC. sapidus using RT-PCR, possibly explaining the widespread presence of E2. The relatively high expression levels of all steroidogenic genes in the brain were consistent with the ELISA results, wherein the brain had the highest E2 levels among tested tissues. Similarly, most of the enzymes involved in steroidogenesis were highly expressed in the central nervous system of mammals ; 3βHSD (MT013238); 17βHSDa (MT762163); 17βHSDb (MT762164); 583 and ERR (MT013240)).TW: Data acquisition, Analysis, Visualization, Writing – Original draft preparation, Writing & Editing. KH: LC-MS/MS analysis and Writing–Review & Editing. LB: LC-MS/MS analysis and Writing–Review & Editing. JSC: Conceptualization, Data acquisition, Project Administration, Resources, Supervision, Validation, Original draft Writing – Review & Editing. All authors contributed to the article and approved the submitted version.This research was supported by an NSF grant (NO. 1146774) to JC and the China Scholarship Council (CSC201906330108) to TW.We are grateful to the staff of the aquaculture research center  for maintaining the closed recirculating system and Zooquatic Laboratory for daily water analysis. All experimental animals were produced in the Chung laboratory . The broodstock females for juvenile production were collected onboard Miss Paula in the mid-upper Chesapeake Bay with the help of Captain CJ Candy and his crews. This research was supported by an NSF grant (NO. 1146774) to JC and the China Scholarship Council (CSC201906330108) to TW.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Metastatic breast cancer to the lungs is a serious, life-threatening complication that is difficult to cure. Circulating tumor cells play a key role in the metastatic spread of breast cancer to the lungs via the lymphatic or circulatory system. Palmatine is a protoberberine alkaloid, identified as an active component of traditional African herbal preparations. Palmatine has antimetastatic and antiproliferative effects. The inhibitory activity of palmatine on the metastatic colonization of triple negative breast cancer cells in the lungs was investigated in this study.Methods: 4T1 triple breast cancer cells were transplanted synergically to the thoracic duct of the female balb/c mice via the lymphatic system. Palmatine 1, 5 and 10 mg/kg were administered for 28 days. The lungs were analyzed for levels of arterial blood gas, histological damage, immunohistochemical expression of the metastasis-associated protein 1 (MTA1) and tumor suppressor p53 (p53).Results: Administering palmatine 1–10 mg/kg dose dependently improved hypoxemia, ameliorated metastasis associated lung injury; histology score of 3.33 ± 0.33, 1.67 ± 0.33, 1.33 ± 0.33, decreased lung MTA1  and increased p53 expression  respectively.Conclusion: Palmatine preserved lung morphology and demonstrated therapeutic potential in aiding the treatment of lung metastasis. Breast cancer is a disease frequently diagnosed among women marked by an abnormal growth of cells in the breast . Lung meDespite progress in breast cancer therapy, once breast cancer cells develop in secondary organs the disease essentially enters an incurable phase . Effortsin vitro and in vivo antiproliferative and antimetastatic properties against various forms of cancer. Notable examples include taxol analogs, vinca alkaloids and camptothecins currently used in cancer therapy , Coptis chinensis Franch. (Ranunculaceae), Corydalis yanhusuo W.T.Wang ex Z.Y.Su & C.Y.Wu (Papaveraceae), Tinospora cordifolia (Willd.) Hook.f. & Thomson (Menispermaceae), Tinospora sagittata (Oliv.) Gagnep. (Menispermaceae) and Stephania yunnanensis H.S.Lo (Menispermaceae) .Palmatine is a protoberberine alkaloid found in several medicinal plants including rmaceae) . In vitrll lines . Howeverl-glutamine , Fetal bovine serum, Penicillin-streptomycin , MTA1  and p53 Antibodies .Palmatine , Doxorubicin, Trypan blue . Roswell Park Memorial Institute 1640 medium, Trypsin/EDTA, l-glutamine 2 mM. The cells were kept at 37°C in a 5% CO2 humidified atmosphere. Cell viability was determined with a trypan blue assay before the experiment.The 4T1 triple negative breast cancer cell line (ATCC CRL-2539) was purchased from AddexBio . The 4T1 cells were cultured in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum, penicillin 100 U/mL, streptomycin 100 g/ml and Female balb/c mice weighing 20–30 g and aged 6–8 weeks were purchased from the Noguchi Memorial Institute for Medical Research at the University of Ghana, Legon and kept at the animal house of the Department of Pharmacology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi. The mice were housed in stainless steel cages with delicate wood shavings as bedding, with free access to chow , and water. The room was maintained at a temperature of 24–28 C with a relative humidity of 60–70%, and a light-dark interval of 12 h. Before the experiment, the animals were acclimatized for a week.6) were transplanted via the lymphatic thoracic duct into each recipient mouse , Doxorubicin , Lung metastasis  and Negative control . After 28 days, the following assessments were made;Animals were administered Palmatine .The lung tissues were excised and embedded in paraffin after being fixed in 10% neutral-buffered formalin. 4 µm thick deparaffinized sections were stained with hematoxylin and eosin (H&E) and analyzed at 10 × magnification. The histology sections were evaluated for metastatic lesions, morphological changes and necroinflammation according to a modified histology activity index . The sec2O2 in methanol. Tissue sections were dehydrated in graded alcohols before performing heat-induced epitope antigen retrieval with 10 mM sodium citrate. Sections were washed with tris borate saline tween-20 (TBST) and then blocked for 1 h with 5 percent bovine serum albumin. The sections were incubated with the mouse polyclonal primary antibody. After washing for 5 min in TBST, the sections were incubated for 1 h with a horseradish peroxidase-conjugated rabbit anti-mouse IgG secondary antibody mixed with TBS in a 1:200 ratio. The sections were incubated with 3,3′-diaminobenzidine and counterstained with hematoxylin. Images of the slides were captured under a 10 × light microscope Leica DM2500 M . The manifestation of a dark, brown-colored, intracytoplasmic precipitate is indicative of the presence of MTA1 and p53. The antibody stain intensity of the images obtained was analyzed with an IHC Profiler plugin in ImageJ. The Immunohistochemistry optical density score (IODS) was calculated  followed by Dunnett’s post hoc tests. The criterion for significant difference between groups was set at aO2) 190.70 ± 2.19, 207.0 ± 3.51, 218.5 ± 3.75 and decrease in the partial carbon dioxide pressure (PaCO2) 40.75 ± 0.58, 35.10 ± 0.92, 30.00 ± 0.68; bicarbonate (HCO3) 16.30 ± 0.17, 14.00 ± 0.20, 11.75 ± 0.20; base excess (BE) −1.45 ± 0.02, −0.50 ± 0.03, −0.37 ± 0.01 and anion gap 20.46 ± 0.27, 16.41 ± 0.70, 14.19 ± 0.32 respectively compared to a decreased PaO2 185.70 ± 1.89, increased PaCO2 43.83 ± 0.51; HCO3 17.23 ± 0.20, BE −1.47 ± 0.02 and anion gap 21.98 ± 0.78 in the untreated lung metastasis group consistent with hypoxemia and respiratory distress. This resulted in reduced activity levels and labored breathing. There were no significant changes in values recorded for potential of hydrogen (pH) and chloride (Cl−) for all groups in the experiment  demonstrated a dose dependent increase in the partial oxygen pressure . Tissue sections from the untreated lung metastasis group showed positive immunoreactivity in the cytoplasm for the MTA1 protein; IODS 3.11 ± 0.07 , 1L compTissue sections from palmatine treated groups 1–10 mg/kg showed positive immunoreactivity in the cytoplasm for the p53 protein; IODS 1.99 ± 0.06, 2.27 ± 0.12, 2.34 ± 0.12 respectively , 1M compFrom the “seed and soil hypothesis”, colonization of organs requires complex adaptations by cancer cells to various host components. Intercommunication between cells is steered by a dynamic network of cytokines, chemokines, growth factors, inflammatory and matrix remodeling enzymes against a background of major perturbations to the physical and physiological functions of the tissue .Breast cancer cells interact with the lung stroma such as the mesenchymal stem cells, fibroblasts and myeloid cells  through several paracrine signaling cascades that allow the formation of immunotolerant niches to promote cancer cell proliferation . To a siIn vitro experiments have also demonstrated the efficacy of palmatine against tumor migration and invasion , the arterial partial pressure of carbon dioxide (PaCO2), and acid-base disturbances. An ABG test assesses gas exchange through the alveolar-capillary membrane and lung function. Such knowledge is important to evaluate the severity of respiratory disease in lung metastasis. Mice in the untreated lung metastasis group showed high levels of PaCO2 while PaO2 was low with impaired acid-base homeostasis consistent with hypoxemia and underventilation from impaired lung function. These observations were however improved in palmatine-treated mice.To investigate the effects of palmatine on metastatic lung colonization, an arterial blood gas test (ABG) was performed. The ABG test analyzed the arterial partial pressure of oxygen (PaOThe formation of lung metastases constitutes a variety of genes and pathways. The Metastasis associated protein 1 (MTA1) is a member of the nuclear remodeling histone deacetylate complex, a critical modulator of chromatin remodeling. The MTA1 gene is upregulated in several cancers . OverexpPalmatine greatly preserved lung morphology and restored respiratory function demonstrating therapeutic potential in attenuating metastatic colonization of breast cancer cells in the lungs.


