A function of PG lytic transglycosylases MltA and MltD as “space makers” was proposed through multiple gene deletions. These findings unveil novel roles for NlpI-Prc in the regulation of both PG synthesis and degradation, shedding light on the previously undiscovered function of lytic transglycosylases as “space makers” in PG expansion.Balancing peptidoglycan (PG) synthesis and degradation with precision is essential for bacterial growth, yet our comprehension of this intricate process remains limited. The NlpI-Prc proteolytic complex plays a crucial but poorly understood role in the regulation of multiple enzymes involved in PG metabolism. In this paper, through fluorescent D-amino acid 7-hydroxycoumarincarbonylamino-D-alanine (HADA) labeling and immunolabeling assays, we have demonstrated that the NlpI-Prc complex regulates the activity of PG transpeptidases and subcellular localization of PBP3 under certain growth conditions. PBP7 (a PG hydrolase) and MltD (a lytic transglycosylase) were confirmed to be negatively regulated by the NlpI-Prc complex by an in vivo degradation assay. The endopeptidases, MepS, MepM, and MepH, have consistently been demonstrated as redundantly essential “space makers” for nascent PG insertion. However, we observed that the absence of NlpI-Prc complex can alleviate the lethality of the  N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) glycan strands crosslinked by short peptides [Peptidoglycan (PG), a net-like polymer provides structural support and protects bacterial cells from osmotic pressure . The PG peptides . In Grampeptides . Howeverpeptides ,4.For the synthesis of new PG, glycosyltransferases (GTases) are required to polymerize the glycan strands, while transpeptidases (TPases) are responsible for establishing D-Ala-Dap (4-3) or Dap-Dap (3-3) crosslinks . Class AE. coli, hydrolytic endopeptidases, carboxypeptidases, amidases, and lytic transglycosylases are involved in peptidoglycan metabolism [E. coli, eight hydrolytic enzymes exhibit endopeptidase activity. Among these, endopeptidases MepM, MepS, and MepH were found to be redundantly essential for peptidoglycan expansion and tightly regulated during cell growth [The insertion of new peptidoglycan units into the existing layer is essential for PG expansion and bacterial integrity. Due to the net-like structure of PG, hydrolytic enzymes have been predicted to be essential for the insertion of nascent PG ,22,23. Itabolism . In 2012tabolism . Endopepl growth . These tl growth . MeanwhiThe periplasmic protease Prc and the lipoprotein NlpI are involved in the negative regulation of endopeptidases, including MepS and MepH ,28. NlpIE. coli encodes eight lytic transglycosylases, including six outer membrane-bound lytic transglycosylases (LTs) designated MltA to MltF, and a soluble periplasmic LT called Slt70 [Lytic transglycosylases have a crucial function in cleaving the glycan strands within peptidoglycan. The genome of ed Slt70 ,37,38,39ed Slt70 . Previoued Slt70 ,31,40. Ied Slt70 . In theonlpI strain cultured in LB medium without NaCl at 42 °C. In addition, new substrates (PBP7 and MltD) for the NlpI-Prc proteolytic complex were confirmed. A novel function of lytic transglycosylases as “space makers” in PG expansion is proposed in this work. These findings highlight the importance of the regulatory role of NlpI-Prc in PG synthesis and degradation and reveal the “space-makers” function of lytic transglycosylases.The delicate balance between PG synthesis and its degradation is crucial for bacterial growth. The NlpI-Prc proteolytic complex plays an important but less well-known role in this process ,42,43. IE. coli. Consistent with previous findings [E. coli leads to cell filamentation in LB medium without NaCl at 42 °C  can be incorporated into PG by transpeptidases and used as a tool for the assessment of new PG synthesis ,48,49. A reduced c,d. The prc cultured in LB medium without NaCl at 42 °C did not decrease but showed rather an increase . The abundance changes in existing proteins in cells over time can be tracked by immunoblot. After addition of 400 μg/mL spec, samples were collected at indicated time points and the abundance changes in PBP7 in different strains were analysed by immunoblot.As shown in nlpI strains, a clear decrease in the amount of PBP7 was observed over time within an hour after the adding of spec  was absent, the mepH, mltA, or mltD genes were deleted in the ΔnlpI prc mutant background. As shown in nlpI prc background did not exhibit increased sensitivity to EDTA.Knocking out  to EDTA a. ImplyiPtrc promotor) and the cleavable DsbA signal sequence (Dsbass), respectively. The fused protein is transported into the periplasm by the cleavable Dsbass [prc mutant  between the WT and ΔnlpI prc mepS strains could be attributed to the degradation activity of NlpI-Prc in WT. However, it is important to consider that the differential abundance of HA-MltD between the WT and ΔnlpI prc mepS strains could also be influenced by differences in the expression levels of the HA-mltD gene.The pXWL058 plasmid expressing HA-MltD was introduced into both WT and ΔnlpI prc mepS strain is much slower compared to that in WT  in the ΔmepS strain. The expression of wild-type MltD is under control of the same IPTG-inducible promotor (Ptrc promotor). As shown in mepS mutant. This suggests that the function of MepS can be substituted by the overexpression of MltD, further supporting the hypothesis that MltD is another substrate of the NlpI-Prc proteolytic system.The ΔPG expansion requires both PG synthesis and PG hydrolysis. One of the functions of hydrolysis is to create enough space for the insertion of nascent PG. In Gram-positive and Gram-negative bacteria, approximately 40–50% of the PG is degraded per generation by hydrolases. In order to prevent bacterial lysis due to excessive degradation of PG, the synthesis and degradation of PG during bacterial PG expansion are tightly regulated. The NlpI-Prc complex has been reported to be involved in this crucial regulatory process. However, the specific mechanisms by which the NlpI-Prc complex mediates PG synthesis enzymes and PG degradation enzymes are still largely unknown.prc is unable to grow under the same conditions [nlpI, and Δprc cells. As shown in nlpI and Δprc mutants do not exhibit a noticeable decrease compared to wild-type cells, implying the growth defects in ΔnlpI and Δprc mutants are not caused by the absence of some PBPs.The absence of NlpI results in cell filamentation when cultured in hypotonic media at high temperatures . Δprc isnditions . NlpI prnditions . Suggestnditions ,17,54. InlpI mutant cultured in LB medium without NaCl at 42 °C exhibited filamentous growth, that indicates the divisome is dysfunctional , lytic transglycosylases  ,28,30,31nlpI and Δprc are affected. The dysfunctional divisome in the ΔnlpI mutant might is the reason for its filamentous growth. Furthermore, delocalization of PBP3 in the ΔnlpI mutant has been confirmed, the mechanisms through which NlpI influences PBP3 assembly within the divisome require further investigation. The cessation of growth observed in the Δprc mutant is likely cause by multiple factors. The absence of MepS in the Δprc mutant relieved partly its growth defect. The growth stagnation observed in the Δprc mutant may be attributed to the imbalance between PG synthesis and hydrolysis. In addition, a novel function of the NlpI-Prc complex in mediation growth between the outer membrane and peptidoglycan was recently proposed [The activity and subcellular localization changes in some PG transpeptidases in the Δproposed . The NlpE. coli strains used in this work are listed in E. coli strains used in this work are derivatives of BW25113. Strains were cultured in LB medium , 5 g yeast extract , and 10 g NaCl  per litre at 37 °C or in LB medium without NaCl at 42 °C. Strains were cultured in minimal glucose (GB4) medium , 2.95 g KH2PO4 , 1.05 g (NH4)2SO4 , 0.10 g MgSO4·7H2O , 0.28 mg FeSO4·7H2O , 7.1 mg Ca (NO3)2·4H2O , 4 mg thiamine , 2 mg uracil , 2 mg lysine , 2 mg thymine , and 0.5% glucose , per litre, pH 7.0) at 28 °C. When necessary, the following antibiotics were introduced to the medium: 25 µg/mL chloramphenicol , 50 µg/mL kanamycin , 10 µg/mL tetracycline , 400 μg/mL spectinomycin , and 100 µg/mL ampicillin .E. coli deletion strains were constructed by λ-Red recombination as described previously [E. coli genome at the regions of homology. The red recombination protein was induced with 0.2% L-arabinose . Recombinants were selected on LB plates containing 25 µg/mL chloramphenicol or 50 µg/mL kanamycin. If required, the resistance gene in the genome were removed by temperature-sensitive plasmid pCP 20. For plasmids construction, all primers used in this work are listed in the eviously . The pri600 of ≈0.3. Cells were collected by centrifugation (8000× g for 2 min at room temperature). After washing with 1 mL PBS, the pellet was resuspended in 50 µL PBS containing 5 µg/mL Bocillin-FL  and incubated for 10 min. The pellets were washed 2 times with 100 µL PBS and then resuspended in 100 µL PBS to which 20 µL 5X Protein Loading Buffer  at pH 8.3, 10% SDS , 500 mM DTT  and 50% Glycerol from Biosolve Chimie, Dieuze, France) was added. The samples were heated for 10 min at 99 °C to denature the proteins and 10 µL was loaded on a 10% SDS-PAGE gel. The gel was scanned using a LICOR Odyssey M Imager  at 520 nm.The Bocillin-binding assay was carried out as described previously and modifications were made to the details . Strainsg for 2 min and then resuspended in pre-warmed LB medium with and without NaCl, supplemented with 250 µM fluorescent D-amino acid HADA , for a 10-min incubation at 37 °C and 42 °C. Cells were collected by centrifugation (8000× g for 2 min). The pellets were fixed in 70% ethanol  for 10 min. After washing 2 times with PBS, samples were immobilized on an object glass slide coated with 1% agarose and imaged with an Olympus BX-60  equipped with a Hamamatsu ORCAFlash-4.0LT CMOS camera  fluorescence microscope through a 100×/N.A. 1.35 oil objective. The filter used was U-MWU  longpass . Images were taken using the program ImageJ 1.53f  with MicroManager 1.4.22 .A modified protocol of HADA labelling was carried out . The strThe strains used for immunolabeling were cultured in GB4 medium to steady state at 28 °C and fixed by 2.8% formaldehyde  and 0.004% glutaraldehyde  for 15 min. The immunolabeling experiments were carried out as described before . A rabbiE. coli strains to EDTA, cells were cultured overnight in LB medium at 37 °C. Cells were serially 10-fold diluted from 10−1 to 10−6 before loading 2 μL samples onto LB agar and LB agar containing 1 mM EDTA . The expression of wild-type MltD from pAG003 was induced with 20 or 50 µM isopropyl ß-D-1-thiogalactopyranoside . The LB agars were incubated overnight at 30 °C.To examine the sensitivity of Samples were separated by SDS-PAGE and transferred onto nitrocellulose  in a semi-dry transfer manner as described previously . The memhttp://imagej.nih.gov/ij/, accessed on 25 October 2020), were merged into hyperstacks and linked to the Coli-Inspector project file, running in conjunction with the ObjectJ-1.05n plugin . Each cell in the images can be delineated, and background fluorescence can be subtracted before analysis. Image analysis was carried out as previously described [The phase-contrast and fluorescence images, captured using the ImageJ 1.53f program . Statistical significance was indicated as follows: * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001. p-values less than 0.05 were considered statistically significant.For the HADA labelling and in vivo degradation assay of PBP7, The NlpI-Prc complex regulates the activity of PG transpeptidases and subcellular localization of PBP3. The NlpI-Prc complex negatively regulates PBP7 and MltD. The absence of NlpI-Prc complex can alleviate the lethality of the mepS mepM mepH mutant. Based on multiple gene deletions, we propose a function of the PG lytic transglycosylases MltA and MltD as “space makers”. It is clear that NlpI-Prc functions as a central hub for the regulation of the availability of a multitude of proteins involved in PG synthesis and degradation. The exact nature of how it functions requires further investigation.


