Escherichia coli infection, Resolvin D5 (RvD5) was found to enhance bacteria phagocytosis and reduce the production of inflammatory mediators, contributing to the resolution of infection. LPS (lipopolysaccharide) is a gram-negative bacterial structure product which activates the immune system and, at high doses, leads to endotoxemia. To our knowledge, the effect of RvD5 against LPS endotoxemia has not been investigated to date. Female Swiss mice received an i.p. treatment with RvD5 . After 1 h, they were stimulated with LPS , and samples were collected after additional 6 h. The resulting data demonstrated that RvD5 protected the kidneys (urea and creatinine serum levels) from tissue injury. These effects were related to an improvement in histopathological parameters and a reduction of enzymatic markers of leukocyte infiltration, pro-inflammatory cytokine  production, and oxidative stress. Antioxidant markers were also increased by RvD5, but IL-10 (an anti-inflammatory cytokine) levels were unaltered. We also observed that RvD5 reduced the infiltration of CD45+ hematopoietic cells into the kidneys, reduced the activation of NFκB and promoted the Nrf2 pathway by reducing Keap-1 levels. Our data indicate that RvD5 may be a therapeutic possibility to reduce kidney lesions in LPS endotoxemia.In self-revolving gram-negative  Sepsis is a major disease in health care, since approximately 50% of affected patients do not survive ,2. SurviResolvins are SPMs derived from the omega-3 fatty acids EPA (Eicosapentaenoic acid) and DHA (Docosapentaenoic acid), which are primary substrates of these biomolecules. Resolvins can be subdivided according to their precursor molecule into Resolvins of the E (RvE) series for those derived from EPA, and Resolvins of the D (RvD) for those derived from DHA . DHA-derS),17(S)-diHDHA has been identified in the inflammatory milieu of Escherichia coli infection in a bacterial load that produces self-resolving inflammation. Interestingly, RvD5 was shown to enhance bacteria phagocytosis, which is an important function to promote the killing of bacteria. RvD5 also reduced the production of pro-inflammatory cytokines, contributing to the reduction of inflammation [Citrobacter rodentium infection of infant mouse as well as reinfection. Normally, this infection would be characterized by high C. rodentium load that increases rapidly and remains sustained, resulting in intestinal inflammation with necrosis and reaching the blood stream; however, RVD5 reversed this scenario [Resolvin D5 (RvD5) or 7. We used a pharmacological approach to test RvD5 as a drug; however, the selected dose range was based on literature data that was obtained using up to 10 ng of RvD5 with positive activities , as wellLeukocytes activation and recruitment to affected tissue are crucial points in the inflammatory process, as well as in inflammatory tissue injury. We determined the levels of MPO and NAG activities in the kidneys. We observed a significant increase in MPO and NAG activities after systemic administration of LPS . All tesLPS endotoxemia induced an increase of IL-1β A, TNF-α The ABTS ) and the FRAP (Ferric Reducing Antioxidant Power) assays are methods to evaluate the total antioxidant capacity of a sample. Systemic administration of LPS decreased antioxidant capacity as per ABTS A and FRASuperoxide anion production (NBT) and lipid peroxidation levels (TBARS) increased in the kidneys upon LPS endotoxemia . RvD5, aKidneys samples were processed and stained with H&E. Compared to the negative control group  are two transcription factor pathways that are sensitive to oxidative stress. The difference is that NFκB upregulates inflammation while Nrf2 increases antioxidant and anti-inflammatory responses ,55. The RvD5 has an analgesic effect in chemotherapy-induced neuropathy and formalin nociception. This analgesic activity occurred in male, but not in female mice . Taking ® Equipamentos, Ribeirão Preto, Brazil) with a maximum of 10 animals per cage. All procedures were performed in accordance with local, national, and international standards of ethics. Animal handling and welfare were approved by the Universidade Estadual de Londrina Ethics Committee for animal experimentation . Animal studies are reported in compliance with the ARRIVE guidelines [The present study was conducted with pathogen-free female Swiss mice . Animals were provided by Universidade Estadual de Londrina animal facility  and were housed in a climatized room (21 ± 1 °C) with free access to water and food, and a light/dark cycle (12/12 h). The animals were kept into standard polypropylene cages measuring 41 × 34 × 16 cm (Insightidelines . The totE. coli—Santa Cruz Biotechnology) or