The use of the asymmetric NP2PhOHAr ligand resulted in the formationof both bidentate and tridentate products depending on the presenceof base. The tridentate Re(V) complex [ReOCl2(κ3-NP2PhOAr)] has providedthe basis of a new reactive “metal-fragment” for furtherfunctionalization in [3 + 2] mixed-ligand complexes. The synthesisof [3 + 2] complexes with catechol-based π-donors could alsobe achieved under one-pot, single-step conditions from Re(V) oxo precursors.Analogous complexes can also be synthesized from suitable 99Tc(V) precursors, and these complexes have been shown to exhibithighly similar structural properties through spectroscopic and chromatographicanalysis. However, a tendency for the {MVO}3+ core to undergo hydrolysis to the {MVO2}+ core has been observed both in the case of M = Re and markedlyfor M = 99Tc complexes. It is likely that controlling thispathway will be critical to the generation of further stable Tc(V)derivatives.N-Triphos derivatives corefor the formation of mixed-ligand [3 + 2] complexes. Characteristiclineshapes arising from an AA’BB’XX’ spin systemare diagnostic of bidentate vs tridentate coordination modes of theligand.An N-centered tripodal heterofunctionalizedphosphine ligandwas used to generate a reactive “metal-fragment” basedon the {M However,these sets have seen only limited success in, or indeed attemptedtranslation to, 99mTc radiopharmaceutical formulation.Notable examples with Re include the use of tridentate ligands derivedfrom Schiff bases,58 diolates and dithiolates;62 N-heterocycles;64 and heteroscorpionates,65 but very few have used heterofunctionalizedphosphines incorporated into tridentate ligands  radiopharmaceuticals includinga mono-oxo unit generally incorporate tetradentate N ligands 1c.16,66 chnetium 1d.67,68VO}3+ core is to revisit a [3 + 2]strategy with a new class of heterofunctionalized phosphine ligandfor Re(V) and Tc(V). The NP3R and NP2RXR′ ligands are employed as potentiallytridentate chelators due to their perceived favorable properties forstabilizing {MVO}3+ complexes. These includethe presence of strongly coordinating phosphine donors; a bridgeheadstructure predisposed toward facial coordination to a metal centerto limit isomer formation, and, in the case of asymmetric variants,an oxygen donor to exploit the well-established preference for oxygendonors trans to the oxo group in such complexes.72 {MVO}3+ complexes of this nature are desirabledue to the inherent ease of access of oxidometallates(V) from thepertechnetate anion [99mTc]-[TcO4]− with common reducing agents, such as SnCl2, and the potentialextension of this approach to the “kit”-based formulationof 99mTc radiopharmaceuticals.73 In a single previous study, NP2RXR′ ligands have been applied to [ReI(CO)3]+ units, albeit in a bidentate coordination mode, and withoutextension to radioactive analogues.36Our approachto produce a substitution-inert “metal-fragment”for the {M3Ph and NP2PhOHAr – VO}3+ and {99TcVO}3+ precursors are studied,and their products were characterized. In particular, the use of aphenolate functionality has been shown to facilitate facial tridentatecoordination to {ReVO}3+ complexes63 and has been used here in the formation of thenovel reactive “metal-fragment” complex, [ReOCl2(NP2PhOAr)] (4). The reactivity of this “metal-fragment” has alsobeen explored with several bidentate π-donating ligands to producestable [3 + 2] mixed-ligand Re(V) complexes. These [3 + 2] complexeshave also been successfully synthesized through one-pot reactionsfrom M(V) precursors, illustrating a preference for [3 + 2] heterocomplexformation, and extended to coordination chemistry with the long-lived 99Tc radioisotope.Herein, the reactions between model phenyl-substituted phosphineligands of this type 2],74 (NBu4)[ReOCl4],75 and (NBu4)[TcOCl4]76 were prepared as described in theliterature. Other reagents were commercially available and used asreceived. [99Tc]-(NH4)[TcO4] waskindly donated by Professor Philip Blower of Kings College London.All reactions were performed under an N99Tc is a weak β– emitter. All manipulations with thisisotope were performed in a laboratory approved for the handling ofradioactive materials. Normal glassware provides adequate shieldingagainst low-energy β– emissionof the technetium compounds. Bremsstrahlung is not a significant problemdue to the low energy of the β– particles involved and the low amounts of 99Tcused. However, normal radiation safety procedures must be used atall times to prevent contamination and inhalation.1H, 1H{31P}, 31P{1H}, and 13C NMRspectra were acquired on Bruker AV-400, AV-500, or DRX-400 spectrometers. 1H, 1H{31P}, and 31P{1H} NMR spectra for samples containing 99Tc wereacquired on a Bruker Avance III 400 spectrometer equipped with a BBOprobe or a Bruker Avance III 700 spectrometer equipped with an AVIIIconsole and a quadruple-resonance QCI cryoprobe. Chemical shifts arereported in ppm and referenced to the solvent for 1H and 13C{1H} NMR spectroscopy. 31P chemicalshifts were referenced (δ = 0) externally to 85% H3PO4 (aq). Peak multiplicities are abbreviated as s = singlet,m = multiplet, d = doublet, t = triplet, q = quartet, qu = quintet,sx = sextet, spt = septet, dd = doublet of doublet, td = triplet ofdoublet, and br = broad. Mass spectrometry analyses were conductedby the Mass Spectrometry Service, Imperial College London. Infraredspectra were recorded on a PerkinElmer Spectrum FT-IR spectrometer.Flash silica column chromatography for Re compounds was performedon a Biotage Isolera Prime advanced automated flash purification unitusing SNAP KP-Sil or Sfar Duo cartridges. Details of the single crystalX-ray diffraction (XRD) analysis can be found in the SI.xI, yI, zI and the identity matrix E to givea 64 × 64 matrix for a six-spin system taking care to retainthe ordering of the spin indices 1–6. The Zeemann Hamiltonianfor each spin was given by 1H and 31P) were assumed to be weakly coupledwith a Hamiltonian givenby 1H and 1H′ or 31P and 31P′) werestrongly coupled with a Hamiltonian given by t)I–) followed by application of a phenomenologicalline broadening factor and Fourier transformation to yield the spectrum.NMR lineshapeswere simulated in Matlab. Briefly, the Hamiltonian and density matrixfor an AA’BB’XX’ system were calculated as theKronecker tensor products of the corresponding Cartesian Pauli matrices 3(PPh3)2]  and NP3Ph  were dissolved in toluene (30 mL) andheatedto 100 °C for 18 h. Over this time, the reaction mixture turnedfrom light green to dark green. The solvent was removed invacuo to yield a dark green solid. This was redissolved inminimal CH2Cl2 and precipitated using hexane.The precipitate was collected by cannula filtration and the 31P{1H} NMR analysis indicated the presence of both 1 and its oxidized analogue 2. Subsequent attemptsto separate the complexes on silica column chromatography resultedin the oxidation of compound 1 to 2. 31P{1H} : δP /ppm = – 29.4 , – 29.8 . + at 900.0893, foundat 900.0883.Re-NP3(PPh3)2]  and NP2PhOH  were dissolved in MeCN (30 mL)and heated to60 °C for 18 h. Over this time, the reaction mixture turned frombright green to darker green. The solvent was removed in vacuo, and the green solid redissolved in minimal CH2Cl2 and precipitated from hexane (50 mL). The precipitate wascollected by filtration and dried under high vacuum to give a palegreen solid . The product was found to be poorly solublein chlorinated solvents but highly soluble in acetonitrile and methanol.Green crystals suitable for X-ray analysis were grown by vapor diffusionof acetone with hexane. 1H NMR :δH /ppm = 7.76–7.64 , 7.55–7.43 , 7.40–7.33 , 7.14 , 7.10 , 6.91 , 6.73 , 6.65 , 4.99–4.86. 31P{1H} NMR : δP =– 29.1 . FT-IR: νRe=O = 993(s). HR-ESMS: m/z calc. for [M + Na]+ at836.0194, found at 836.0201.[ReOCl3(PPh3)2]  and NP2PhOH were suspended in MeCN (30 mL). Thereaction mixture was stirred for 10 min, and then DIPEA  added. The reaction mixture was then heated to 60 °Cfor 18 h, over which time the solution turned from bright green tobrown. The reaction mixture was filtered, and the solvent was removed in vacuo. The resulting solid was dissolved in minimal CH2Cl2, precipitation was induced with hexane (50mL), and the precipitate was collected by filtration. A pure yellowproduct was obtained by purification using silica column chromatography(95:5 CH2Cl2:MeOH) . Crystals suitablefor single-crystal X-ray diffraction were grown by vapor diffusionof CH2Cl2 with hexane.Method A: + at 777.0608, found at 778.0617, m/z calc. for [M-Cl+ MeCN]+ at 783.1107, found at 783.1094.Method B: 5 wasobtained in a low yield as a byproduct from the preparation of 4, described above. The pure product could be obtained followingpurification using silica column chromatography (90:10 CH2Cl2:MeOH) .Method A: 5 could also be obtained from hydrolysis of 3. 