While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity.The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2–8% of irinotecan can be transformed enzymatically in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models.Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 , which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38–5 and SPESN38–8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and 0–∞ of 0.0548 and 4.5007 (nmol × h/mL) for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 82:1. However, PK of SPESN38–5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0–∞ of 18.80 and 27.78 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.48:1. Antitumor efficacy of SPESN38–5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38–5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38–8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38–8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX’s insensitivity to SK-LMS-1 with high toxicity.The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38–5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38–8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38–5 by PO at 250 mg/kg gave mouse plasma AUCSPESN38 complexes provide a water soluble SN38 formulation. SPESN38–5 and SPESN38–8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX. Chromatographic separation was achieved using a Waters CORTECS C18 + LC column  maintained at 50 °C and sample elution carried out at flow rate of 300 μL/min with a biphasic gradient (water with 0.1% acetic acid and acetonitrile with 0.1% acetic acid). SN-38 and SN-38G were detected by multiple reaction monitoring (MRM) using an AB SCIEX 5500 mass spectrometer with an electrospray ionization source in positive ion mode controlled by AB SCIEX Analyst® software, version 1.6.2. All sample results were obtained within one analytical run. Samples above the calibration range were diluted to be below the point of saturation of the detector.For PK study, mouse plasma samples were analyzed for SN-38 and SN-38G by LC-MS/MS using a previously described method  over themax), Area Under the plasma Concentration-time curve (AUC), elimination half-life (t1/2), apparent clearance (CL/F), and clearance (CL). AUC values were calculated using the linear-up log-down method  was performed utilizing mouse plasma concentrations of SN38 and SN38-G that were obtained by LC-MS/MS. Plasma samples that were included in the NCA were collected at t = 0, 1, 2, 4, 8, 12, and 24 h post-dose for PO route and at t = 0, 0.083, 1, 2, 4, 8, and 24 h post-dose for IV route. The PK parameters were calculated using Phoenix WinNonlin software: maximum plasma concentration (Cn method .6 per injection) were suspended in 200 μl of a 1:1 solution of ice-cold PBS and Matrigel  solution. HCT-116 cancer xenograft tumors were first generated by injecting 2 × 106 cancer cells into the flank area of severe combined immunodeficiency (SCID) mice . After the tumors grew to 800–1200 mm3, they were isolated, and approximately 50 mg of non-necrotic tumor masses were subcutaneously implanted into the flank area of individual female SCID mice. When the implanted xenograft tumors grew to 250 to 350 mm3 on Day 7 after tumor transplantation, mice were randomly divided into 4 groups for intravenous injection: 1) vehicle , 2) SPESN38–5 , 3) Irinotecan , 4) SPESN38–5 . The intended dose for irinotecan  at 100 mg/kg was attempted on 2 SCID mice bearing HCT-116. Surprisingly, both died immediately. Other doses at 75 mg/kg and 50 mg/kg were tried on healthy CD-1 mice. Both mice from 75 mg/kg IV died instantly, but 2 mice from 50 mg/kg IV were safe, which is consistent with the literature report. Therefore, irinotecan treatment group only had 6 female mice for this study. Mice in group 1 and group 2 on Day 10 were sacrificed due to the large tumor size with diameter ≥ 20 mm. Tumor volume (TV) and BW were measured three times per week or daily depending on the condition of the mouse. TV was calculated using the formula: v = 0.5 (L × W2). Progression at the endpoint was a tumor size with diameter ≥ 20 mm or a moribund condition.HCT-116 CRC xenograft model study was performed at Roswell Park Comprehensive Cancer Center Animal Facility following the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC). HCT-1116 cell line (CCL-247) was purchased from ATCC. After growing in Eagle’s Minimum Essential, HCT-116 cells were harvested by trypsinization and washed twice with PBS. HCT-116 cells (2 × 106 per injection) were suspended in 200 μl of a 1:1 solution of ice-cold PBS and Matrigel  solution. SK-LMS-1 cancer xenograft tumors were first generated by injecting 1 × 106 cancer cells into the flank area of SCID mice . After the tumors grew to 800–1200 mm3, they were isolated, and approximately 50 mg of non-necrotic tumor masses were subcutaneously implanted into the flank area of individual mice. Equal number (12) of female and male mice were used in this experiment. When the implanted xenograft tumors grew to 250 to 350 mm3 on Day 7 after tumor transplantation, mice were randomly divided into 6 groups for intravenous injection: 1) vehicle , 2) DOX , 3) SPESN38–8 , 4) vehicle , 5) DOX , 6) SPESN38–8 . The intended schedule for drug or vehicle treatment was weekly for 3 doses. However, mice in groups 2 and 5 with DOX at 5 mg/kg after 2 doses lost > 20% BW, indicating severe toxicity. Mice in group 1, 2, 4 and 5 on Day 9 were sacrificed due to the large tumor size with diameter ≥ 20 mm and severe BW loss (> 20%). One male mouse from group 6 had some health issues early on and was sacrificed on Day 14. Tumor volume (TV) and BW were measured three times per week or daily depending on the condition of the mouse. TV was calculated using the formula: v = 0.5 (L × W2). Progression at the endpoint was a tumor size with diameter ≥ 20 mm or a moribund condition.Human SK-LMS-1, leiomyosarcoma (sub-type of STS) tumor model study was performed at Roswell Park Comprehensive Cancer Center Animal Facility following the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC). SK-LMS-1 cell line (HTB-88) was purchased from ATCC. After growing in Eagle’s Minimum Essential, SK-LMS-1 cells were harvested by trypsinization and washed twice with PBS. SK-LMS-1 cells  for 20 minutes using a DAKO PT Link. With an Autostainer, the following steps and reagents were used for IHC analysis2O2 for 15 minutes; 2) Incubation with 10% normal goat serum 10 min (Thermo Fisher #50062Z) 10 min; 3) Incubation with Avidin/Biotin block (Vector Labs Cat#SP-2001); 4) Incubation with primary KI67 antibody (Abcam #ab15580 or Cleaved Caspase-3 (Asp175) antibody  diluted in 1% BSA for 30 min; 5) Incubation with secondary Goat anti Rabbit (Vector labs #BA-1000) for 15 min; 6) Incubation with ABC reagent (Vector Labs Cat #PK 6100) for 30 min; 7) Incubation with DAB substrate (Dako Cat #K3467) for 5 minutes; 8) Counterstained with DAKO Hematoxylin for 20 seconds; 9) Coversliped slides.1) Incubation in 3% HFig. S1-S3 and Tables S4-S10.Statistic analyses of TV and tumor weight changes are described in Supplemtary Material. Detailed statistic results are shown in Two SPESN38 complexes, SPESN38–5 and SPESN38–8 , were prin vivo efficacy study and a dose of 250 mg/kg for PK study.Using CD-1 mice, we conducted MTD determination for SPESN38–5 in both oral (PO) and IV routes and SPESN38–8 in IV route. In the PO route, the first dosing at 300 mg/kg was fed to two female CD-1 mice, but one mouse died in less 72 h, indicative of exceeding MTD. The second dose at 250 mg/kg and the third dose at 200 mg/kg were evaluated and the female mouse body weight (BW) change vs the days after treatment were shown in in vivo antitumor efficacy study.In the IV route for SPESN38–5, an initial dose at 80 mg/kg was attempted, both mice died immediately, meaning that the dose was too high. Lower doses at 55 mg/kg, 50 mg/kg, 45 mg/kg and 35 mg/kg were evaluated using female CD-1 mice or male/female mice. The mouse BW changes vs days after treatments were shown in in vivo antitumor study, we conservatively chose a dose at 33 mg/kg for 3 weekly injections in order to ensure that the body weight loss was < 20% after three doses.In the IV route for SPESN38–8, two doses at 45 mg/kg and 35 mg/kg were evaluated using both male and female CD-1 mice. The mouse BW changes vs days after treatments are shown in max), Area Under the plasma Concentration-time curve (AUC), elimination half-life (t1/2), apparent clearance (CL/F), and clearance (CL)  .0–∞ for SN38 (FW = 392.40 g/mole) and SN38G (FW = 568.53 g/mole) are 21.5 and 2558.8 ng × h/mL, respectively. When converted into nmol, AUC0–∞ for SN38 and SN38G are 0.0548 and 4.5007 nmol × h/mL, with the molar ratio of SN38:SN38G = 1:82 in mouse plasma. It is surprising that most SN38 was glucuronidated to SN38G, an inactive form from SN38 metabolism. For the IV route at a dose of 55 mg/kg, AUC0–∞ for SN38 and SN38G are 7377.6 and 15794.8 ng × h/mL respectively. When converted into nmol, AUC0–∞ for SN38 and SN38G are 18.80 and 27.78 nmol × h/mL, with the molar ratio of SN38:SN38G = 1:1.48 (i.e. 40:60%). Therefore, the IV route at a much lower dose afforded much higher AUC0–∞ for SN38 and much better SN38:SN38G ratio, compared with the PO route. Based on the AUC values, the oral bioavailability of SPESN38–5 was estimated to be ~ 3%. Therefore, it is expected that antitumor efficacy of SPESN38–5 would be higher by IV over the PO route.For the oral route at a dose of 250 mg/kg, AUCin vivo efficacy study with a total of 6 mice per group. On Day 10, tumor growth inhibition (TGI) for SPESN38–5 PO, irinotecan IV and SPESN38–5 IV, are 24.1% , 82.3% , and 96.6% , respectively, compared to the control group (Table S1). While SPESN38–5 via PO