vehicle  intravenously . Animals were divided into five equal sized groups consisting of six animals per group. Group 1: 0.1% ethanol in saline (i.p.) + saline (i.v.); Group 2: 0.1% ethanol in saline (i.p.) + LPS 10 mg/kg ; Group 3: RvD5 0.1 ng/animal  + LPS 10 mg/kg (i.v.); Group 4: RvD1 1 ng/animal (i.p.) + LPS 10 mg/kg (i.v.); and Group 5: RvD1 10 ng/animal (i.p.) + LPS 10 mg/kg (i.v.). For all assays, samples were collected 6 h after LPS administration.RvD5 , Cayman Chemical, Ann Arbor, MI, USA, 0,1; 1 and 10 ng/animal) or vehicle  were administrated intraperitoneally (i.p.). After 60 min, mice received LPS , followed by an increase in isoflurane concentration for euthanasia (5% v/v in oxygen). Blood samples were placed in microcubes containing 50 µL of porcine sodium heparin  and centrifuged (3500 rpm × 10 min at 4 °C) for plasma separation. The determination of plasma urea levels was quantitated by the enzymatic method (Laborlab—São Paulo—Brazil), while creatinine levels were quantitated by the kinetic method (Gold Análise Diagnóstica—Minas Gerais—Brazil) using Selectra automated equipment. Results were expressed in mg/dL [Blood was collected by cardiac puncture under isoflurane anesthesia (3% in mg/dL .2HPO4 buffer solution (pH 6.0), containing 0.5% of HTAB, and were homogenized with a tissue homogenizer. Subsequently, samples were centrifuged  and the supernatant was separated. The kidney homogenate supernatant was added to 200 µL of 50 mM phosphate buffer solution (pH 6.0), containing 0.167 mg/mL of o-dianisidine dihydrochloride and 0.015% of hydrogen peroxide. Absorbances were recorded with a microplate spectrophotometer  at 450 nm. MPO activity was expressed as myeloperoxidase activity (number of neutrophils × 103/mg of tissue) compared to a neutrophil standard curve [MPO activity was used as an indirect measure of neutrophils in the kidneys. Samples were collected in 200 µL of Krd curve .N-acetyl-β-d-glucaminide, followed by incubation at 37 °C for 10 min. Finally, 100 µL of 0.2M glycine buffer (pH 10.6) was added to the reaction. The enzyme activity was determined spectrophotometrically at 400 nm; the results were expressed as NAG activity (macrophages × 103/mg tissue) compared to the macrophage standard curve.NAG activity was determined as described by Hohmann et al. . BrieflyKidneys were collected in phosphate buffered saline for cytokines level determination. Samples were homogenized, centrifuged (3000 rpm × 4 °C × 15 min), and the supernatant was used to measure the levels of tumor necrosis factor-α (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 by enzyme-linked immunosorbent assay (ELISA). The assay was conducted following manufacturer’s instructions .Kidney samples were collected in 500 µL of 1.15% KCl buffer, homogenized, and centrifuged (1500 rpm/4 °C/10 min). To measure the ability of each sample to resist oxidative stress, the modified ABTS  method primarily described by Re et al.  was usedg, 3 min, 4 °C), the supernatant was reacted with 50 µL of 1% TBA at 95 °C for 60 min or until the color changed to pink. Then, the absorbance was measured at 572 nm and 535 nm. The results were expressed as 572 nm–575 nm OD/mg of protein [Free radicals, when in contact with lipids, destabilize these molecules and produce malondialdehyde (MDA). In turn, MDA amplifies the inflammatory process. Therefore, a TBARS test was used to detect the presence of MDA in samples using thiobarbituric acid (TBA). TBA reacts with MDA, forming a new compound which can be detected spectrophotometrically. Sample were homogenized in 500 µL of 1.15% KCl. Then, 50% trichloroacetic acid (TCA) was added, and after centrifugation . For the assay, 100 µL of the sample supernatant was mixed with 200 µL of 0.4M Tris-HCl pH 8.9 and 10 µL of dithiobisnitrobenzoic acid (DTNB) 10 mM. After 5 min at room temperature, the absorbance was determined at 412 nm. The results are presented as nmol GSH/mg of protein compared to a GSH standard curve .Kidney samples were collected and fixed in 10% buffered formaldehyde. Subsequently, the samples were embedded in paraffin, sectioned, and the tissue sections (5 µm) were stained with hematoxylin-eosin (HE), and examined under an optical microscope  focusing on cortical regions. For histopathological evaluation, a score was adapted, as previously described . The scom/v)  and embedded in optimum cutting temperature (OCT) reagent. The blocks were sectioned using cryostat equipment  at 16 µm of thickness. In a prior phase of the technique, antigenic recovery was carried out through exposure to 90 °C followed by immediate cooling until 30 °C was reached. Then, all samples were blocked with 5% bovine serum albumin (BSA) in phosphate buffered saline with 0.3% Triton 100-X. The kidney was stained with mouse CD45 PE/Cy5 , as well as mouse Phospho-NFκB  and rabbit Kelch-like ECH-associated protein-1 (Keap-1) , followed by goat anti-mouse Alexa Fluor 647  and goat anti-rabbit Alexa Flour 488 , respectively. The nuclei were stained with DAPI . Image acquisition and analysis of fluorescence intensity were performed in the cortex region of the kidneys using a confocal microscope . Scale bars are presented in each representative image contained in the results section. Analyses were focused on cortical regions of samples and always performed by a blinded experimenter and measured using confocal microscope software to provide quantitative data for the experiments.Six hours after LPS stimulus, animals underwent a perfusion process using 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) injected via the ascending aorta artery. Next, kidney was dissected and post-fixed in 4% PFA for 24 h. Kidney samples were incubated in saccharose 30%  to determine the sample size . StatistWe demonstrated that RvD5 reduces LPS endotoxemia-triggered acute kidney injury. RvD5 was shown to reduce biochemical, immune, molecular, and histopathological alterations in the kidneys of LPS endotoxemia in female mice. The mechanism of action of RvD5 was related to the inhibition of NFκB activation and reduction of Keap-1 that ultimately facilitated Nrf2 activity. Because of these molecular mechanisms, RvD5 reduced the endotoxemia-triggered production of cytokines and oxidative stress, thereby reducing the recruitment of leukocytes and kidney damage, reestablishing urea and creatinine to normal levels. Thus, RvD5 deserves consideration for further development as a treatment for endotoxemia-triggered inflammatory kidney injury. Future studies might investigate whether the activity of RvD5 is restricted to LPS endotoxemia or whether it could be used to treat kidney injury caused by other conditions such as toxic agents, auto-immune diseases, non-steroidal anti-inflammatory drugs, some antibiotics, severe burns, allergic reactions, and the breakdown of tumor cells.


Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo ( Colorectal cancer (CRC) is the third most commonly diagnosed cancer in women and men worldwide, with prevalence increasing even in traditionally low-risk countries . NeverthThe agri-food industry produces a large amount of waste and residues along the entire supply chain that still contain a considerable number of bioactive compounds such as polyphenols , saponins, tannins, alkaloids, sterols, triterpenes, peptides, and carbohydrates ,7. In reBubalus bubalis) milk has been demonstrated in in vitro CRC cell models , In our previous report, we documented an involvement of sirtuins, enzymes belonging to the NAD+-dependent histone deacetylases family and involved in several biological processes including apoptosis , in the Increasing evidence suggests that CRC tumour progression is regulated by key factors involved in glycolysis ,36,37. LIn conclusion, by revealing whey-induced cell death in a CRC xenograft model, this study paves the way for the potential reuse of this dairy industry byproduct in potential clinical and nutritional strategies against CRC. Considering the ethical debate on the use of chemotherapy in pets, the nutritional strategy against CRC would also be beneficial for fighting cancer in pets.Bubalus bubalis) was obtained from 30 commercial buffalo farms located in the Caserta region of southern Italy. The milk was delactosed by enzymatic hydrolysis with the enzyme lactase . Subsequently, the whey residue obtained from the ricotta was subjected to reverse osmosis to obtain a concentrated solution of 60% delactosed milk whey (DMW).Milk from Italian Mediterranean dairy buffaloes (v/v) and then injected directly into the LC-MS system for metabolic profile analysis. This used an Agilent high performance liquid chromatograph (HPLC) 1260 Infinity Series  equipped with a DAD (diode array detector) system (Agilent Technologies) and coupled to a quadrupole-time of flight (Q-TOF) mass spectrometer (Agilent Technologies) with a Dual ESI source (Agilent Technologies). Chromatographic separation used a Zorbax Extend C-18 column , maintained at a constant temperature of 30 °C. Elution was carried out at a flow rate of 0.6 mL/min using 0.1% (v/v) formic acid (FA) in water (H2O) as phase A and 0.1% formic acid (FA) in acetonitrile (ACN) as phase B. The gradient was as follows: starting condition 5% B, ramping to 70% B in 4 min, held at 70% B for 1 min, ramping to 80% B in 3 min, and to 100% B in 2 min; finally, lowering to starting condition (5% B) in 5 min and held at 