3  was dissolved in MeCN in air and stirredat RT for 10 min. DIPEA (0.065 mmol) was added, and the reaction mixtureheated to 60 °C for 16 h. The solvent was removed invacuo, and the crude mixture was purified by silica columnchromatography (90:10 CH2Cl2:MeOH) to give theproduct as a brown solid . 1H NMR : 7.45 , 7.32 , 7.13 , 6.87 , 6.64 , 6.31 , 5.60 ,4.36 . 31P{1H} NMR : δP /ppm = – 36.7 .FT-IR : νRe=O = 999 (m). HR-ESMS: m/z calc. for [M-Cl]+ at 724.1180, found at 724.1180.Method B: 4  was dissolvedin CH2Cl2 (20 mL), and the bidentate ligand (0.025 mmol) was added to the solution underN2. The reaction mixture was stirred for 10 min, afterwhich two drops of triethylamine were added. The mixture was stirredat 60 °C for between 18 and 36 h and monitored by thin-layerchromatography (95:5 v:v CH2Cl2:MeOH). Upondisappearance of the starting material by TLC, the solvent was removed in vacuo. The brown residue was redissolved in minimal CH2Cl2 and precipitated using hexane (50 mL). Theprecipitate was collected and further purified by silica column chromatography(95:5 v:v CH2Cl2:MeOH) to give the productsas dark brown/red solids.1H NMR: δH /ppm = 7.83–7.74, 7.47–7.26 , 7.04–6.98 , 6.74 , 6.71–6.66, 6.63 , 6.34 , 6.29 , 5.17–5.09 , 4.88–4.79 . 13C NMR : δC /ppm = 162.9 (s), 162.5 (s), 144.4(s), 138.0 (s), 134.0 (t), 132.9 (t), 131.1 (s), 130.8 (s), 128.8(t), 128.4 (t), 128.1 (s), 123.6 (s), 120.5 (s), 120.2 (s), 119.9(s), 115.6 (s), 115.0 (s), 57.7 (t). 31P{1H}NMR : δP /ppm = –32.9 . HR-ESMS: m/z calc.for [M + H]+ at 816.1443, found at 816.1464.17 mg (82%). 1H NMR : δH /ppm = 7.61–7.43, 7.43–7.28 , 6.89 , 6.83 , 6.59 , 6.47 , 5.23–5.14, 4.88–4.78. 31P{1H} NMR : δP /ppm= – 29.6 . HR-ESMS: m/z calc. for [M + H]+ at 796.1028, found at796.1050.15 mg (74%). 1H NMR: δH /ppm= 7.77–7.66 , 7.48–7.38, 7.37–7.21 , 6.70 , 6.66 , 6.31 ,6.24 , 5.18–5.11, 4.92–4.84 4.78–4.68 . 31P{1H} NMR : δP /ppm = – 37.1 . HR-ESMS: m/z calc.for [M + H]+ at 768.1437, found at 768.1411.12 mg (60%). 1H NMR: δH /ppm= 7.76–7.66 , 7.63, 7.56–7.44 , 7.43–7.27 , 6.94 , 6.92 , 6.79 , 6.68 , 6.41 , 6.22 , 6.12 , 5.28–5.20 , 5.09–4.91 , 4.80–4.71 . 31P{1H} NMR : δP /ppm = – 30.0 , – 33.3 .HR-ESMS: m/z calc. for [M + H]+ at 977.1719,found at 977.1723.15 mg (64%): 1H NMR: δH /ppm = 7.82–7.73, 7.46–7.37 , 7.36–7.24 , 6.95–6.90 , 6.76–6.72 ,6.67–6.58 , 6.38–6.30, 5.17–5.08 , 4.89–4.79 , 3.78–3.73 , 3.65–3.60 . 31P{1H} NMR : δP /ppm = – 32.7 , −32.9. HR-ESMS: m/z calc. for [M + H]+ at 888.1654, found at 888.1636.18 mg (79%). 3(PPh3)] , NP2PhOHAr (0.21 mmol), and the bidentate ligand (2-mercaptophenolor N-acetyldopamine) (0.21 mmol) were dissolved inMeCN (30 mL) and stirred for 10 min. DIPEA (0.03 mL) was added bysyringe under N2. The reaction mixture was heated to 60°C for 16 h. Volatiles were removed in vacuo, the resultant residue was dissolved in minimal CH2Cl2, and the product mixture was precipitated using hexane. Thebrown precipitate was collected by filtration and purified by silicacolumn chromatography (95:5 v:v CH2Cl2:MeOH)to give the pure product. Compounds 6, 7, and 10 were also prepared through this method.+ at 901.1970,found at 901.1982. m/z calc. for [M + Na]+ at 923.1789, found at 923.1816. m/z calc. for [M+ K]+ at 939.1529, found at 939.1531.52 mg (34%) 1H NMR (CDCl3): δH /ppm = 7.85–7.77 , 7.70–7.62 , 7.54–7.46 , 7.44–7.22 , 6.93–6.86, 6.79–6.76, 6.75–6.69 , 6.43–6.35 , 5.24, 5.04 , 4.84 , 4.76 . 31P{1H} NMR (CDCl3): δP /ppm = – 29.1 ,– 40.6 . HR-ESMS: m/z calc. for [M+ H]+ at 832.1214, found at 832.1195.17 mg (12%). 4)[TcOCl4]  and NP2PhOHAr (1 equiv)were added to a sealed glass vial equipped witha stirrer bar and purged under N2. MeCN (1 mL) was addedunder N2, and the vial was heated to 60 °C for 2 hwith a vent needle. The vial was opened, and volatiles were removedunder a jet of N2. The residue was dissolved in minimalCH2Cl2, precipitated using hexane (25 mL), andcollected by centrifugation. This process was repeated three timesto yield the product mixture as a purple solid. The entire samplewas dissolved in d3-MeCN (600 μL) analyzed by NMRspectroscopy. Tc-3 was confirmed as the major speciesin the reaction mixture. Selected analytical data; 1H NMR: δH /ppm = 5.13–5.04, 4.87–4.74. 31P{1H} NMR (CDCl3): δP /ppm = + 27.1.(NBu4)[TcOCl4] , NP2PhOHAr (1 equiv), and catechol (1.1 equiv) wereadded to a sealed glass vial equipped with a stirrer bar and purgedunder N2. MeCN (1 mL) was added under N2 followedby NEt3 (3 equiv), and the vial heated to 60 °C for2 h with a vent needle. The vial was opened, and volatiles were removedunder a jet of N2. The residue was dissolved in minimalCH2Cl2, precipitated using hexane (25 mL), andcollected by centrifugation. This process was repeated three timesto yield the product mixture as a purple solid. The presence of Tc-6 could be postulated by TLC , 1H NMR spectroscopy and low-resolution mass spectrometry data(see text).: δH /ppm = 4.31. 31P{1H} NMR (CDCl3): δP /ppm = + 26.9 .Thepresence of 3Ph, was prepared accordingto the published method.37 This ligandwas selected due to its ease ofsynthesis and its known propensity toward forming facial κ3-coordination geometries with transition metals in a numberof different oxidation states.41The phenyl-substitutedN-triphos ligand, NP3(PPh3)2] and the NP2PhXR ligands. Reaction of NP3Ph with [ReOCl3(PPh3)2] intoluene at 373 K resulted in the formation of the diamagnetic complex[ReOCl3(κ2-NP2PhPPh)] (1), in which two PPh3 ligandshave been displaced by one unit of NP3Ph. Duringworkup of 1, a second Re species was also produced, [ReOCl3(κ2-NP2PhP(O)Ph)] (2), resulting from the oxidationof the free third phosphine arm of the coordinated NP3Ph ligand. The oxidized arm displayed a singlet at δP = + 25.3 ppm (relative to the uncoordinated phosphine armin 1 at δP = – 29.8 ppm) in the 31P{1H} NMR spectrum; a P=O absorption wasobserved at 1093 cm–1 in the FT-IR spectrum.1 to 2. Nevertheless, crystallographic characterization enabled confirmationof the structure of 2, in which the NP3Ph ligand coordinates in a bidentate fashion to the metal center,alongside three chloride ligands to complete the pseudo-octahedralcoordination sphere. Single crystals of 2 were obtainedby slow evaporation of a CDCl3 solution of the complex Å) bond trans to theoxo group, relative to the equatorial Re–Cl bond (2.3737(17)Å), which is typical of rhenium oxo complexes due to the substantial trans influence of the oxo donor.77Attempts to separate thesetwo species by silica column chromatographyresulted in further conversion of  complex1a. The c2 distorted octahedral geometry, which is commonlyobserved with Group 7 M(V) complexes containing an oxo unit.78 Therefore, 1H and 31P{1H} NMR spectroscopy can be used to effectively characterizethese species in solution. The 1H NMR spectrum exhibitsdistinctive multiplets in the δH = 3.5–4.5ppm region of the spectrum corresponding to the chemically inequivalentmethylene protons of the coordinated ligand. Upon coordination ofthe phosphines to the Re metal center, a six-membered chelate is formed,as confirmed by the chair conformation of this ring observed in thecrystal structure of 2 2-Re- unit. The unusual natureof the lineshapes observed in all the{MVO}3+ complexes synthesized in this study led us to explore the spin systemin greater detail through NMR simulations, which will be discussed vide infra.The 31P{1H} NMR spectrafor the complexes showcharacteristic singlets, reflecting the equivalent phosphorus environmentsthat arise from the sC symmetry of themolecules. The coordinated phosphine groups of 1 exhibita singlet at δP = −29.8 ppm and the free phosphinearm at δP = −29.4 ppm in a 2:1 integral ratio,indicating two different phosphorus environments within the molecule. 