did slow down tumor growth (24.1% TGI on Day 10) relative to the control group, SPESN38–5 via IV shrank TV by 73.2% over the same period, indicating potent anticancer activity. In comparison, the standard CRC treatment drug irinotecan had 82.3% TGI, but the tumor still increased by 20.6% by Day 10. TV in the irinotecan group continued to increase by 135.5% from Day 10 to Day 21. In stark contrast, SPESN38–5 IV group reduced TV slightly by 3.3% (Table S1). The toxicity of the treatment agents was evaluated by body weight (BW) change over time, which is an established method for early-stage preclinical studies. The normalized BW changes for the above testing groups are not different from each other and all BW changes are within the acceptable ranges (< 10%) (Table S1). Therefore, SPESN38–5 at 55mg/kg via IV exhibited potent anticancer activity with low systematic toxicity.Irinotecan, a prodrug of SN38, is the standard treatment of CRC. Therefore, we wanted to test the antitumor efficacy of SPESN38–5 in a CRC mouse model. The HCT-116 CRC xenograft model was chosen for the following reasons: i) its low FcRn level  ; ii) itsol group . By compp = 0.0058), irinotecan at 50 mg/kg IV group on Day 21  and SPESN38–5 at the 55 mg/kg IV group on Day 21  was significantly less than that in the control group on Day10. SPESN38–5 IV group significantly reduced tumor growth  relative to the irinotecan group on Day 21, resulting in the average tumor weight ratio of irinotecan:SPESN38–5 = 7.7:1, indicating superior SPESN38–5 efficacy over irinotecan in the CRC mouse model.The tumor tissues from all mice at their ending points were dissected and weighed . In agrePhotographic images of the tumors removed at the end of experiments for each treatment group are shown in in vivo efficacy evaluation, in comparison to DOX against SK-LMS-1 xenograft tumors using 4 male and 4 female SCID mice per study group. This study also intended to demonstrate the superior antitumor efficacy of SPESN38–8, just like SPEDOX-5. Due to the fast growth rate of SK-LMS-1 tumors, all mice in the vehicle control group and 5 mg/kg DOX had to be euthanized on Day 9 (p = 0.0039), 33 mg/kg SPESN38–8 had 68.5% TGI . On Day 9, DOX and SPESN38–8 exhibited respective 25.3%  and 86.2%  TGI, and the difference between DOX and SPESN38–8 treatment were very significant . On Day 21, 6 out of 7 mice in the SPESN38–8 treatment reached tumor-free status . On Day 9, TV change were 280.9% (increase) for the control group, 142.4% (increase) for the DOX treatment group, and − 40.1% (reduction) for the SPESN38–8 treatment group (Table S2). While 5 mg/kg DOX treatment slowed the tumor growth rate ~ 2X in TV relative to the control group, the treatment showed severe toxicity, and the mice in both the control group and the DOX treatment group had to be sacrificed on Day 9 due to the high tumor burden and unacceptable toxicity. From Day 9 to Day 21, TV in the 33 mg/kg SPESN38–8 treatment group continued to shrink to reach 95.9% (average) reduction (Table S2). Six of the 7 mice were tumor-free  at the end of the experiment (Day 21).To further explore the anticancer activities of SPESN38 complexes, we chose STS given it has limited treatment options. SK-LMS-1 as an established human leiomyosarcomas cell line is insensitive to DOX, the standard treatment for STS patients, with a high DOX IC50 of 0.49 uM  and has on Day 9 . On Day Table S2). In contrast, 33 mg/kg SPESN38–8 treatment did not show any systemic toxicity  with excellent anticancer activity (Table S2). The combined data from BW change and TV confirm that DOX is not suitable for treating SK-LMS-1 (leiomyosarcoma). SPESN38–8 with a different mechanism of action (Topo I inhibitor) can overcome the resistance of some subtypes of STS toward DOX (Topo II inhibitor) treatment.The normalized BW change for 5 mg/kg DOX treatment group is < 80% (75.4%), indicating unacceptable toxicity without effective antitumor activity (p = 0.0005) on Day 9 and in SPESN38–8 group  on Day 21 was significantly less than that in the control group on Day 9. In addition, the SPESN38–8 group on Day 9 significantly reduced the tumor growth , relative to the DOX group on Day 9. At the end of the experiment (Day 21), SPESN38–8 treatment led 4 mice free of tumor, with other 2 mice having tumor appearance but later proved to be mouse scar tissues without tumor cells by immunohistochemical (IHC) staining (see below section).The tumor tissues from all mice at their ending point were dissected and weighed . In agrePhotographic images of tumors and heart removed at the end of experiments for each treatment group are shown in Taken together, TV, tumor weight, photographic tumor images, and IHC staining results convincingly demonstrate that SPESN38–8 at 33 mg/kg  dose is very effective in suppressing SK-LMS-1 STS tumor growth with low systemic toxicity, eliminating the implanted tumor from 6 out of 7 mice while significantly reducing the tumor size of the other . Therefo1/2 for the ring opening reaction and reach the equilibrium state with ~ 20/80% L/C forms in PBS buffer at 37°C. However, the presence of HSA significantly changes the kinetics and thermodynamics of the ring opening reaction, due to differential HSA binding to L/C forms of different CPTs. While HSA binds the L/C forms of CPT with a 157-fold higher affinity for the C form, SN38 binding to HSA is reversed, with 4.3-fold stronger binding for the L form. As a result, HSA increases the ring opening of CPT with decreasing t1/2 and < 0.5% L form at the equilibrium. On the contrary, SN38’s t1/2 and the L form at the equilibrium both increase substantially in the presence of HSA, from 20 min to 35 min and 13–38%, respectively [CPTs belong to class of TOP1 inhibitors , 45–51. ectively . For theConsequently, the prodrug approach led to an irinotecan approved by the FDA, generating active metabolite SN38 by a biotransformation , 14–18. in vivo antitumor evaluation. Based on the similar preparation procedure except for different SN38/HSA ratios, SPESN38–5 and SPESN38–8 are expected to display similar pharmacological properties such as MTD, PK, and antitumor efficacy. Toxicity study in mice indicated SPESN38–5 and SPESN38–8 with respective 55 and 45 mg/kg MTD, confirming their similar but not identical properties. We thus proceeded to conduct PK and different tumor model studies with either SPESN38–5 or SPESN38–8. SN38 AUC0–∞ of SPESN38–5 at a single IV dose of 55 mg/kg (Table S3) [0–∞ value for SPESN38–5 is estimated to be 1.8 times higher than that for equivalent irinotecan. Since the carboxylesterase activity is much lower in humans than in mice [0–∞ value of irinotecan compared to SPESN38–5 in human plasma. Unlike the prodrug irinotecan, SPESN38–5 does not need biotransformation to SN38 by carboxylesterases, minimizing insistency among different patients. As demonstrated for SPEDOX-6 [0–∞ value relative to irinotecan in humans than in mice.The current study represents the first example of developing unmodified SN38 in soluble and stable forms for 55 mg/kg  is similable S3) . Based o in mice , much smSPEDOX-6 , deliverSPEDOX-6 , leadingTable S1). Separately, in the SK-LMS-1 STS model, excellent antitumor activity was achieved by SPESN38–8 at 33 mg/kg, resulting in 6 of 7 mice tumor-free. In stark contrast, conventional DOX at 5 mg/kg (MTD) was ineffective. Due to a fact that each HSA molecule in SPESN38–8 carries 60% more of SN38 than SPESN38–5 without lowering anticancer efficacy and higher toxicity, the proposed plan of SPESN38–8 (IND #: 164346) in the IV route via a 505 b (2) regulatory pathway was green-lighted by the FDA and the IND-enabling studies are under way. Furthermore, SPESN38–5 in the PO route did not provide a viable option for treating cancer because of low oral bioavailability.As expected from the PK values, both SPESN38–5 and SPESN38–8 demonstrated excellent antitumor efficacy in 2 mouse models. In the HCT-116 CRC model, SPESN38–5 at 55 mg/kg showed superior antitumor activity compared to irinotecan . Furthermore, drug molecules are often linked to the carrier through covalent conjugation. In contrast, the SPE technology has the following unique properties: 1) The formulation process involves no chemical steps. HSA encapsulation of drug molecules are achieved through multiple non-covalent interactions between HSA and drug molecules under a specific set of conditions; 2) The binary system contains a single native HSA molecule that encapsulates a predefined number of a specific drug molecule in its unmodified form; 3) The resulting SPEDRUG complex is uniform in size (monodisperse) and has the same size of a native HSA molecule; 4) The number of drug molecules per HSA molecule may be adjusted according to specific application; 5) The HSA-drug binding strength is tunable by adjusting formulation conditions to effect PK and antitumor efficacy. The successful development of SPEDOX-6 , SPESN38Using the newly developed SPE technology, we prepared SPESN38–5 and SPESN38–8, demonstrating the first examples of unmodified SN38 in clear, stable, and injectable solution. Compared with irinotecan and UF DOX in animal models, SPESN38–5 and SPESN38–8 showed favorable pharmacokinetic values, superior antitumor efficacy against CRC and STS, and lower systemic toxicity. The successful development of SPEDOX-6, SPESN38–5, and SPESN38–8 has validated the SPE platform in drug formulation. These SPEDRUG complexes represent a new uniform macromolecular nanodrug that may be used to target low FcRn expressing cancer cells, further improving their antitumor efficacy while reducing side effect toxicities. These promising preclinical results have prompted these SPEDRUG complexes to be aggressively pursued for their clinical applications.


Preliminary phytochemical analysis of ASAB extract indicates the presence of polyphenolics such as phenols, alkaloids, and flavonoids that can act as potential capping agents in NPs synthesis, which was later confirmed in FTIR analysis of pure and Pd-doped Mn3O4 NPs. XRD, Raman, and XPS analyses confirmed the Pd doping in Mn3O4 NPs. FESEM and HRTEM study reveals the mixed morphologies dominated by nanocorns appearance. Zeta potential investigation reveals high stability of the synthesized NPs in colloidal solutions. The developed Pd-doped Mn3O4 NPs were tested against two fungal phytopathogens, i.e., Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, known for causing great economic losses in yield and quality of different plant species. The antifungal activity of synthesized Pd‐doped Mn3O4 NPs displayed a dose‐dependent response with a maximum of ~92%, and ~72% inhibition was recorded against S. sclerotiorum and C. gloeosporioides, respectively, at 1000 ppm concentration. However, C. gloeosporioides demonstrated higher sensitivity to Pd‐doped Mn3O4 NPs upto 500 ppm) treatment than S. sclerotiorum. The prepared NPs also showed significant antibacterial activity against Enterococcus faecalis. The Pd-doped Mn3O4 NPs were effective even at low treatment doses, i.e., 50–100 ppm, with the highest Zone of inhibition obtained at 1000 ppm concentration. Our findings provide a novel, eco-benign, and cost-effective approach for formulating a nanomaterial composition offering multifaceted utilities as an effective antimicrobial agent.Microbial pathogens are known for causing great environmental stress, owing to which emerging challenges like lack of eco-friendly remediation measures, development of drug-resistant and mutational microbial strains, etc., warrants novel and green routes as a stepping stone to serve such concerns sustainably. In the present study, palladium (Pd) doped manganese  oxide (Mn With the advent of nanobiotechnology, nanostructures' biological and physiochemical properties are tuned to serve the most relevant areas of human welfare, like medicine and agriculture5. Among different types, metallic nanoparticles (MNPs) are one of the widely exploited antimicrobial nanomaterials against phyto- and animal pathogens12. Researchers have recently witnessed a growing interest in synthesizing biocompatible metal-based NPs, utilizing green chemistry and bioinspired fabrication routes21.Nanobiotechnology integrates biotechnology and nanotechnology, dealing with applications of nanomaterials in biological sciences22 and is preferred over conventional methods23. Different biogenic sources for the synthesis of MNPs, like bacteria, plants, algae, fungi, yeasts, and actinomycetes, cause considerable modifications in the properties of their corresponding metals24. Among such sources, plant-based bioinspired fabrication of NPs is one of the preferred approaches25. The bioactive compounds in plant extracts can act as potential reducing and capping agents in synthesizing MNPs25. Green synthesis of MNPs like silver, copper, gold, iron, titanium, zinc, platinum, palladium, etc., has been extensively investigated28. However, limited investigations for green synthesis of manganese (Mn) NPs have been reported so far24, despite various applications in catalysis, biomedicine, electronics, electrochemistry, energy, optics, biosensors, food, pharmaceutical, cosmetics, textile industries, etc.29.Biological synthesis offers an eco-friendly and cost-effective method for the fabrication of NPs2 and MnO etc. have also a great potential to have excellent antimicrobial activity19. Chamaecostus cuspidatus extract is used to green synthesis CeO2 and ZnO nanoparticles (NPs) and effective antibacterial activities. The anticancer effects of CeO2 and ZnO nanoparticles were investigated in human breast cancer cell lines21. Similarly, Green synthesis is used to prepare Cerium oxide nanoparticles (CeO2 NPs) from Artabotrys hexapetalus leaf extracts. The prepared NPs exhibit excellent antibacterial activity against a variety of bacterial species. The anticancer potential of the compound was studied against the (MCF-7) human breast cancer cell line22. In addition to that, Zinc oxide nanoparticle (ZnO NPs) was prepared utilizing starch in a single step green synthesis and had highly porous, novel hollow multi-sphere in morphology. Because of their morphology and porosity, the synthesized ZnO NPs can be employed in a variety of drug delivery applications19. Mn has been reported as the transition element with the third highest abundance on earth followed by iron and titanium30. Among various 3d transition metal-oxides, Mn-oxides  have obtained key attention owing to their compositional and structural diversity31. Mn-oxides NPs also possess structural adaptability with varying physicochemical qualities32. Mn-oxide NPs have excessive potential for sustainable-nanotechnology research and innovation30. Mn-oxides can have applications in optoelectronics, magnetic storage devices, imaging contrast agents, magnetic materials, drug delivery, catalysis, wastewater treatment, solar cells, etc.24.Metal oxides such as ZnO, CuO, TiOSclerotinia sclerotiorum is a necrotrophic phytopathogen that harbors a broad host range and causes stem rot disease in different crops including soybean, oilseed rape, sunflower, tomato, etc., resulting huge losses of agricultural produce33. On the other hand, C. gloeosporioides follows the hemibiotrophic infection mode and is known for causing anthracnose in fruits like papaya, mango, avocado, apple, guava, banana, papaya, cashews, grapes, pitaya, etc., resulting in serious postharvest losses35. Although chemical fungicides have been utilized for their control, their indiscriminate utilization has serious environmental consequences that necessitate the search for novel and eco-friendly alternatives36. E. faecalis is known to colonize the human intestine, and its occurrence in aquatic bodies implies fecal contamination37. These bacteria have been reported as multidrug-resistance microbial pathogens associated with hospital-acquired infections38.3O4 NPs through the green chemistry route as a potential tool to offer a broad spectrum of antimicrobial activity.To combat such underlying issues, present study focused on developing nanoformulation comprised of Pd-doped Mn3O4 NPs was completed by using Syzygium aromaticum bud extract as a potential reducing and capping agent. Adding novelty to this work, attempts were made to modify the structural attributes of Mn3O4 NPs through Pd doping to prioritize their multifaced applications in sectors such as agriculture, environment and medicine. To the best of our knowledge and available literature, this is the first report on the bioinspired fabrication of nanocorn-like Pd-doped Mn3O4 NPs using ASAB extract.In the current study, green synthesis of Mn3O4 NPs against S. sclerotiorum, C. gloeosporioides, and E. faecalis.This present work investigated the antimicrobial potential of bioinspired Pd-doped MnAll the chemicals used in the present study  were of analytical grade and utilized without any further purifications. The solutions and reagents were prepared in double-distilled water (DDW).S. aromaticum were obtained from the nearby local market of Meerut . The taxonomical evaluation was performed by Prof. Vijai Malik, Head, Department of Botany, CCS University, Meerut (UP) (letter reference no. Bot/PB/380). Plant material was procured as per applicable Institutional, International, and National guidelines. The specimens were deposited in the University Herbarium at the Department of Botany (Accession no. Bot. 26 V2L4).The evenly looking dried flower buds of The procured flower buds were thoroughly washed with distilled water to remove dirt particles and dried at 40 °C for 48 h. The dried flower buds were homogenized into a fine powdered form and stored in an air-tight container until used. To prepare the extract, powdered flower buds were macerated in DDW  at 60 °C for 2 h. After cooling at room temperature, the aqueous solution of phytoextract was filtered using Whatman filter paper number 1 at stored at 4 °C temperature until use.39, assisted by the addition of phytoextract as a potential source of capping agent  followed by drop-wise addition of 10 ml aqueous Syzygium aromaticum bud extract under continuous stirring at 650 rpm at 90 °C for one hour. After getting the light brown color of the solution, 25 mM aqueous solution of NaOH was added drop-wise to adjust the pH. The alkali-mediated synthesis provides rapid precipitation leading to the mixture of manganese dihydroxide (Mn (OH)2) and manganese trihydroxide (Mn (OH)3)41. The appearance of a dark brownish-black precipitate indicated the formation of Pd-doped Mn3O4 NPs. The precipitate was collected using centrifugation at 5000 rpm for 10 min. The recovered precipitate gave multiple washings with ethanol and DDW to remove impurities, then dried at 150 °C for 3.5 h in a hot air oven. The finally dried residue was transformed into fine powder through mechanical grinding. The obtained Mn3O4 NPs were stored in an airtight bottle for further characterization. Except for the dopant addition, all other synthesis steps for pure Mn3O4 NPs were the same as those mentioned for Pd-doped Mn3O4 NPs. The key reaction steps in synthesizing Mn3O4 NPs are mentioned below40.The NPs were synthesized using a sol–gel methodent Fig. . Briefly3O4 NPs. The optical properties of prepared NPs were determined by UV–Visible spectroscopy.XRD analysis was performed to study the crystallinity and phases of prepared NPs . The phase transitions and chemical composition of synthesized NPs were examined by Raman spectroscopy. The morphology and elemental composition were studied by FESEM , and EDAX, respectively. FTIR analysis conducted on ASAB extract and synthesized NPs to investigate the role of bioactive compounds in the development of NPs. The zeta potential and structural properties were determined using Zetasizer  and HRTEM/SAED, respectively. XPS analysis was conducted to study the composition, and oxidation states of Pd-doped MnS. sclerotiorum and C. gloeosporioides using the poisoned food technique. The fungal cultures were procured from Indian Type Culture Collection (ITCC), Division of Plant Pathology, Indian Agriculture Research Institute, New Delhi, India. The potato dextrose agar (PDA) media was prepared for fungal growth with following composition: dextrose (2% w/v), potato starch (0.4% w/v), and agar (1.5% w/v). The pH of 5.6 ± 0.2 was adjusted using 0.1N KOH/0.1N HCl. The synthesized NPs were dispersed in PDA media to get the desired concentrations (up to 1000 ppm). The media containing NPs was poured into a Petri plate. At the next step, a ~ 8 mm piece of actively growing mycelia from 5 to 8 days old pure cultures of S. sclerotiorum and C. gloeosporioides were placed in the middle of each plate and incubated for five days at ~ 25 ± 2 °C and ~ 28 ± 2 °C temperature, respectively. The media plates without NPs treatment served as the negative control, and plates with 2 mg/ml of carbendazim + mancozeb were designated as the positive control. The % of growth inhibition was calculated using the below formula:The antifungal activity of NPs was tested against E. faecalis was inoculated to freshly prepared nutrient broth media (NBM) and maintained at 37 °C for ~ 18 h. The composition of NBM was Yeast extract (0.2% w/v), Beef extract (0.1% w/v), Peptone (0.5% w/v), NaCl (0.5% w/v), and pH ~ 7.4 ± 0.2. 