2 exhibits a singlet at δP = −30.6ppm for the coordinated phosphine groups, a very slight lower frequencyshift relative to its unoxidized analogue, and a singlet at δP = +25.3 ppm for the phosphine oxide unit.3Ph in the abovecomplexes is likely precluded by both the strong trans influence of the oxo donor enhancing the lability of the site oppositeto it and the documented preference of this site for hard-type donors.78 Attempts to promote tridentate coordinationof the NP3Ph ligand, including the use of morepolar solvents, elevated temperatures, and increased reaction times,were unsuccessful and either returned 1 or decompositionproducts.Tridentatecoordination of NP2PhOHAr was synthesized accordingto the literature.34 Smith and co-workershave successfully coordinated this ligand in a bidentate mode to anumber of transition metals,80 but tridentate coordinationof this ligand has not been observed prior to this study. This ligand,containing a phenol unit as a coordinating third arm, was envisionedto promote tridentate coordination to rhenium through deprotonationand coordination of the anionic oxygen donor to the coordination site trans to the oxo group. The extraordinary preference ofthis site for anionic oxygen donors is well-documented, and stabilityof complexes containing oxygen at this site is likely promoted throughthe presence of a stabilizing interaction of π symmetry acrossthe [O=Re-OR]2+ fragment.72In order to facilitatetridentate coordination to the metal center, the asymmetric ligandNP2PhOHAr and [ReOCl3(PPh3)2] in toluene,in the absence of base, resulted in formation of the diamagnetic d2 complex [ReOCl3(κ2-NP2PhOHAr)] (3), which could be purifiedand isolated by silica column chromatography. This complex had lowsolubility in chlorinated solvents but reasonable solubility in MeOHand MeCN, most likely due to the presence of the polar free phenolarm. Crystallographic characterization of the species confirmed the cis bidentate coordination of the phosphine groups and theprotonated phenolic arm (Reaction between NPolic arm 2a.2PhOHAr and the more labile Re(V)precursor (NBu4)[ReOCl4] in d3-MeCNwas also conducted and was expectedto proceed without the need for elevated temperatures. Indeed, thisreaction, conducted at room temperature, also produced 3 in very good yield and high purity, as determined by 1H and 31P{1H} NMR spectroscopy. This was consideredadvantageous for the translation of such chemistry to 99Tc, as the complex [TcOCl3(PPh3)2] is unknown, whereas (NBu4)[TcOCl4] can besynthesized readily from [TcO4]− throughthe action of concentrated hydrochloric acid.76The reaction betweenNP2PhOHAr and [ReOCl3(PPh3)2] in the presence of a tertiaryamine base (such as NEt3 or DIPEA) conducted in MeCN at60 °C resulted in the formation of the diamagnetic air-stablecomplex [ReOCl2(NP2PhOAr)] (4). This complex could be purified by silica columnchromatography in CH2Cl2:MeOH and produces adeep yellow color when dissolved in organic solvents. X-ray analysisof single crystals of 4 grown by vapor diffusion of CH2Cl2 with hexane confirmed the tridentate coordinationmode of the  donor ligand. X-ray crystal analysis illustratedthe reciprocal trans configuration of the oxygenatoms, and the boat-like conformation adopted bythe six-membered ring upon coordination of the phosphorus atoms tothe metal center °, close to 180°,reflecting the considerable π interaction across the dioxygenunit.81 Perhaps more telling of the extentof the π interactions in this unit is the C–O–Reangle, which reflects the distortion away from the idealized anglefor sp2 hybridization (120°). Thisangle has a value of 156.8(4)°, a considerable distortion towardlinearity. This distortion toward sp hybridization likely enablesthe 2p orbitals of the oxygen atom to participate in bonding orbitalsencompassing the whole [O=Re–O–Ar]2+ unit. This distortion is likewise present in the synthesized [3+ 2] complexes. VO}3+ complexes synthesized in this study.The reactionbetween NPl center 2b. The O1H NMR spectrum. Whereas in the chair-like conformationof the bidentate complexes, the resonances corresponding to the methylenecarbons of the complex exhibit a highly distorted line shape withsignificant second-order couplings, the tridentate 4 complexinstead exhibits smaller 2JHP coupling constants.The variation in magnitude of this coupling constant inducing changesin the lineshapes has been further explored through NMR simulations. The measuredvalue of 2JHH in the case of 3 wasfound to be 2JHH = 15.0 Hz. In the case of 4, a smaller coupling is removed, one which has induced asmaller splitting compared to the non-decoupled case. The geminalcoupling in 4 was measured as 2JHH = 15.4 Hz. The difference in lineshape between 2JPP’ = 0) clearly suggests the presence of a strong,passive coupling between the magnetically inequivalent cis-configured phosphorus atoms.The lineshapes in this region of the 4 could also besynthesized by initial deprotonationof NP2PhOHAr in MeCN solution using1 equiv of KOtBu. The progress of this deprotonation couldbe monitored visually by a color change from colorless to green. Additionof the solution containing the deprotonated ligand to a solution of[ReOCl3(PPh3)2] and subsequent heatingof the reaction mixture resulted in formation of 4 butin a reduced yield due to the significant formation of a byproduct,the identity of which is likely to be the dioxo Re(V) complex [ReO2Cl(NP2PhOHAr)] (5) ] (5) was identified by the presenceof a molecular ion peak for [M-Cl]+ in the ES mass spectrumat m/z = 724.1180 (expected at m/z = 724.1180). While this does not confirm the presence of a chloridein the coordination sphere, it does strongly suggest a dioxo unitwith a single NP2PhOHAr ligand coordinated.Ar)] (5) 2c. Notab2PhOHAr and (NBu4)[ReOCl4] in the presence of basewas also conducted in the hope of observing formation of 4. However, this reaction generally resulted in a crude mixture ofproducts in which varying proportions of 3, 4, and 5 could be observed by 31P{1H} NMR analysis. A cleaner one-pot variant of this reaction, achievedthrough the addition of a bidentate dioxygen ligand to the reactionmixture, was able to circumvent this issue of multiple product formationand is discussed in the next section.The reaction between NP3 washeated in MeCN at 60 °C overnightin the presence of NEt3, then the identity of the productformed was dependent on the presence of water in the system. Use ofanhydrous solvents resulted in the formation of 4 asthe major species; however, the use of standard grade solvents resultsin the formation of 5, likely due to hydrolysis of the[O=Re-Cl]2+ unit.82 Thiseffect has been observed as even greater in the case of 99Tc complexes and is discussed vide infra.When VN(PNP)}2+  “metal-fragment”is often combined with a “soft” sulfur-based donor inthe bidentate component of mixed-ligand [3 + 2] complexes.43 Conversely, for {MVO}3+ cores, “hard” oxygen donors such as catechols, 1,2-diols,and 1,2-dicarboxylic acids are often preferred.78 In the case of the tridentate scorpionates dithiolateshave been used to form [3 + 2] complexes with the [ReVO]3+ core.84 These ligands havebeen shown to be compatible with β-diketones and diamines, althoughwith only bidentate coordination of the scorpionate exhibited in theselatter cases.86 Papadopoulos and co-workers haveused imino-pyridines as bidentate ligands to form complexes with an/ donor set.62 Pyrazole-derived ligands have been shown to stabilize [3 + 2] complexes withcatechol and ethylene glycol,63 while [3+ 2] complexes of the closely related pyridyl-derived  ligandpublished by Abrahams et al. have been stabilized using an oxalicacid ligand.87 The trio of catechol, ethyleneglycol, and oxalic acid has been used effectively with a number ofdifferent tridentate ligands and were selected as the starting pointfor exploring further ligand substitution chemistry with 4.88 More specifically,a study using catechol as a bidentate ligand alongside a heterofunctionalizedphosphine was conducted by Sigouin et al.89 The coordination behavior of [ReVO]3+ withdppe and catechol was explored, with the catechol only observed asbinding to the metal center in the equatorial plane; the site trans to the oxo group was instead occupied by a halideion. This suggests that the catechol is not expected to compete forbinding to the labile trans site, despite phenolicoxygens being known to have an affinity for this position. A stronglyπ-donating dioxygen ligand may be able to better accommodateπ-acceptor phosphine ligands with which it will be sharing dorbitals in the equatorial plane.The {M4. When 4 was dissolved in MeCN andheated in the presence of one equivalent of catechol and three equivalentsof DIPEA, the complex [ReO(NP2PhOAr)] (6) was formed as the major product and couldbe purified by silica column chromatography in good yield. This air-stablediamagnetic [3 + 2] mixed-ligand complex displayed a single resonancein its 31P{1H} NMR spectrum at δP = −32.9 ppm, a slightly lower frequency from its dichloride 4 analogue, perhaps due to the improved π-donation bythe oxygen donors. Complexes [ReO(NP2PhOAr)] (7) and [ReO(NP2PhOAr)] (8) were also synthesizedby the reaction between 4 and corresponding bidentateoxalate or ethylene glycolate ligand, respectively, in the presenceof a tertiary amine base with CH2Cl2 as thereaction solvent. Monitoring the course of these reactions by 31P{1H} NMR spectroscopy showed that the reactionwith catechol reached completion after 24 h, oxalic acid after 36h, while ethylene glycol took several days before appreciable amountsof the product were formed. The rate of complex formation could beincreased by using MeCN and a higher temperature of 60 °C. Comparedto the catecholate and oxalate analogues, 8 was relativelyunstable in solution, and attempts to obtain single crystals wereunsuccessful. However, the crystal structures of 6 and 7, confirming [3 + 2] complex formation, are depicted in 6, several additionalbidentate ligands, also bearing a 1,2-dihydroxyphenyl group, wereadded to 4 to investigate the impact of other functionalgroups on the formation of the [3 + 2] complexes (2PhOAr)] (9) and [ReO(NP2PhOAr)] (10) were also synthesized by the routeoutlined above and fully characterized. Initial attempts to use 3,4-dihydroxyphenylaceticacid as a bidentate ligand were unsuccessful, potentially due to thecompetitive coordination behavior of the free acid group.Due to the ease ofsynthesis of omplexes 2. 