2% (w/v) agar was added to the nutrient broth for preparing solid media plates (SPM). The pure culture of E. faecalis at the active log phase was uniformly spread on SPM. The sterile filter paper discs of about 6 mm diameter, each dipped in various concentrations of NPs (0 to 1000 ppm), were placed on SPM. The DDW-dipped discs were served as a negative control. The Petri plates were placed in an incubator overnight, and the ZOI was measured in millimetre. All the experiments on antimicrobial activity were performed in triplicates under aseptic conditions in a laminar airflow chamber. The nutrient media, glassware, and other utilities were autoclaved at 121 °C for 15 min at 15 psi pressure before use to maintain aseptic conditions throughout the assay.The antibacterial activity of NPs was determined using agar disc diffusion assay (ADDA). The pure culture of All of the experiments were completed in triplicates and recorded data presented as mean ± standard deviation using Microsoft Excel®.3O4 NPs were synthesized using an aqueous Syzygium aromaticum bud (ASAB) extract. Preliminary phytochemical analysis indicated the presence of phenols (ferric chloride test)42, flavonoids (lead acetate test)43, alkaloids (Wagner test), carbohydrates (fehling's test), and tannins (ferric chloride test)44. The results were in concordance with the findings of Jimoh et al.43, which established the suitability of the tested plant as a potential substrate for developing Phyto inspired nanoparticles47. Through FTIR analysis, Rajesh et al.45 predicted the role of metabolites present in S. aromaticum bud extract, such as flavonoids, tannins, alkaloids, and carotenoids, in the green synthesis of CuNPs. We have compared the FTIR spectra of ASAB extract, pure and Pd-doped Mn3O4 NPs to validate the capping and stabilizing potential of bioactive compounds present in phytoextract , and C–O ester group was observed at ~ 2931.95 and ~ 1711.92 cm−1, respectively48. A sharp peak at ~ 1611.99 cm−1 belongs to –C = C aromatic stretching vibrations and C = O stretching vibrations of proteins denoting amide linkages 45. The aromatic groups were indicated at ~ 1511.36 cm−148, while two separate peaks at ~ 1366.60 cm−145 and ~ 1051.25 cm−148 denoted the C–O group. These characteristic FTIR spectral peaks suggest for eugenol presence in ASAB extract48. The peak at ~ 640 cm-1 are attributed to the Mn–O in synthesized nanoparticles49. The peaks common to ASAB extract, pure and Pd-doped Mn3O4 NPs  depicted the capping potential of bioactive compounds in phytoextract.FTIR spectrum of ASAB extract revealed a broad band at ~ 3431.62 cm3O4 samples at 2θ values of 31.04, 32.36, 36.13, 44.47, 53.83, 58.57, 59.90, and 64.69 matched well with the (200), (103), (211), (220), (312), (321), (224), and (400) crystal planes of Mn3O4 phase respectively50. The above (hkl) planes correspond to the Hausmannite phase of the Mn3O4 crystal structure (JCPDS 24–0734)51. The intensity of XRD peaks was decreased in the case of Pd doping, indicating peak shifting towards lower diffraction angles and crystalline lattice expansion52, suggesting the successful incorporation of Pd in Mn3O4. There is no shifting of XRD peaks in the case of Mn3O4 NPs, indicating that as-synthesized NPs are comprised of the tetragonal Hausmannite phase53. The sharp peaks confirmed the highly crystalline nature of NPs54. There were no other peaks in the XRD pattern that demonstrated the phase purity of the produced NPs55.The XRD confirms the crystalline structure of the prepared manganese oxide NPs with two distinct phases, as shown in Fig. 3O4 NPs, respectively57. The Pd-doped Mn3O4 NPs were smaller than the pure Mn3O4 NPs, which could be owing to the fact that ionic radii of Pd (0.137 nm) are much larger than that of Mn (0.082 nm)58.D is crystallite size, λ is the wavelength, θ is Bragg’s angle, and β is FWHM.The crystallite size of synthesized NPs were calculated by Scherrer’s formula at a extremely intense peak Eq. , and the3O4 NPs in Fig. 3O4 NPs with the likely appearance of nanocorn-like structures. The morphological changes from rod (Mn3O4 NPs) to nanocorn-like nanocorn (Pd-doped Mn3O4 NPs) could be owing to the decoration of Mn3O4 with Pd2+. EDAX analysis revealed Pd, Mn, and O in their suitable stoichiometric proportion, as given in Fig. 59.The surface morphology of the pure Mn3O4 NPs were recorded at room temperature using a UV–visible spectrophotometer. In absorption spectra, the optical absorption edge was shifted to a higher wavelength region with Pd doping; consequently, the red shift was noticed in Pd-doped Mn3O4 NPs. The energy band gap of developed NPs was calculated by Tauc’s relation  and Pd (IV) states68. Moving on to the Mn 2p core-level spectrum, two distinct peaks were observed at binding energies of 654.94 eV, 653.56 eV and 643.53 eV, 642.13 eV for pure and Pd-doped Mn3O4 nanocorns, respectively. These peaks were associated with Mn 2p1/2 and Mn 2p3/2 in Mn3O4 NPs and indicating a spin-orbital splitting of 11.4 eV  and Pd (IV) and specific Mn 2p states characteristic of Mn3O4.The oxidation states of Pd-doped Mn3O4 NPs in Fig. 3O4 NPs were composed of nearly uniform types of particles. The SAED patterns of Mn3O4 NPs in Fig. 71. Figure 3O4 NPs, and the magnified calibrated lattice fringes of Mn3O4 NPs for the crystal plane of (103) and (211) revealed an interplanar spacing (d-spacing) of 2.7 and 2.4 Å in Fig. 3O4 NPs, which was also consistent with the XRD patterns73.The TEM micrograph of the green synthesized Mn3O4 NPs. Two characteristic peaks at ~ 629 and ~ 630 cm−1 were observed, corresponding to the skeletal vibrations for pure and Pd-doped samples, respectively. The strongest peaks at ~ 629 and ~ 630 cm−1 are consistent with the reported data74 for both materials. These sharp peaks can be assigned to the A1g mode, representing the Mn–O breathing vibration of divalent manganese ions in tetrahedral coordination. This mode is a characteristic feature of Hausmannite76. The comparison of the Raman spectra between pure and Pd-doped Mn3O4 NPs reveals similarities in the characteristic peaks, indicating that the introduction of Pd did not significantly alter the skeletal vibrations and Mn–O breathing vibrations in the tetrahedral coordination. The Raman spectra analysis of pure and Pd-doped Mn3O4 NPs confirmed the presence of specific vibrational modes and provides key insights into the structural properties of these materials. The similarities in the Raman spectra between pure and Pd-doped samples suggested that the Pd-doping did not cause significant changes in the observed vibrational features.Figure 77, which may also influence its bactericidal potential78. Without proper surfactants or capping agents, nanoparticles tend to agglomerate, and their surface area-to-volume ratio decreases due to their increased size79. The zeta potential studies allow us to investigate nanoparticles' surface charge and stability in colloidal solutions80 81, with recorded values of −33.2 ± 0.404 and −36.6 ± 1.74 mV for pure and Pd-doped Mn3O4, respectively. The NPs with greater ZP values (negative or positive) prevent agglomeration via electrostatic repulsion, hence conferring stability80.A particle's surface potential substantially impacts its dispersion stability3O4, NPs, we have observed maximum antifungal activity at 1000-ppm concentration with over 50% inhibitions in the growth of S. sclerotiorum and C. gloeosporioides was recorded at 500 ppm dose . Hence, doping Mn3O4 NPs with Pd favoured their antifungal potential66, which is highly explicitly recommended in the case of C. gloeosporioides to decrease the effective dose. Overall, the bioinspired fabrication of nanocorn-like Pd doped Mn3O4 NPs can be used as an effective antifungal nano-pesticide against different necrotrophic and hemibiotrophic phytopathogens, known for causing enormous loss to agricultural food crops globally.The developed NPs showed mycelium growth inhibition in a dose-dependent manner. In the case of Mnins Fig. . This co3O4 NPs was also investigated against E. faecalis to establish their broad spectrum of antimicrobial potential, in terms of pathogen diversity, i.e., phytopathogens and animal pathogens. As stated earlier, E. faecalis is a well-known human pathogen known for hospital acquired infections. This bacterium colonizes the intestine of animals including humans, and its presence in waterbodies is an indicative of fecal contamination. The resistance of E. faecalis against various antibiotics necessitated the search of novel materials possessing significant antibacterial potential against such nosocomial pathogens. In the present work, both pure and Pd-doped Mn3O4 NPs showed dose-dependent increment in ZOI , causing overlapping of Fermi levels, variation in cellular redox potential, promotes ROS generation  in mitochondria (Eqs. 88. ROS causes alteration in protein structures, oxidative stress, lipid peroxidation, and DNA damage88.In general, NPs have direct and indirect effects on microbial cells. The direct damage occurs via the electrostatic interaction of NPs with cell membrane resulting in membrane depolarization and loss of membrane integrity leading to the disruption in ETC and cell lysision Eqs. , 11, 123ion Fig.  and can ria Eqs. , 11, 1233O4 NPs via utilizing an aqueous extract of S. aromaticum buds. Adding Pd in Mn3O4 resulted in significant changes to their structural attributes, including morphology, crystallite size, and lattice defects. The Pd-doped Mn3O4 NPs exhibited antimicrobial activity in a dose-dependent manner and provides higher inhibitory effects than pure Mn3O4 NPs against S. sclerotiorum, C. gloeosporioides, and E. faecalis. The outcome of this study provides a novel, cost-effective method to develop Pd-doped Mn3O4 based nanomaterials for highly effective antimicrobial applications against tested microbial pathogens. This breakthrough opens up new possibilities in the area of green nanotechnology to develop sustainable and multifaceted antimicrobial agents.The present investigation demonstrates a successful green chemistry approach to synthesis pure and Pd-doped Mn


Pinus tabuliformis (P. tabuliformis) seedlings. We found that four transcription factors were commonly induced by all tested stress treatments, while PtNAC3 and PtZFP30 were highly up-regulated and co-expressed. Unexpectedly, the exogenous hormone treatment assays and the content of the endogenous hormone indicates that the upregulation of PtNAC3 and PtZFP30 are mediated by ethylene. Time-course assay showed that the treatment by ethylene immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), activated the expression of PtNAC3 and PtZFP30 within 8 hours. We further confirm that the PtNAC3 can directly bind to the PtZFP30 promoter region and form a cascade. Overexpression of PtNAC3 enhanced unified abiotic stress tolerance without growth penalty in transgenic Arabidopsis and promoted reproductive success under abiotic stress by shortening the lifespan, suggesting it has great potential as a biological tool applied to plant breeding for abiotic stress tolerance. This study provides novel insights into the hub nodes of the abiotic stresses response network as well as the environmental adaptation mechanism in conifers, and provides a potential biofortification tool to enhance plant unified abiotic stress tolerance.The proper response to various abiotic stresses is essential for plants' survival to overcome their sessile nature, especially for perennial trees with very long-life cycles. However, in conifers, the molecular mechanisms that coordinate multiple abiotic stress responses remain elusive. Here, the transcriptome response to various abiotic stresses like salt, cold, drought, heat shock and osmotic were systematically detected in  Global warming and extreme weather make abiotic stress as an important factor affecting world food security . Plants Abiotic stress normally refers to adversity growth conditions like drought, salt, heat, and cold, as well as ultraviolet (UV) which can affect plant growth . Since tCPKs and MAPKs family genes were regarded as responders that responded to multiple abiotic stress by the abscisic acid (ABA) signaling pathway [To realize this ideal, the regulatory mechanisms of response to abiotic stress in model plants have been extensively studied. In angiosperm,  pathway . To unde pathway . However pathway . The intPinus tabuliformis (P. tabuliformis) found C-repeat/DREB binding factors (CBFs), which played an important role in abiotic stress in angiosperm, were lacking in conifer [Conifer serves as one of the most widely distributed trees in the northern hemisphere and has crucial ecological and economic value. As a gymnosperm, conifer accounts for 39% of the global forests and possesses 615 extant species due to its super adaptability . For con conifer , indicatOsNAC5, OsNAC6, OsNAC9, and OsNAC10 responded to drought, high salinity, and ABA [OsNAC6, OsNAC9, and OsNAC10 enhanced drought stress resistance and overexpressed OsNAC5, OsNAC9, and OsNAC10 increased grain yield under both normal and drought conditions [OsNAC5 responded to almost all abiotic stress, overexpressed OsNAC5 increased plants tolerance to multiple abiotic stress [OsNAC5 was regarded as a potential tool for biofortification strategies in rice [RhNAC3 and JUNGBRUNNEN1 (JUB1) were reported to enhance dehydration tolerance in rose [HaNAC1, VvNAC17, ZmSNAC13, PeNAC034, PeNAC045, and PeNAC036 in Arabidopsis enhanced multiple abiotic stresses tolerance in an ABA pathway-dependent manner [NAC transcription factors (TFs) especially the ATAF subfamily have been widely reported as stress-related TFs . In rice and ABA . Overexpnditions , 25. Comc stress , and owi in rice . The NAC in rose  and drou in rose . Ectopict manner . Taken tPpNAC2 and PpNAC3 were isolated in Pinus pinaster and were found in response to multiple abiotic stress like salt, cold, and wounding treatment [Picea abies), PaNAC03 was found induced by stress and participated in stress defense by secondary metabolite production [Picea wilsonii, PwNAC2 was reported to enhance plant tolerance under abiotic stress [P. tabuliformis, PtNAC3 was found to be related to cold stress [P. tabuliformis, and their role in adversity requires more research.In gymnosperm, SNAC family members participated in abiotic stress response in many cases. In recent years, reatment . In Norwoduction . In Picec stress . In P. td stress . AlthougP. tabuliformis seedlings. The core TFs that respond to multiple abiotic stresses were selected, and a NAC family member was highly induced by all test treatments. Furthermore, overexpression of the PtNAC3 enhanced unified abiotic stress tolerance without growth penalty in transgenic Arabidopsis. Interestingly, we show that the activation of PtNAC3 was mediated by ethylene (ET) rather than ABA. These results provide novel insights into the hub nodes of the abiotic stresses response network as well as the environmental adaptation mechanism in conifers, and provide a potential genetic tool to enhance plant unified abiotic stress tolerance.In conifers, the molecular mechanisms that coordinate multiple abiotic stress responses remain largely unknown. Here, we systematically detected the transcriptomic response to various abiotic stresses like salt, cold, drought, osmotic, and heat shock in P. tabuliformis were treated with cold (4°C), heat (40°C), drought, salt (2 M NaCl), and osmotic (300 mM mannitol). RNA-seq analysis showed that different stress treatments tended to induce specific transcriptome response profiles, such as, 3222/3137 genes were specifically induced/repressed by salt, 1760/1824 genes were specifically induced/repressed by heat, 1244/510 genes were specifically induced/repressed by osmotic, 1209/341 genes were specifically induced/repressed by cold, 532/692 genes were specifically induced/repressed by progressive drought. Meanwhile, the most significant differential gene induced/repressed by different abiotic stress shared less similarity , PtNAC5 (Pt2G26090), and PtMYB175 (Pt3G25430) also show a similar response profile albeit to a lesser extent. Compared to other treatments, including osmotic stress induced by mannitol, NaCl treatment induced the expression of these genes to the greatest extent  assay [PtZFP30. The results showed that PtNAC3 binds to the PtZFP30 promoter in yeast and digested X-gal and let yeast turn blue . Taken together, these results indicate that PtNAC3 serves as the core responsor in the upstream of PtZFP30 in the abiotic stresses response pathway in P. tabuliformis.n plants   assay , dual-luH) assay , electrourn blue . To confon assay . The resactivity . MoreoveA motifs . An in vPtNAC3-PtZFP30 abiotic stress response pathway, 2-month-old P. tabuliformis seedlings were treated with ABA, 1-amino cyclopropane carboxylic acid (ACC), methyl jasmonate (MeJA), salicylic acid (SA), strigolactones (GR24), indole-3-acetic acid (IAA), trans-zeatin (TZ), brassinolide (BR), gibberellin A3 (GA3), gibberellin A4 and A7 (GA4 + 7), and paclobutrazol (PAC). Based on RNA-seq analysis, the expression level of both PtNAC3 and PtZFP30 was significantly upregulated under ACC and MeJA treatment, but not response to ABA  did not exhibit any typical negative traits like growth retardation and lower reproductive yields phenotype of overexpressing stress response TFs under normal conditions. On the contrary, compared with wild-type (WT), OEs were more vigorous, such as growing faster and higher  and grew in the greenhouse  for two months. The ecotype Columbia Arabidopsis (WT) and 35S::PtNAC3 transgenic lines were planted in pots with the turfy soil mixed with the nutrient soil Fangjie (2:1 v/v). N. benthamiana was planted in pots containing turfy soil mixed with vermiculite (1.2:1 v/v).The P. tabuliformis seedlings were transformed to a 4°C, 10°C, 20°C, 30°C, and 40°C climate chamber for 8 hours during the light. Drought treatment raw data was from Tariq Pervaiz's study [P. tabuliformis seedlings were irrigated daily with 2 M NaCl or 300 mM mannitol for 3 days; UV treatment raw data was from Xu J's study [P. tabuliformis seedlings were sprayed daily with 100 μM ABA, 2 mM ACC, 250 μM MeJA, 1 mM SA, 300 μM GR24, 100 μM IAA, 10 μM TZ, 10 μM BR, 50 μM GA3, 50 μM GA4 + 7, and 50 μM PAC for 3 days, each treatment has three repeats. After the treatment mentioned above, 2 g needles were collected.For temperature treatment, two-month-old 's study ; each trArabidopsis, the WT and 35S::PtNAC3 grew in the greenhouse for 4 weeks before flowering, and then treatments were started; for salt treatment, 1 L 200 mM NaCl was watered in a tray twice a week for two weeks; for drought, treatment kept the tray without water for two weeks; for cold treatment, transfer the Arabidopsis to a cold treatment greenhouse  for two weeks; for hot treatment, transfer the Arabidopsis to a cold treatment greenhouse  for a week.For abiotic stress treatment of P. tabuliformis genomic DNA was extracted from the needles following the protocol of the Plant Genomic DNA kit . Total RNA from different tissues of P. tabuliformis or Arabidopsis was extracted following the protocol of the Vazyme Plant Total RNA Isolation Kit  and stored at −80°C. 1 μg total RNA was used for first-strand cDNA samples generated by the Vazyme kit .P. tabuliformis reference genome [The RNA-seq analysis followed the protocol of , 59. Thee genome  and the e genome . The RNAwww.PremierBiosoft.com) was used to develop the primers listed in The qRT-PCR was performed by QuantStudio 6 Rex (Thermo Fisher Scientific) with the protocol of TSINGKE qPCR Mix. Primer Primer5 . SNAC and C2H2-B subfamily proteins conserved domains were identified by NCBI CCD Tools, and their motifs were predicted by MEME which selected classic mode to find five motifs, multiple alignments used DNAMAN v8 (https://www.lynnon.com/), phylogenetic analysis using MEGA software with ML tree which was based on the JTT model, the bootstrap values were 200 bootstrap replicates. Microsoft Office (https://www.microsoft.com/zh-cn), R program and GraphPad Prism 8 were used to analyse the experimental data.Upset figure, heat map and gene structure analysis used the TBtools software (PtNAC3 CDS regions and the 500 bp promoter regions of PtZFP30 were amplified by PCR with the NEB Polymerase (#M0515), the primer listed in PtNAC3 was recombined into the vector pBI121, pGBKT7 which used OK Clon DNA Ligation Kit , and the CDS of PtNAC3 was recombined into the vector pHBT-mCherry, pB42AD, pGEX-4 T-1 and pGreenII 62-SK; and the promoter of PtZFP30 was recombined into the vector pLacZ2μ and pGreenII 0800-LUC by the protocol of BP Clonase II Enzyme mix (11789–020) and LR Clonase II Enzyme mix (11791–020).The recombined plasmid pHBT-NAC3-mCherry was extracted using the CsCl gradient method to get purified plasmid, and the protocol was from the Sheen lab website. The protoplast transient expression assay followed the protocol as described previously , 70. ThePtNAC3, pGBKT7-PtNAC3, and pLacZ2μ-PtZFP30p were extracted by GoldHi EndoFree Plasmid Maxi kit .The recombined plasmid pB42AD-PtNAC3 and pLacZ2μ-PtZFP30p were co-transformed into yeast EGY48 strains  and grew on the SD-Trp/-Ura medium for 4 days at 28°C and transfer to SD/−Trp/-Ura + Gal+Raf + X-gal for 3 days at 28°C. The pLacZ2μ and the pB42AD, pLacZ2μ and the pB42AD-PtNAC3, the pLacz2μ-PtZFP30p and the pB42AD were used as the negative control.The recombined plasmid pB42AD-PtNAC3 and pGADT7-T were co-transformed into yeast Y2HGold strains  and grew on the SD-Leu/−Trp medium for 2 days at 30°C. The single colonies were transferred to SD-Trp-Leu-His-Ade medium for 5 days at 30°C.The recombined plasmid pGBKT7-PtZFP30p and pGreenII 62-SK-PtNAC3 were extracted by GoldHi EndoFree Plasmid Maxi kit ; Nanodrop 8000 was used to measure plasmids quality and quantity.The recombined plasmid pGreenII 0800-LUC-PtZFP30p, pGreenII 62-SK-PtNAC3, pGreenII 0800-LUC and pGreenII 62-SK were transformed into GV3101 Agrobacterium strain  separately, the Agrobacterium GV3101 cells harbored the recombined plasmid were cultured overnight and transferred 1 mL Agrobacterium GV3101 cells into 20 mL LB broth  for approximately 10 hours at 28°C (OD600 = 0.5). The Agrobacterium GV3101 cells were then resuspended (OD600 = 1.0) by infiltration buffer with pH = 5.6 and containing 10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone for 3 hours in the dark. Equal volumes of the suspensions were mixed and infiltrated into N. benthamiana leaves, the transgenic N. benthamiana were placed in the dark for 24 hours and then transferred to the greenhouse for 48 hours. Before fluorescence was detected by LB 983 NightOWL II (Berthold Technologies), leaves were sprayed with a solution containing 0.32 mg/mL D-Luciferin and 0.1% Triton X-100 .The recombined plasmid pGreenII 0800-LUC-PtZFP30 promoter probes containing ACACGTAA motifs (The GST and PtNAC3-GST proteins were expressed by BL21(DE3) cells following the protocols . The PtZP. tabuliformis was carried out according to the method of Liu [The transient transformation assay in the hypocotyl of d of Liu . The celAgrobacterium strain EHA105 , and 35S::PtNAC3 overexpressed lines were generated by the floral-dip method [35S::PtNAC3 lines were used for further research, and 30 events were used to measure WT and transgenic lines’ lifespan.The recombined plasmid pBI121-PtNAC3 was transformed into p method , 73, andWe thank Meiqin Liu and the Testing and Analysis Center of Beijing Forestry University for assisting in confocal microscopy analysis.This work was supported by the Fundamental Research Funds for the Central Universities .P. tabuliformis, wild-type Arabidopsis, screened transgenic lines, and observed the phenotype, H.Z. and Q.Z. assisted with CUT & Tag assay, X.C. and Y.S. participated in analysing data. Y.L. and M.G. contributed to designing and supervising various parts of the research. All authors discussed and approved the final manuscript.S.N. conceived the research, participated in the design of the experiments, headed and managed the project and edited most of the manuscript. F.H. designed part of the experiments, carried out most of the experiments, analysed data and drafted the manuscript. P.W., X.C. and Y.N. planted http://db.cngb.org/search/project/CNP0002179; the RNA-seq data of UVB treatment can be found at: https://www.ncbi.nlm.nih.gov/bioproject/557580; the RNA-seq data of light treatment can found at: http://db.cngb.org/search/project/CNP0000737. Any additional information required to reanalyze the data reported in thiswork paper is available from the Corresponding Author upon request.The authors declare that all data supporting the findings of this study are available within the article and supplementary material or are available upon request from the corresponding author. The RNA-seq data of drought treatment can be found at: None declared.Horticulture Research online.Web_Material_uhad130Click here for additional data file.