3,4-Di2O complexes (X = O or S) could also besynthesized via one-pot reactions from the [ReOCl3(PPh3)2] and (NBu4)[ReOCl4] precursorsand showed improved yields and purification procedures compared tothe two-step method. Notably, this also indicates a preferential formationof the completed [3 + 2] ligand set relative to any intermediate complexescontaining monodentate ligands such as chloride or triphenylphosphine.Homocomplexes, which would result from coordination of multiple ligandsof the same type, were also not observed in these reactions.44Further [3+ 2] Re-ar-X,O-NPN-acetyl dopamine wassynthesized readily from its free amine counterpart. Such derivativescould be coordinated to the [ReVO(NP2O)]2+ unit under one-pot conditions very readily. Use of the freeamine was avoided to prevent any competitive coordination behaviorand to improve ease of purification. The resultant complex [ReO(NP2PhOAr)] (11) was fullycharacterized by 1H NMR, 31P{1H}NMR, and HR-MS and further illustrates the compatibility of additionalfunctionality on the 1,2-dihydroxyphenyl unit chelating unit ] (12), obtained through this route, contains a coordinated2-hydroxythiophenol ligand and is to our knowledge the first exampleof a {ReVO}3+ metal center stabilized by thisunique combination of donor atoms: /. Due to the breakingof the sC symmetry of the [3 + 2] complexby the coordination of an asymmetric 2-hydroxythiophenol ligand, inwhich sulfur has replaced oxygen in the coordination sphere of themetal, the phosphorus donors are no longer chemically equivalent anda pair of resonances are seen in the 31P{1H}NMR spectrum. Such a pair of doublets were observed with a 2JPP coupling constant value of 2JPP = 15.0 Hz, which could be used to estimate the 2JPP coupling constant used in the NMR simulations for the sC-symmetric {ReVO}3+ complexes.One-pot reactions also enabled access to novel [3 + 2] complexesthat were otherwise difficult to obtain with a two-step reaction procedure.The complex [ReO] (Tc-3), was synthesized by the reaction between (NBu4)[99Tc]-[TcOCl4] and NP2PhOHAr in d3-MeCN at RT in the absenceof a base. The related bis(triphenylphosphine) complex [TcOCl3(PPh3)2] is unknown,46 and its use precluded in the synthesis of Tc-3, despite the favorable reactivity of its Re analogue toward theligands described above. Nevertheless, the reaction using (NBu4)[TcOCl4] proceeded relatively cleanly with Tc-3 formed as the major product.The Tc-3 bearsa strong structural similarity to 3, as evidenced bythe highly similar coupling pattern observed for the methylene protonsin its 1H NMR spectrum in d3-MeCN. 1H NMR and 1H{31P} spectrafor Tc-3 corresponding to the methylene resonances. Thesespectra are suggestive of a AA’BB’XX’ spin systemin which highly similar lineshapes to that of the Re analogue areobserved for the bidentate coordination mode of the ligand. Upon broadband 31P decoupling, only the 2JHH′ geminal coupling between the methylene hydrogens in Tc-3 is observed with a value of 15.2 Hz (compared to 15.9 Hz for 3). The presence of these lineshapes in the 1HNMR spectrum is strongly indicative of a bidentate coordinated phosphineligand. The 31P{1H} spectrum for Tc-3 contains a lone singlet at +27.5 ppm, which is considerably higherfrequency relative to the analogous Re complex 3. Consideringthe similar chemical shifts observed for the methylene hydrogens in 3 and Tc-3, this large shift to higher frequencylikely results from inherent differences between Tc(V) and Re(V),rather than any significant structural dissimilarity.1H NMR spectrum indicated the presence of this speciesby the presence of a singlet resonance in the methylene region ofthe spectrum at δH = +4.28 ppm, and by analogy tothe Re case, this can ascribed to the presence of a {TcVO2}+ species analogous to 5, [TcO2Cl(κ2-NP2PhOHAr)] (Tc-5). Such a species is not expected to exhibita complicated lineshape corresponding to an AA’BB’XX’spin system due to the chemical equivalence of the methylene hydrogens.A singlet at δP = +26.9 ppm is observed in the 31P{1H} NMR spectrum for Tc-5. Consideringthat formation of this species is observed even using a room temperaturesynthesis, it suggests that the hydrolysis pathway to {MVO2}+ from {MVO}3+ forTc in such systems is more facile than in the case of Re, as expectedfrom the rates of substitution reactions on 2nd vs 3rd row transitionmetals.90 Indeed, if elevated temperaturesare employed or a base is added to the reaction mixture for this synthesis,in order to promote the formation of a tridentate complex, analogous to 4, then no such species is observedbut rather the relative amounts of Tc-3 and Tc-5 are altered to favor the latter. This is consistent with the behaviorof Re(V) derivatives in which hydrolysis of 3 to 5 is promoted in the presence of base when sufficient wateris present in the system. Using anhydrous solvents without base favorsthe formation of Tc-3.A second species was formed as a minor product in the samereaction.The Tc-5. Consequently, a one-pot synthetic procedure was attemptedusing catechol. Although this reaction resulted in the formation of Tc-5 as the major product, the presence of another Tc-containingspecies observed in the 1H NMR, indicated by the presenceof a distinctive methylene region lineshape. 6.It is possible that thepresence of a stabilizing bidentate catecholor other dioxygen ligand could favor the formation of a [3 + 2] Tc(V)complex in preference to the hydrolyzed dioxo species such as 2Cl2 alongsidea pure sample of 6 facilitatedchromatographic comparison between the components of the Tc reactionand a pure [3 + 2] complex. A yellow band was observed with Rf = 1.0 in both cases (see the SI), strongly suggesting the presence of isostructural Tc-6 in the crude reaction mixture. Additionally, low-resolutionmass spectrometry also indicated the presence of Tc-6 through observation of a signal at m/z = 728.1 .Thin-layer chromatographyanalysis of this mixture in 5% MeOH:CHTc-6 by silicacolumn chromatography resulted in further decomposition of the product,and appreciable material for further analysis could not be recovered.It is likely that the hydrolysis pathway necessitates rigorous conditionsto promote formation of Tc-6 during reaction and purification,if stable [3 + 2] complexes derived from Tc(V) are to be isolated.Unfortunately, attempts to isolate VO}3+ complexes containing bidentate and tridentate coordinationmodes. A tridentate [ReOCl2(NP2PhOAr)] complex can function as a reactive “metal-fragment”toward further functionalization with oxygen (and sulfur) donors toform [3 + 2] mixed-ligand sets. Control over bidentate and tridentatecoordination can be achieved through the choice of third arm donorof the ligand and the presence of base in the reaction medium. A distinctiveAA’BB’XX’ spin system formed through cis coordination of the diphosphine to the metal centeraids rapid diagnosis of the binding mode through a large change inlineshapes for the 1H NMR resonances corresponding to thebridging methylene groups of the ligand. These lineshapes have beensuccessfully recreated by NMR simulation for a 6-spin system, withthe magnitude of 2JPH coupling constants beinga critical parameter in their appearance and highly second-order lineshapesarising due to the passive 2JPP′ couplingmediated via the metal center.The present study demonstrates that N-centeredphosphine ligandscan be used to prepare air and moisture-stable {Re2PhOAr)]2+ units with well-definedcoordination spheres. Further work in our group is looking at thecoordination chemistry of cysteine residues with this “metal-fragment”.The successful formation of [3+ 2] complexes under one- or two-stepreaction conditions from Re(V) oxo precursors has been shown to becompatible with a range of functionalities on the bidentate unit.The functionalities selected in this study represent a useful startingpoint from which further targeting units could be appended to [ReO(NPVO}3+ is complicated by hydrolysisto {TcVO2}+ units under a range ofconditions. It is anticipated that this may be improved through furtherstudy toward the selection of a bidentate ligand with a rapid tendencyto coordinate to {TcVO}3+ units and stabilizea [3 + 2] mixed-ligand set.The extension of such ligand systems to the formation of [3 + 2]complexes with {Tc2PhXR ligands to the formulation of mixed-ligand[3 + 2] complexes based on mono-imido {MV(NR)}3+  cores, with the potential for greaterstability toward hydrolysis. Likewise, the recurrent appearance ofthe {MVO2}+ core in our syntheticprocedures has led us to consider the use of this related core inthe formulation of heterofunctionalized bis(diphosphine) complexestoward targeted nuclear imaging.Further studies in our group arelooking at the extension of NP