Nothopoda todeica n. sp., collected in South Africa from a fern, Todea barbara. This fern has an ancient distribution in Africa and Australasia and belongs to the Gondwanan royal fern family Osmundaceae. We show that the new species is not in a basal position in Nothopodinae. It is very similar and closely related to other members of Nothopoda, associated with derived groups of Asian flowering plants. This contradicts the expectation that the ancient host plant (fern) should be associated with a primitive mite. We also obtained a complete sequence of the mitochondrial DNA of N. todeica n. sp. And demonstrated that it has the same, but differently ordered, mitochondrial genes that were previously found in other eriophyoid mites. Our study contributes to the problem of the history of symbiotic relations of eriophyoid mites with plants, and provides new data that are important for better understanding their evolution.Plant-feeding gall mites of the superfamily Eriophyoidea are serious pests in agriculture because they are capable of transmitting viruses and causing growth abnormalities in plants. Evolutionary archaic gall mites are associated with ancient conifer hosts, while more derived forms inhabit flowering plants. In this study, we investigate the morphology and phylogeny of a new species of the subfamily Nothopodinae, Nothopoda todeica n. sp. , associated with a disjunct Afro-Australasian fern Todea barbara (Osmundaceae) from South Africa. Our analyses (1) determine new erroneous sequences  wrongly assigned to Nothopodinae instead of Phyllocoptinae, (2) confirm the basal position of Nothopodinae in Eriophyoidea s.l., (3) question the monophyly of the Colopodacini and Nothopodini tribes, and (4) show the nested position of African fern-associated Nothopoda within a clade dominated by Asian nothopodines from angiosperms, which implies (a) a secondary association of nothopodines with ferns and (b) no relation between geography (continents) and the phylogenetic relationships of Nothopodinae species. Finally, we obtained a first complete mitochondrial genome for Nothopodinae and revealed a new gene order in the mitogenome of N. todeica n. sp., notably deviating from those in other investigated eriophyoids. Our results contribute to resolving the phylogeny of Eriophyoidea and provide an example of an integrative study of a new taxon belonging to an economically important group of acariform mites.Eriophyoidea is a group of phytoparasitic mites with poorly resolved phylogeny. Previous studies inferred Eriophyidae s.l. as the largest molecular clade of Eriophyoidea, and Nothopodinae as the basal divergence of Eriophyidae s.l. We investigate the morphology and molecular phylogeny of  Cox1, 12S, 16S) and nuclear  genes [Eriophyoidea is an ancient group of greatly miniaturized and morphologically simplified acariform mites associated with higher vascular plants ,2,3. ForS) genes ,11,12,13S) genes . MoreoveS) genes ,16. Pentasetacus and Loboquintus) associated with ancient conifer hosts (Araucaria and Cupressus) and tentatively combined in the family Pentasetacidae [The consensus, at present, among all the published molecular cladograms of Eriophyoidea is a polytomy  comprisietacidae ,18,19 reetacidae , which aetacidae ,12,13. TFragariocoptes setiger), two full genomes , and six mitochondrial genomes  of eriophyoid mites in GenBank (accessed on 1 May 2023) [This deadlock might be broken using a phylogenomic approach through the analyses of larger sets of gene sequences from full nuclear and mitochondrial genomes of Eriophyoidea. To date, there exist only one transcriptome ay 2023) ,20,21,2223 ,20,21ay 2023) . All pubay 2023) ,12,20,221b. Nothopodinae are absent from gymnosperms and early angiosperms . In the boreal zones of Eurasia and North America, nothopodines have not been recorded; although, several species (including introduced ones) are known to be from southern Europe and USA. In Oceania, South America, and Africa, Nothopodinae are limited and comprise, in total, about 25 species.Colopodacus, Solenidiversum, Cosella, Disella, Floracarus, Nothopoda, and Nonthaburinus) cause various galls and discolorations on plants; most of them (~92%) belong to the tribe Nothopodini. No Nothopodinae species induce the formation of galls containing a true chamber sensu [Similar to the entire superfamily Eriophyoidea , most sper sensu , similarer sensu . Most daTodea barbara (L.) T. Moore in the Western Cape, contained nothopodine mites. Todea barbara has a disjunct Afro-Australasian distribution and belongs to the Triassic leptopteroid clade of the ancient late-Paleozoic fern lineage Osmundaceae [T. barbara, are considered “living fossils” [In 2013–2017, several expeditions focusing on exploring the diversity of eriophyoid mites associated with indigenous Southern African plants were organized by the ARC Plant Protection Research Institute . During these expeditions, numerous samples containing eriophyoids were collected from four provinces in South Africa . Among them, only one sample that was collected from an indigenous fern, undaceae ,28. Thisfossils” ,30.Nothopoda from T. barbara, provide the sequences of Cox1 and rDNA genes, describe the complete mitogenome, and compare it to other published mitogenomes of Eriophyoidea. We also reconstruct a molecular phylogeny of Nothopodinae using the data available in GenBank in order to test three hypotheses: (1) species of Nothopodinae cluster according to the presence/absence of coxal seta 1b and form two clades corresponding to the Nothopodini and Colopodacini tribes, (2) the phylogenetic relationships of Nothopodinae species is related to the continent on which they occur, and (3) cladogenesis of Nothopodinae follows the divergence of higher vascular plants into large superclades, specifically–ferns and angiosperms.In this paper, we describe a new eriophyoid mite species of the genus Collection and morphological measurements. The fronds of the fern Todea barbara (L.) T. Moore were sampled in South Africa in November 2016. They were examined under a stereo microscope and the mites were collected using a minuten pin. Some mites were slide-mounted in modified Berlese medium with iodine [h iodine  and cleah iodine ,14, resph iodine , scannedDNA extraction and sequencing. For DNA extraction, four females from two different populations  before being heated 3 times (5 min at 95 °C) in a thermostat with intermediate short vortexing. The solution above the Chelex® granules was used as the DNA template for PCR to amplify the fragments of the Cox1 gene. For the PCR and sequencing, we applied the protocol and primers detailed by [Cox1 sequences were obtained using BigDye Terminator v.3.1 chemistry  and a 3500xl Genetic Analyzer (Applied Biosystems). Sequences of 18S and 28S rDNA genes were obtained through genomic sequencing  and added three species from Eriophyidae s.l.  for which complete Cox1, 18S, and 28S sequences are available. We then removed all identical sequences of the same species. We also removed all sequences of Floracarus perrepae (Nothopodini) and Nonthaburinus (Colopodacini), because they were too short and crushed the analysis because of too few numbers of common sites. The sequences of one species of Pentasetacidae (Loboquintus subsquamatus) and five species of Phytoptidae s.l.  were used, respectively, as distant and close out-groups in our analyses. We combined them with the previously mentioned sequences of Eriophyidae s.l. and obtained three final FASTA files that included 28/29/19 sequences of 18S/28S/Cox1 genes. All sequences, except those of the new species, were generated in previous studies focusing on the molecular phylogenetics of Eriophyoidea [phyoidea ,12,20.18S and 28S genes were aligned with the E-INS-i MAFFT algorithm [Cox1 gene were treated as codons. Maximum likelihood analyses were conducted in IQ-tree 2 [18S + 28S datasets and the GY + F3X4 + I + G4 model was selected for the Cox1 dataset using ModelFinder [Sequences of lgorithm  through lgorithm  using delgorithm  as referlgorithm ,37, withlgorithm . SequencQ-tree 2 . For theelFinder , as implMitogenomics. In order to obtain the complete mitochondrial genome of the new nothopodine species, we extracted DNA from 400 ethanol-preserved mite specimens using a LumiPure Genomic DNA from AnySample Kit  and prepared genomic libraries using a Nextera DNA Flex Library Prep Kit. Genomic sequencing (150 bp paired reads) was performed using Illumina Hiseq 6000 in Eurogen (Russia). FastQC was used to calculate and visualize the sequence-quality metrics of raw and filtered reads [Cox1 gene (see above) by lastz v.1.04.00 . Additionally, the de novo assembly of the mitochondrial genome was performed by GetOrganelle v.1.7.7.0 [bedtools getfasta v.2.29.2 [seqtk subseq . BLASTn v.2.13.0 [Nothopoda todeica n. sp. was manually annotated in Unipro UGENE v.46 [ed reads . Raw reaed reads  to removed reads . Mitocho.1.7.7.0  and Mito.1.7.7.0 . All mit.1.7.7.0 . Ribosom.1.7.7.0 . Scaffolv.2.29.2  or seqtkv.2.13.0  against v.2.13.0 . Then, t12S and 16S) and protein genes (translated into amino acids) of eight eriophyoid taxa  were aligned using MAFFT and the resulting alignments were modified using Gblocks, as described above. Sequences of the ATP8 gene were excluded because Gblocks failed to find reliable blocks in the alignment of this gene. Maximum likelihood analyses were conducted in IQ-tree 2 [12S + 26S datasets, mtZOA + F + G4 was selected for merged ATP6 + COX2 + COX3 + CYTB + NAD1 + NAD2 + NAD3 + NAD4 + NAD4L + NAD5 + NAD6 datasets, and the mtART + I + G4 model was selected for the Cox1 dataset using ModelFinder [For the molecular phylogenetic analysis, mitochondrial rRNA .FEMALE , 60 (59–64) wide at level of setae c2. Prodorsal shield subtriangular, 36 (32–38), 75 (72–85) wide, without frontal lobe, anterior margin of prodorsal shield distinct, broadly rounded. Prodorsal shield with distinct net-like pattern formed by longitudinal and transverse ridges. Median and admedian lines complete, entire, admedians converging in anterior part of shield. Submedian line I indistinct or fragmented in posterior half of prodorsal shield and situated between admedian line and tubercle of sc. There are four rows of cells. Anterior-most row includes 9–10 cells formed by thin lines; these cells are open anteriorly, ending almost on the anterior margin of the shield. Second row includes six cells; among them two median cells are subrectangular and the remaining four cells are subtriangular or of a more irregular shape. The two posterior rows have one uniform rectangular cell on each side of the median line. Posterolateral areas of the prodorsal shield with distinct curved ridges that are dorsal continuations of ventral opisthosomal semiannuli. Tubercles of setae sc. large, situated anterior to prodorsal shield rear margin, sc. 15 (15–17), 25 (22–26) apart, directed upward and convergently posteriad. Gnathosoma projecting downward, palps 14 (12–15). Gnathosomal setae: seta ν 1 (1–2); pedipalp genual seta d absent in all studied specimens (n = 8); and pedipalp coxal seta ep 2 (1–3). Suboral plate is smooth and 6 (6–7), 8 (8–10) wide.Leg I 23 (22–24), tarsus 7 (7–8), u′ 3 (2–4), ft′ 22 (20–26), ft” 17 (15–21), ω 5 (5–6) with large spherical knob; empodium 4 (4–5), 4-rayed, all rays except terminal pair with one  or two (in other rays) additional subrays; tibia absent; genu 5 (4–5), l” 23 (21–26), femur 8 (7–9), bv 5 (5–6), with ventro-distal microgranulations. Leg II 21 (21–24), tarsus 7 (7–8), u′ 3 (2–3), ft′ 6 (6–8), ft” 18 (16–21), ω 7 (6–7) with large spherical knob; empodium 4 (4–5), 4-rayed, similar to empodium I; tibia absent; genu 4 (4–5), l” 10 (8–11), femur 8 (8–9), bv 7 (6–8), with ventro-distal microgranulations. Coxal plates I and II with distinct spine-shaped microtubercles situated mainly around tubercles of setae 1a and 