These wide-ranging applications could result in direct or indirect exposure of these NPs to humans and the environment. Accordingly, conflicting levels of toxicity has been associated with these NPs. Therefore, the risk associated with these reports and for TiO2NPs produced using varying methodologies should be measured. This study aimed to investigate the effects of various media on TiO2NP properties  and the effects of TiO2NP exposure on human colorectal adenocarcinoma (Caco-2) epithelial cell viability, inflammatory and cell stress biomarkers and angiogenesis proteome profiles. The NPs increased in size over time in the various media, while zeta potentials were stable. TiO2NPs also induced cell stress biomarkers, which could be attributed to the NPs not being cytotoxic. Consequently, TiO2NP exposure had no effects on the level of inflammatory biomarkers produced by Caco-2. TiO2NPs expressed some anti-angiogenic properties when exposed to the no-observed-adverse-effect level and requires further in-depth investigation.Titanium dioxide nanoparticles (TiO The rutile form has been studied extensively due to its electrical, optical and thermal properties -3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate) (XTT) assay (Sigma-Aldrich). The assay monitors the conversion of the XTT tetrazolium salt to a soluble formazan salt in metabolically active cells . TherefoThe amount of nitrite produced by the Caco-2 cells exposed to the respective nanoparticle concentrations was assessed in the cell culture supernatant as an indication of NO production. This assay is based on the Griess reaction . The amoDouble antibody sandwich enzyme linked immuno-sorbent assays (DAS-ELISA) were run according to the manufacturer’s instructions. Both the IL-6 (Invitrogen) and IL-8 (R & D Systems) assays were run using undiluted supernatants.The cells were harvested after NP exposure using lysis buffer solution . The cells were then scraped with a scrapper after which cells were then sonicated  on ice for short burst for 20 secs and centrifuged at 12.1 rcfs for 1 min. Protein concentration of the cell homogenate was quantified using Bradford reagent. The cell homogenates at 300 µg/mL protein were used to evaluate cell stress biomarkers potentially induced by the nanoparticle.The production of cell stress biomarkers induced by exposing the cells to the nanoparticles was assessed by performing SOD, Phospho-HSP27 and HSP70 ELISAs . The experiments were all performed per the manufacturer’s instructions. All samples were run at a cell homogenate protein concentration of 300 µg/mL. The exposure concentration range selected was 0 and 31.25–500 µg/mL TiO2NPs.2NPs. The concentration selected represented the control and the no observed adverse effect level (NOAEL) (100 µg/mL TiO2NPs). The membranes were subjected to an ultra-sensitive chromogenic 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate  to show sample–antibody complexes labelled with streptavidin-horseradish peroxidase (HRP). Pictures were taken of the membranes after substrate exposure.The angiogenesis proteome profiler (R & D Systems) contained 4 membranes, each spotted in duplicate with 55 different angiogenesis antibodies. The assay was performed per the manufacturer’s instructions. This was a qualitative assay that indicates the relative expression of the angiogenesis markers. The concentrations selected were 0 and 100 µg/mL TiOp < 0.01 deemed significant.All experiments were performed in triplicate and the data calculated using Microsoft Excel. The data are represented as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) using SigmaPlot 12.0  was used to determine statistical differences, with 2NPs  card no.: 21-1272) and rutile (Power Diffraction File (PDF) card no.: 21-1276). The (XRD) pattern of TiO2NPs , (110), (004), (200), (105), and (211) which are peculiar to TiO2. The (XRD) also showed the different peaks pattern of TiO2NPs  from 297 ± 43.55 to 647.53 ± 56.18 nm (14 days) (The NPs in the presence of a 150 mM NaCl solution indicated a steady increase in size across the period assessed. The hydrodynamic size over the 2-week incubation period significantly increased (14 days) . However14 days) .2NPs were incubated in the presence of PBS, it was noted that size did not change across the 24 h period. It was also notable that the size of the NPs at 0 h were the same for PBS and DMEM in the absence and presence of serum (787.33 ± 65.24 nm). Subsequently, TiO2NPs in PBS for 14 days exhibited a significant increase (p < 0.014) in size from 787.33 ± 65.24 (0 h) to 1055.8 ± 86.95 nm (14 days) (p < 0.001) from −26.03 ± 2.29 to −16.05 ± 2.77 mV for PBS over the 24 h period. It then decreased to −27.87 ± 1.97 mV at day 7 and then another significant increase (p < 0.001) at day 14 with a charge of −16.48 ± 1.29 mV. Thus, 0 h and 7 days had similar surface charges and 24 h and day 14 have comparable zeta potentials for PBS (When the TiO14 days) . Nonethe for PBS .2NPs in the presence of DMEM without serum displayed a notable increase (p < 0.014) in size from 0 h to 7 days, with sizes ranging from 787.33 ± 65.24 to 1442.35 ± 491.99 nm respectively (p < 0.014) from −14.1 ± 0.87 to −8.29 ± 1.9 mV between 0 h and day 7 respectively  from 787.33 ± 65.24 to 1121.26 ± 216.6 nm at day 7 and another subsequent increase (p < 0.003) to 1821.8 ± 450.9 nm after 14 days (2NPs notably increased (p < 0.014) at day 14 (−16.45 ± 1.1 mV) compared to incubation period 0 h to 7 days  in NO released from the cells at concentrations ≥62.5 μg/mL TiO2NPs  at concentrations ≥31.25 μg/mL TiO2NPs. Phospho-HSP-27 levels doubled at 31.25 μg/mL TiO2NPs and tripled at 125 μg/mL TiO2NPs, compared to the 0 μg/mL TiO2NP control, and then increased to 7-fold at 250 and 500 μg/mL TiO2NPs (The cell stress biomarkers phospho-HSP-27 and HSP-70 notably increased ( TiO2NPs a. Howeve TiO2NPs b. HSP-70p < 0.001) at 250 μg/mL TiO2NPs, with an approximate 1.8-fold increase in its production in comparison to the control  and NOAEL (100 μg/mL TiO2NPs) supernatants assayed for potential angiogenic biomarkers revealed the same proteins except persephin, which was only evident on the control membrane  and L-alanine , was used to evaluate how complete DMEM impacted the size and surface charge of TiO2NP. The authors of the aforementioned study noted an increase in size when placed into cell culture media for 24 h, with hydrodynamic size being 1842.6 ± 263 nm and 1296 ± 662 nm for gly-TiO2NP and ala-TiO2NP, after an initial size of 85.5 and 72.8 nm respectively [2NP and ala-TiO2NP was −7.9 ± 0.4 and −8.2 ± 0.2 mV respectively. These results imply that the reducing agent does not impact size once in culture medium as can be seen when comparing the data found in this study.The initial size of TiO65.24 nm  which wa ± 69 nm . However ± 69 nm ,30. In c ± 69 nm . This waias used . A studyectively . The sur2NPs were not cytotoxic to the cells at the assessed concentrations  elucidated that TiO2NPs activates IL-8 and IL-8-related pathways [2NPs resulted in an increase of IL-8 levels after a 24 h exposure period [2NPs exposure found no change in their expression levels, which is in agreeance with our data. However, they did find an increase in other inflammatory cytokines such as IL-4, IL-12, TNF-α and IFNγ [The TiO or IL-8 b,c. As ccytokine ,44. The pathways . This wae period . This tre period . An in vand IFNγ .2NPs  [The angiogenesis proteome profile revealed very little differences between the control and 100 μg/mL TiO2NPs . There w process ,48,49,50ogenesis . The uprogenesis ,53. Thesr (VEGF) . This har (VEGF) . However2NPs and could be a potential candidate for use in cancer therapy. This, with the lack of inflammatory cytokine production and cytotoxicity makes this NP a good candidate. However, further comprehensive investigations are required as the exposure period, dosage and size of the NP could greatly impact the above-mentioned factors.Caco-2 cells express anti-angiogenic markers  upon exposure to 100 μg/mL TiO