2a; coxal setae 1b absent; 1a 9 (8–9), 10 (9–11) apart; 2a 22 (20–25), 22 (21–24) apart. Prosternal apodeme indistinct; 4 (3–5) incomplete coxigenital annuli between coxae II and epigynium. External genitalia. Genital coverflap posteriorly rounded and slightly notched midway, with two curved, fragmented transverse ridges in two rows separating thin, smooth, distal part of coverflap and thicker microtuberculated basal part, 10 (9–10), 20 (19–23) wide; setae 3a 7 (6–7), 17 (16–19) apart. n = 3).Internal genitalia  annuli, ventrally with 60 (59–64) annuli between posterior margin of coxae II and caudal lobes. Dorsal annuli smooth, except posterior-most 5–10 annuli with small, irregular, round microtubercles. Ventral annuli with spine-shaped microtubercles in anterior half of opisthosoma and more elongated, gradually changing their shape and becoming more elongated and eventually ridge-like, posterior to tubercles of setae f. Setal lengths: c2 23 (22–31), d 46 (42–48), e 6 (6–7), f 14 (14–15); h1 4 (3–5); h2 38 (37–45); 9 (9–10) annuli from rear shield margin to c2; 11 (10–12) annuli between c2–d; 13 (13–15) annuli between d and e; 18 (18–19) annuli between e and f; and 8 (8–9) annuli between f and h2. Small subrhomboid plate about 3–4, 3–4 wide present behind last ventral opisthosomal annulus. This plate is dotted. The dots may be small, round microtubercles or pores. Indistinct cuticular tubes and small rectal sac  were obType material. Holotype female from slide E4719 and paratype females in slide series E4251, E4320, and E4322, and in vials filled with 96% ethanol collected on 7 November 2016 by P. Chetverikov, C. Craemer, and S. Neser on fronds of Todea barbara (L.) T. Moore in Cederberg Wilderness Area, near the Algeria Waterfall (32°21′41.8″ S 19°04′19.4″ E), Western Cape Province, South Africa (h Africa . Type maHost and relation to host. Mites live on the lower surface of fronds of Todea barbara (L.) T. Moore (Osmundaceae), causing no visible damage.Remarks. Paleobiogeography of Todea, the host genus of N. todeican. sp., suggests a largely disjunctive Gondwanan distribution of this fern genus across the Southern Hemisphere in the past with different species that were common in South America, Africa, and Australasia in the Cenozoic [Todea is absent in South America and comprises only two extant species: T. papuana and T. barbara. The first species is a New Guinea endemic, whereas T. barbara has a remarkable Afro-Australasian distribution with two relictual populations, one distributed in Southern Africa  and the other in Oceania  [N. todeica n. sp. on T. barbara in South Africa presents interesting questions for the future research: (1) does Nothopoda occur on Todea in Australasia and (2) which extant eriophyoid taxa are primary and which are secondary  symbionts of ferns?Cenozoic . At presZealand) . Our finAdditional material. Females and males from slide series E4721–E4723, F184, F186, and F187 were collected from the same host, by the same collectors on right bank of the Lower Palmiet River, in the Kogelberg Nature Reserve, Western Cape Province, South Africa (34°19′42.8″ S 18°58′50.5″ E) (50.5″ E) .GenBank data. OQ737112 , OQ737113 , OQ737114 , OQ720953 , OQ720954 , and OQ934080 .Remarks. Females of N. todeica n. sp. from the type (n = 8) and additional (n = 9) material morphologically were very similar, except that the females from the type locality were smaller (length of body 156–196 vs. 186–234), had narrower and shorter prodorsal shields (33–36 × 45–52 vs. 37–40 × 60–63), and differed slightly in the shape of the cells in the second row on the prodorsal shield .MALE . Leg I 21–23, tarsus 7–8, u′ 2–3, ft′ 19–24, ft″ 18–20, ω 6–8 with large spherical knob; empodium 4–5, 4-rayed, similar to that in females; tibia absent; genu 4–5, l″ 20–26, femur 7–8, bv 4–5. Leg II 19–21, tarsus 6–8, u′ 2–3, ft′ 6–7, ft″ 17–19, ω 6–8 with large spherical knob; empodium 4 (4–5), 4-rayed, similar to empodium I; tibia absent; genu 4–5, l″ 8–11, femur 7–9, bv 5–7. Coxal plates I and II with distinct spine-shaped microtubercles situated mainly around tubercles of setae 1a and 2a; coxal setae 1b absent; 1a 7–16, 10–11 apart; 2a 14–17, 20–22 apart. Prosternal apodeme indistinct, 7–8 coxigenital annuli between coxae II and external genitalia. Genital area 15–18 wide, setae eu 0.5–1, 3a 6–7, 13–14 apart, cuticle between tubercles of 3a with small, round microtubercles. Opisthosoma dorsally with 58–65 annuli and ventrally with 57–66 annuli between posterior margin of coxae II and caudal lobes. Opisthosomal annuli similarly microtuberculated to those in females. Setal lengths: c2 18–25, d 34–39, e 4–6, f 13–15; h1 3–4; h2 34–42; 12–13 annuli from rear shield margin to c2; 8–13 annuli between c2–d; 12–13 annuli between d and e; 17–18 annuli between e and f; and 7–8 annuli between f and h2. Etymology. The species name, todeica, is a feminine adjective o in the nominative case. It is derived from the generic name of the host plant and means “associated with Todea”.Differential diagnosis. Among all presently known species of the genus Nothopoda, the new species is morphologically closest to Nothopoda camelliae [N. todeica n. sp. and N. camelliae are in (a) the shapes of microtubercles on coxal plates, (b) pattern of distribution of microtubercles on opisthosomal annuli, (c) presence/absence of microgranulations on femora I and II, and (d) presence/absence of short thin lines between the thicker ridges of the prodorsal shield that form cells , the only other Nothopoda species associated with ferns. Both species can be separated from N. todeica n. sp. based on the ornamentation of the prodorsal shield and female genital coverflap, and the presence/absence of microtubercles on dorsal opithosomal annuli  and additional (OQ720954) material were identical, except for one synonymous nucleotide substitution (A vs. R) in the 468 position of the Cox1 gene in a codon corresponding to serin. Cox1 sequences of Nothopoda spp. were absent from https://www.ncbi.nlm.nih.gov/nucleotide/ (accessed on 23 March 2023). A Blastn search for Cox1 sequence OQ720953 of N. todeica n. sp. against Eriophyoidea returned as the best hits the sequences of two nothopodine species Floracarus perrepae (AY823158) and Cosella viburniae (MZ274943) when sorted by percentage of identity. Blastx returned sequences of Cosella parvifoliae  and C. viburniae  when filtered by coverage (>50%) and sorted by percentage of identity.18S (OQ737112) and 28S (OQ737114) sequences of N. todeica n. sp. against Eriophyoidea returned two sequences of Nothopoda sp., MZ279931  and KF782476  as the best hits, when filtered by E-value.A Blast search for ITS1-5.8S-ITS2 sequences of Nothopodinae were absent from GenBank (accessed on 23.03.2023). A Blast search for the ITS1-5.8S-ITS2 sequence OQ737114 of N. todeica n. sp. against Eriophyoidea returned as the best-hits various sequences of Eriophyinae  and Cecidophyinae (Cecidophyopsis).18S, 28S, and Cox1 sequences produced a tree topology  were inferred in different clades of Nothopodinae  indicating (a) a putative homoplastic loss of coxal setae 1b in Nothopodinae and (b) polyphyly of the tribes Colopodacini and Nothopodini. Nothopoda todeica n. sp., a single African taxon from a fern host in our analyses, was nested within a large group of Asian nothopodine taxa associated with angiosperms in clade A. Sequences of two nothopodine genera, Cosella and Nothopoda, did not form separate genus-specific groups in our tree, but were mixed in a moderately supported clade NC that was sister to Floracarus.Our analyses of topology  showing Remarks. After inferring Kuangella theae in the unexpected phylogenetic position out of Nothopodinae, we performed a Blast search for the three sequences of this species  obtained by [K. theae, isolate 28sd2-5r7 28S)—about 85% similarity with sequences of various phyllocoptines, KF782375 —99.39% similarity with sequence MZ279804 of Fujianacarus wisterianis (Eriophyidae: Phyllocoptinae: Phyllocoptini), and KF782586 —99.61% similarity with sequence MZ326423 of F. wisterianis and 80%–90% with sequences of some other phyllocoptines. These findings are in accordance with the results of our molecular phylogenetic analyses , and six genes code tRNAs . The GC content is 16%. All protein-coding genes are terminated with the stop-codon TAA, except NAD4 (TAG). The control region (CR) is located between genes Cox1 and Cox2. In comparison to the other six eriophyoid species investigated to date [CR in N. todeica n. sp. Is, on average, six times longer (651 bp vs. 114 bp) and contains poly-AT repeats. The mitochondrion genome of l region . Ten gen to date ,20,21,22Cox1-Cox2), II (ATP6-Cox3-G-NAD3-A-R), and III . Blocks I, II, and III were separated by three variable zones: A, B, and C , 8 or 9 tRNA genes, and 2 rRNA genes (12S and 16S). Contrary to all members of Eriophyidae s.l., in Fragariocoptes setiger (Phytoptidae s.str.), rRNA genes were situated on the negative chain of the mitochondrial DNA, corresponding to the position of these genes in a hypothetical ancestral mitogenome of Acariformes [All eriophyoids in our analyses possessed three stable blocks of mitochondrial genes: I  and shares the same gene order in zone A (D-ATP8-K) with Epitrimerus sabinae.The main differences in the mitochondrial gene order between The molecular phylogenetic analysis of the mitogenomic dataset revealed a tree topology of Eriophyoidea similar to that obtained in the analysis of the sequences of nuclear rRNA genes reported above  and infeN. todeica n. sp., a new species of the subfamily Nothopodinae associated with a disjunct Afro-Australasian plant taxon, the fern Todea barbara, collected in South Africa. Morphologically, N. todeica n. sp. appeared to be a typical representative of Nothopoda that is very similar to some other members of this genus reported from eudicot trees from Asia, especially N. camelliae. Remarkably, both species share an interesting structure, a small subrhomboidal plate preceding the anal opening  is a plesiomorphy, and the loss of this setae (defining Nothopodini) could have occurred homoplastically in different nothopodine taxa. Our results also show the nested position of the African fern-associated Nothopoda within a clade dominated by Asian nothopodines from angiosperms, which implies: (a) a likely secondary association of nothopodines with ferns and (b) no relation between geography (continents) and the phylogenetic relationships of Nothopodinae species. Considering the high concentration of nothopodine taxa in Asia and the results of our study, we hypothesized that Nothopodinae originated and diversified in Asia and, later (probably quite recently), dispersed to other continents. This hypothesis may be tested in the future, when more sequences of various nothopodine genera associated with larger sets of host plant orders are available for analyses.In this work, we applied a very conservative approach for aligning sequences of nuclear rDNA genes, and, following ,35, we rNAD5 and NAD2 in the mitogenome of N. todeica n. sp. Our mitogenomic analysis showed the separation of Nothopodinae from other members of Eriophyidae s.l., which corresponds with a notably deviating gene order in the mitogenome of N. todeica n. sp. and the basal position of Nothopodinae in Eriophyidae s.l. revealed in the analyses of Cox1, 18S, and 28S sequences. The question of whether the new gene order in the mitogenome of N. todeica n. sp. is a synapomorphy of the whole subfamily Nothopodinae or only an autapomophy of the new species needs further investigations. The results of the relatively recent pioneer works by Xue et al. [Finally, in this study, we obtained a first complete annotated mitochondrial genome for Nothopodinae and found two unique clusters of tRNA genes adjacent to the protein-coding genes e et al. ,20 on the et al. ,22 this