Aspergillus fumigatus is a devastating opportunistic fungal pathogen causing hundreds of thousands of deaths every year. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that converts glucose-6-phosphate to fructose-6-phosphate, a key precursor of fungal cell wall biosynthesis. Here, we demonstrate that the growth of A. fumigatus is repressed by the deletion of pgi, which can be rescued by glucose and fructose supplementation in a 1:10 ratio. Even under these optimized growth conditions, the Δpgi mutant exhibits severe cell wall defects, retarded development, and attenuated virulence in Caenorhabditis elegans and Galleria mellonella infection models. To facilitate exploitation of A. fumigatus PGI as an antifungal target, we determined its crystal structure, revealing potential avenues for developing inhibitors, which could potentially be used as adjunctive therapy in combination with other systemic antifungals. Aspergillus fumigatus is a fungus that is widely distributed in the environment through dissemination of small, airborne conidia  catalyzes the reversible conversion of fructose-6-phosphate (Fru6P) to glucose-6-phosphate (Glc6P). Therefore, PGI is involved in glycolysis and the pentose phosphate pathway (PPP). Furthermore, Glc6P can be converted to UDP-Glc, whereas Fru6P can be converted to UDP-GlcNAc and GDP-Man. These three nucleotide sugars are the main precursor molecules for all cell wall carbohydrates, including chitin (UDP-GlcNAc), galactomannan (GDP-Man), and glucan (UDP-Glc) . As such sources . Our rec. flavus . Apart fynthesis . Bacilluglycerol . PGI def pathway . In Crypsistance . These sin vivo using chemical tools, small molecule inhibitors of PGI, mostly pseudo substrates such as 6-phosphogluconic acid, sorbitol-6-phosphate, ribitol-5-phosphate, erythrose-4-phosphate and 5-phosphate-d-arabinonate, have been extensively studied (–in vivo. Limited numbers of nonphosphate inhibitors of PGIs have been reported (in vivo are still lacking.To target PGI  studied –23. The reported . As suchA. fumigatus PGI (AfPGI) using multidisciplinary approaches, including genetics, structural biology, and biochemistry. This study shows that PGI is required for A. fumigatus survival and maintenance of metabolic homeostasis and possesses a potentially exploitable difference near the active site compared to the human orthologue. Taken together, this study provides a genetic and structural basis for the development of fungal PGI inhibitors.Here, we have characterized A. fumigatus A1163 genome database using S. cerevisiae PGI (GenBank accession No. AAA34862.1) revealed a single putative phosphoglucose isomerase (EDP54506.1) encoded by the pgi gene (AFUB_025630). A. fumigatuspgi is 1,755 bp in length and contains two exons and one intron. The AfPGI protein contains 553 amino acids with 71, 57, and 56% identity to Candida albicans PGI , C. neoformans PGI , and Homo sapiens PGI , respectively.A BLASTp search of the AfPGI possesses phosphoglucose isomerase activity, we overexpressed full-length AfPGI with an N-terminal glutathione S-transferase (GST) fusion tag in Escherichia coli. After purification using glutathione beads, GST tag cleavage by PreScission Protease and size exclusion chromatography, pure AfPGI (9 mg/L) was obtained. A coupled assay with Glc6P dehydrogenase (G6PDH) was used to determine kinetic parameters of the catalysis of Fru6P to Glc6P. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to evaluate the reverse reaction. The mK value of AfPGI for Fru6P is 280 ± 19 μM . Δpgi clones were obtained and confirmed by PCR with multiple pairs of primers and Southern blotting (see pgi mutant was plated on 5-fluoroorotic acid (5-FOA) to obtain the Δpgi ΔpgrG strain. The revertant construct with AfpyrG inserted after the pgi gene was transformed into the Δpgi ΔpgrG protoplasts. PCR and Southern blotting were applied for the wild-type (WT), Δpgi, Δpgi ΔpgrG, and RT strains  of the Δpgi mutant in A. flavus  and product (Fru6P), suggesting that the enzyme activity of PGI is important for A. fumigatus. Taken together, these results demonstrate that pgi is important for A. fumigatus survival. sources , 16, 26.tose see , which ctose see , but thetose see . These r. flavus . Interese growth . The othe growth . These o10.1128/mbio.01426-22.1FIG S1pgi mutant. (A) Diagram illustrating strategies for constructing Δpgi mutant and RT strains. (B) Southern blot analysis using total genomic DNA digested with BglII and NcoI and probed with downstream fragment of pgi and AnpyrG. The Δpgi ΔpgrG strain was simply labeled as Δ′. (C) Confirmation of the mutant and revertant strains by PCR using the primer pairs P1/P2, P3/P4, P5/P6, P7/P8, and P9/P10, as shown in the figure. The Δpgi ΔpgrG strain was labeled as Δ′. Download FIG S1, PDF file, 0.1 MB.Generation of ΔCopyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01426-22.2FIG S2pgi mutant in different carbon sources. (A) 102 to 105 conidia of the WT, Δpgi, and RT strains were inoculated onto MM medium with combinations of (i) 1% Glc with 1 to 10% Fru or (ii) 1% Fru with 1 to 10% Glc. Plate photos were taken after cultivation at 37°C for 48 h. (B) Sole carbon sources such as 1% galactose , 1% maltose , 1% mannose (Man), 1% glucosamine (GlcN), 1% N-acetylglucosamine (GlcNAc), or 1% glycerol were used for growth tests. Plate photos were taken after cultivation at 37°C for 48 h. Download FIG S2, PDF file, 0.2 MB.Growth of the ΔCopyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01426-22.7TABLE S1Table S1, PDF file, 0.1 MB.Primers used in this study. Download Copyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the AfPGI, a minimal medium containing 1% Fru and 0.1% Glc (MMFG) was used for the subsequent experiments to support growth of the mutant. Solid MMFG plates were used to evaluate daily expansion of colony diameter. The Δpgi mutant displayed significantly reduced colony growth compared to the WT or RT strain  in A. nidulans , the cell membrane perturbing agent SDS, the osmotic agent sorbitol, and antifungal drugs affecting the plasma membrane  and cell wall (caspofungin [CAS]). As shown in pgi mutant was sensitive to low concentrations of CR and CAS, suggesting that β-glucan synthesis may be affected in the Δpgi mutant. Analysis of the cell wall components revealed that the α/β-glucan content of the mutant was reduced by 70 to 79%, whereas the content of chitin was increased by 16% together with a 14-fold increase in glycoprotein content , deletion of  content . Althoug content . Collect10.1128/mbio.01426-22.9TABLE S3Table S3, PDF file, 0.1 MB.Monosaccharides and intracellular sugars in the three strains. Download Copyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pgi strain, we next used a high-pressure liquid chromatography (HPLC) approach to trace the levels of intracellular nucleotide sugars , the precursors of cell wall polysaccharides, after growth in MMFG media , while the amount of UDP-GlcNAc was increased by 185% , and the GDP-Man amount was increased by 225%   in A. fumigatus  . These rumigatus .pgi mutant were reduced by 90%  and 70% , respectively , Man6P was increased by 295% , and fructose-1,6-biphosphate (FBP) was increased by 65%  (AfPGI is a key enzyme in maintaining the homeostasis of intracellular phosphate sugars (Phosphate sugars are the building blocks for nucleotide sugar biosynthesis . To dissectively . In the  mutant) . These de sugars .pgi deletion on virulence of A. fumigatus, a recently developed Caenorhabditis elegans-based A. fumigatus infection model was applied (glp-4(bn2); sek-1(km4) worms infected by the WT, Δpgi mutant, and RT strains at 24, 48, and 72 h were recorded and plotted as Kaplan-Meier survival curves. As shown in pgi mutant exhibited a significantly attenuated virulence (P < 0.0001) with a survival rate of 63% ± 4% at 72 h (P < 0.0001) than the rates in the WT and RT strains  as a se of 18.0 . AfPGI w5.7/18.3 .AfPGI monomer in the asymmetric unit contains a large domain and a small domain (HsPGI (PDB code 1IRI [2 buried surface area). During refinement, an unbiased oF-cF density was observed in the interface between two protein monomers and resolved as a Glc6P molecule , with amolecule . The phomolecule . Ser165 molecule . Sequencecule 29–, 34, 35.ecule 29–, 37. Thee chains –40. Strite group . Previoute group . For inste group . By dockoups see , which ioups see , suggest10.1128/mbio.01426-22.3FIG S3AfPGI. (A) Structure of the neutral bisphosphate mimic. (B) Predicted binding mode of the neutral bisphosphate mimic (green sticks) in the AfPGI active site (cyan). Prediction was carried out by AutoDock using the AfPGI-Glc-6P structure as the macromolecule for docking. Representative binding mode of the neutral bisphosphate mimic. Orange dots indicate hydrogen bonds. Download FIG S3, PDF file, 0.3 MB.Prediction of the binding mode of a neutral bisphosphate mimic in the active site of Copyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the HsPGI is essential for vertebrate embryo development  between Ala221 and Gln523 (corresponding to Glu526 in HsPGI) (see HsPGI) is placed into this gap. The intrinsic flexibility of lysine implies that the Lys520 sidechain is likely to move away from the gap, in agreement with the fact that there is no electron density around the Lys520 sidechain in the structure of apo AfPGI. This intrinsic mobility of Lys520 could leave an empty gap between Ala221 and Gln523. In contrast, for the counterparts of AfPGI Ala221/Gln523 in HsPGI (Gln216/Gln526), a hydrogen bond between Gln216 and Glu526, and a salt bridge between Glu526 and Lys523 occupy this gap.elopment , 44, andom Glc6P , is subsatum see . In the PGI) see . As such10.1128/mbio.01426-22.4FIG S4AfPGI. Sequences are from GenBank with the codes EDP54506.1 (A. fumigatus), XP_713513.1 , XP_018229466.1 (Pneumocystis jirovecii), KEY64212.1 (Stachybotrys chartarum), QSS62647.1 (Histoplasma capsulatum), XP_569228.1 (Cryptococcus neoformans), and NP_000166.2 (Homo sapiens). Download FIG S4, PDF file, 0.1 MB.Partial sequence alignment of PGIs. Red triangle indicates Ala221 in Copyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01426-22.5FIG S5AfPGI and HsPGI. (A) Structure of AfPGI in complex with Glc6P (orange sticks). (B) The apo structure of AfPGI. Glc6P (orange sticks) was placed into the active site by superimposing the structure of apo AfPGI onto that of AfPGI-Glc6P complex. The structure of HsPGI in complex with erythose-4-phosphate (PDB code 1IRI). Download FIG S5, PDF file, 0.5 MB.Structures of Copyright © 2022 Zhou et al.2022Zhou et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the AfPGI. A kinetic assay shows that the enzyme activity of Ala221Gln is the same as that of wild-type AfPGI  to Ala221, suggesting that the gateway between the active site and Ala221 is ligandable  and minimal medium (MM)  fructose was supplemented for the Δpgi mutant. Conidia were collected with 0.2% (vol/vol) Tween 20 in double-distilled H2O. Mycelia were incubated in a liquid medium at 37°C for 48 h with shaking at 200 rpm and then washed thoroughly by distilled water, flash-frozen in liquid nitrogen, and stored at –80°C. Whenever required, the frozen mycelia were ground by mortar and pestle for DNA, RNA, and protein extraction.The ium (MM) , and 5 mpgi mutant and revertant strains were constructed by homologous recombination. To generate the Δpgi mutant, the upstream flanking region before the pgi start codon and the downstream flanking region after the stop codon were amplified with two pairs of primers—P11/P12 and P13/P14—and cloned into the modified PMD20T plasmid, which was digested by the FseI-PacI and AscI-NotI, respectively. The fragment neo-pyrG-neo (8.6 kb) was obtained from pCDA14 plasmid with HpaI digestion and cloned onto SmaI-digested PMD20T-up-down to generate the PMD20T-pgi. After linearizing via AscI and PacI sites, the pgi deletion fragment was transformed into KU80ΔpyrG protoplasts. Transformants were screened on MM supplemented with 1 M sorbitol and 1% (wt/vol) fructose, and then PCR was confirmed using the primer pairs P1/P2, P3/P4, P5/P6, and P7/P8.The Δpgi gene  was amplified with the primers P15/P16, the fragment of AfpyrG  was amplified by the primers P17/P18, and the downstream flanking region (800 bp) was amplified by the primers P19/P20. These three fragments were purified and assembled in a pCE-Zero vector. The Δpgi mutant was inoculated on CMU medium containing 1% (wt/vol) fructose, 0.1% (wt/vol) glucose, and 1 mg mL−1 5-fluoroorotic acid to generate the Δpgi ΔpyrG mutant. The assembled fragment amplified by primers P15/P20 was transformed into Δpgi ΔpyrG protoplasts. All constructed strains were first confirmed by PCR and then by Southern blotting, in which NcoI and BglII were used for genomic DNA digestion; the AnpyrG and downstream regions were used as probes.To construct a revertant strain, the fragment of the upstream flanking region and integral 5 were spotted on solid MM supplemented with different concentrations of glucose, fructose, gluconate, and other carbon sources to test the growth of the WT, Δpgi, and RT strains. Radial growth rates were measured by spotting 2 × 105 conidia at the center of the plate and recording the colony diameter every 12 h. After 7 days, conidia were collected and counted by using a hemocytometer. Germination in liquid MMFG was conducted by placing coverslips at the bottom of the plate and inspecting them under the microscope at a specific time. The serial diluted conidia starting from 105 were spotted on MMFG containing the following compounds: 50 μg mL−1 SDS, 50 μg mL−1 Congo red, 100 μg mL−1 Calcofluor White, 1.2 M sorbitol, 2 μg mL−1 itraconazole, 0.1 μg mL−1 voriconazole, 256 μg mL−1 fluconazole, 2 μg mL−1 caspofungin, and 1 μg mL−1 amphotericin B. All of these plates were cultured at 37°C for 48 h and photographed.Serially diluted conidia starting from 108 conidia were inoculated in 100 mL of liquid MMFG, followed by incubation at 37°C with shaking at 200 rpm for 48 h. Mycelia were collected, rinsed with deionized water, and frozen at –80°C. Cell wall extraction, polysaccharide hydrolysis, and quantification were conducted as described previously /pLysS and cultured in Luria-Bertani (LB) medium supplemented with 0.1 mg mL−1 ampicillin at 37°C. Then, 10 mL of overnight culture was inoculated into 1 L of LB medium with 0.1 mg mL−1 ampicillin and grown until it reached an optical density at 600 nm of 0.8. Protein expression was induced by 250 μM isopropyl-β-d-thiogalactopyranoside (IPTG), cultured at 17°C for 36 h, and harvested by centrifugation at 3,500 rpm and 4°C for 30 min. The cells were resuspended in a 25 mL of buffer  and disrupted using a sonicator on ice. After centrifugation at 12,000 rpm for 30 min at 4°C, the supernatant was incubated with preequilibrated glutathione beads on a rotating platform at 4°C for 2 h. The bound fusion protein was cleaved by precision protease to remove the GST tag at 4°C for 2 h. The eluted solution was concentrated to 2 mL and separated by using a Superdex 75 column (Amersham Bioscience) at a flow rate of 1 mL min−1. The collected protein was confirmed by 10% SDS-PAGE, and pure protein was concentrated to 10 mg mL−1 using a 10-kDa cutoff concentrator.The AfPGI for the reverse reaction was determined by coupling with glucose-6-phosphate dehydrogenase (G6PDH) in a reaction mixture containing 50 mM HEPES (pH 7.5), 2.5 mM MgCl2, 0.5 mM NADP+, 1 mM EDTA, 0.5 U of G6PDH, and Fru6P in a final volume of 0.2 mL and incubated at 30°C for 5 min. The concentrations of Fru6P used were 0.1, 0.3, 0.5, 0.75, 1, 1.5, 2.5, and 5 mM. NADPH production was monitored by determining the emission at 460 nm and excitation at 340 nm. One unit of enzyme is defined as 1 mM NADP+ reduced per minute under the experimental conditions.The activity of AfPGI for the forward reaction was directly determined by HPAEC-PAD. In total, 0.2-mL reaction mixtures in 50 mM HEPES (pH 7.5), 2.5 mM MgCl2, 1 mM EDTA, and Glc6P were prepared, followed by incubation at 30°C for 20 min and then 100°C for 10 min to inactivate the enzymes. The concentrations of Glc6P used were 0.1, 0.3, 0.6, 1, 2, 5, 7.5, and 15 mM. Two times the volume of ethanol then was used to precipitate proteins at –20°C for 1 h. After centrifugation at 12,000 rpm for 10 min, the samples were loaded onto a CarboPac PA-1 column for measurement using the same program used for sugar phosphates. The results are presented as the means ± the standard deviations (SD) from three determinations.The activity of AfPGI was incubated with or without 5 mM Glc6P for 2 h on ice before setting up crystal trays using sitting-drop vapor diffusion. Crystals appeared after 2 days in 0.1 M Na-HEPES (pH 7.5)–1.4 M tri-sodium citrate. Using a mixture of 85% mother liquor and 15% glycerol as cryoprotectant, the crystals were flash-frozen in liquid nitrogen. For crystals obtained with Glc6P, X-ray diffraction data were collected at the European Synchrotron Radiation Facility  at 100K and autoprocessed using XDS software (1GZD) as the search model. Manual model building/refinement was applied using Coot (AfPGI-Glc6P complex structure as the search model. Refinement was carried out using PHENIX (Finally, 10 mg/mL of software . The string Coot , REFMAC5ing Coot , and othing Coot . For crying Coot . The phag PHENIX . The fing PHENIX .AfPGI were deposited in the Protein Data Bank under accession codes 7OYL and 7U34.The atomic coordinates and structure factors of