5% B for 1 min as equilibration time. The injection volume was 10 µL. The MS system operated in positive ion mode and MS spectra were recorded in the m/z 50–1700 range as centroid spectra, with a speed of 3.3 spectra/s. The capillary was maintained at 2000 V, fragmentor voltage at 180 V, cone 1 (skimmer 1) at 45 V, Oct RFV at 750 V. Gas flow rate was set at 11 L/min, at 350 °C, and the nebulizer was set at 45 psig. UV spectra were collected by DAD, setting the detection wavelength at 210, 250, and 280 nm. A standard solution was infused by using an isocratic pump  to perform the real-time lock mass correction. The solution consisted of two reference mass compounds: purine  and hexakis -phosphazene . Flow rate was set at 0.06 mL/min, while the detection window and the minimum height were set at 1000 ppm and 10,000 counts, respectively, for reference mass correction. HPLC, UV and MS, and parameters were set using Agilent Mass Hunter Data Acquisition Software, rev. B.05.01 (Agilent Technologies).The DMW sample was diluted in methanol 1:100  software. Compounds were identified by comparing monoisotopic mass values using a freely available electronic database, the Human Metabolome database (HMDB). Of the molecules detected, only those with a mass error of less than 10 ppm and a sufficient score were reported.Mice used in this study were housed in the conventional animal house of the Department of Pharmacy, University of Naples Federico II  and kept in cages equipped with additional environmental conditions. For the xenograft model, 6- to 8-week-old female BALB/c nude mice were used , fed ad libitum with sterile mouse chow and kept under pathogen-free conditions in IVC cages. All animals (n = 22) were anaesthetised by inhalation of enflurane before being sacrificed with carbon dioxide. Every effort was made to minimise the number of animals used and their suffering. The mice were blind randomised, and the experimental procedures and protocols were in accordance with national laws and guidelines (Direttiva 2010/63/UE) and approved by the Italian Ministry of Health.6, 200-μL PBS) into the back of BALB/c nude mice. Ten days after inoculation (once the tumours had reached a size of 150–250 mm3), the mice were randomly assigned to control and treated groups, and treatments were initiated. Tumour size was measured daily with a digital calliper, and tumour volume was calculated using the modified ellipsoid volume formula (volume = π/6 × length × width2). DMW was given orally, at the dose of 0.2 mL/mouse, daily for the duration of the experiment. Eleven days after the start of treatment, the mice were sacrificed, and the tumours were removed, measured by digital calliper, and fixed in formalin or stored at −80 °C for subsequent analyses [The tumour xenograft model of CRC was performed by subcutaneous injection of HCT116 cells , moderate , and severe .Tissue samples were fixed with 10% formalin and embedded in paraffin. Sections were cut at 4 µm, stained with haematoxylin and eosin (H-E), and viewed on microscope  by two independent observers for histopathological examination, which confirmed the diagnosis of colon adenocarcinoma. The extent of tumour necrosis was assessed semiquantitatively, evaluated at low magnification (×40) according to previously published criteria ,40, and Total RNA was purified for each sample using Trizol reagents . The protocol was performed according to the manufacturer’s instructions. The final elution step was repeated, and the purified total RNA was then stored at −80 °C. The amount of total RNA samples was determined using the Thermo Scientific™ NanoDrop™ 2000 spectrophotometer. The level of degradation was analysed by agarose electrophoresis. Samples with distinct 28S and 18S ribosomal RNA bands and band intensities of 28S:18S close to two were selected for this study.® Reverse Transcription Kit  following the manufacturer’s recommendations. Genomic DNA contaminating the RNA samples was removed using 2 μL gDNA wipeout buffer, 1 μg template RNA and RNase-free water to bring the final volume to 14 μL at 42 °C for 2 min. Reverse transcription was carried out using 1 μL Quantiscript reverse transcriptase, 4 μL Quantiscript RT buffer containing Mg2+ and dNTPs, 1 μL RT primer mix, and 14 μL template RNA in a total of 20 μL reaction at 42 °C for 15 min and 95 °C for 3 min. All steps were performed on ice. cDNA samples were stored at −20 °C for later quantitative real-time PCR analysis.First-strand complementary DNA (cDNA) was synthesised with 1 μg total RNA using the QuantiTect® SYBR® Green PCR Kit  on the BIORAD CFX Manager Machine. All primers were designed by the Primer-BLAST software and synthesised by Eurofins Genomics . Samples were prepared according to the manufacturer’s instructions. A total reaction volume of 20 μL contained 10 μL master mix, 2 μL primer, and 8 μL diluted cDNA (1:30). Samples were then centrifuged at 1000× g at 4 °C for 1 min. All cDNA samples were amplified under the following conditions: 95 °C for 5 min, 40 cycles at 95 °C for 10 s, and 60 °C for 30 s. Primers used in this study were: PARP Fw: GCAGAGTATGCCAAGTCCAACAG Rv: ATCCACCTCATCGCCTTTTC, CASP3 Fw: GCAGCAAACCTCAGGGAAAC Rv: TGTCGGCATACTGTTTCAGCA, MLKL Fw: GTGGGAAAGAAGGTGGAAGAG Rv: GCCAAGGGTGATAATATGCTTC, β-ACTIN Fw: CATGTACGTTGCTATCCAGGC Rv: CTCCTTAATGTCACGCACGAT. Data were then quantified using the comparative cycle threshold (CT) method and expression levels were presented as 2−ΔCT normalised to the β-actin housekeeping gene.Quantitative real-time PCR reactions were performed using the QuantiFastThe level of lipid peroxidation was determined by measuring the cellular content of malondialdehyde (MDA) using the colorimetric lipid peroxidation assay kit . According to the manufacturer’s instructions, a solution containing 20 mM Na phosphate buffer at pH 3 and 0.5% TritonX-100 was added to the tissue extracts to avoid non-specific aldehydes that could interfere with absorbance measurements. MDA Colour reagent solution (10 μL) was added to each well and the reaction mixture incubated at room temperature (RT) for 30 min. The reaction solution (40 μL) was also added followed by a second incubation at RT for 1 h. Absorbance was measured at 695 nm using a microplate reader  and total MDA content was calculated by comparing the absorbance of each sample to the standard MDA curve (0–200 μM) and normalising to protein content.g for 10 min at 4 °C. Then, reaction mix (50 μL) was added to pyruvate standard and samples (50 μL) and plate were incubated for 30 min at RT in the dark. The absorbance was measured at 570 nm with a microplate reader  and pyruvate content interpolated from the standard curve.The measurement of pyruvate levels in tissue extracts was performed with Pyruvate Assay Kit , according to manufacturer’s protocol. Deproteinisation was carried out using a 10 kDa spin filter  with centrifugation at 14,000× SIRT3 and SIRT6 fluorometric activity assay kits  were performed according to the manufacturer’s instructions. Briefly, Fluoro-substrate peptide, NAD, developer, and mouse tissue extracts (5 μL) were added to each well of the plate by mixing thoroughly at RT. After 1 h incubation, the reaction was stopped with 20 μL of stop solution to each well, and SIRT3 and SIRT6 activity resulted in fluorescence intensity, measured in a multiplate reader  capable of excitation at a wavelength in the range of 340–360 or 480–500 nm and detection of emitted light in the range of 440–460 or 520–540 nm.Proteins from tissue extract (20–50 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad). Blots were blocked for 1 h at RT in 1× TBS 1% casein blocker  under a gentle shaker. Membranes were then incubated overnight at 4 °C with specific primary antibodies: anti-SIRT3 ; anti-SIRT6 ; antiperoxisome proliferator-activated receptor α ; anti-PPAR-γ ; antilactate dehydrogenase A ; antimixed lineage domain-like protein , antireceptor interacting protein 1 , anti-RIPK3/RIP3 , antinuclear factor kappa B ; cleaved PARP , cleaved caspase-3 , Phospho-p44/42 MAPK Erk1/2 Thr202/Tyr204 , p44/42 MAPK Erk1/2 , Phospho-Histone H3 (Ser10) , c-Myc , anti-α-tubulin ; anti-actin , and anti-glyceraldehyde-3-phosphate dehydrogenase . After 1 h incubation with HRP-conjugated secondary antibodies , the immunocomplexes were visualized, with Excellent chemiluminescent substrate kit , using the ChemiDoc Imaging System with Image Lab 6.0.1 software (Bio-Rad Laboratories). After background subtraction, the densities of immunoreactive bands were obtained by ImageJ 1.52n software  and expressed as arbitrary units (AU). For Phospho-p44/42 MAPK Erk1/2 Thr202/Tyr204, p44/42 MAPK Erk1/2, Phospho-Histone H3 (Ser10), c-Myc, cleaved PARP, and cleaved caspase 3, immunoreactive bands were detected on X-ray films using an enhanced chemiluminescent kit and quantified by densitometry using ImageJ software.Immunofluorescence analysis was performed on deparaffinised sections from mice. Samples were placed in antigen retrieval buffer  and heated in the microwave for 20 min. To reduce background fluorescence, the slides were washed twice in phosphate-buffered saline (PBS) and incubated for 30 min in Tris-buffered saline (TBS) containing 50 mM ammonium chloride. Permeabilisation with PBS containing 0.2% Triton X-100 was carried out for 10 min at RT. The sections were blocked with foetal bovine serum (FBS) and saponin (0.1 g/mL) for 1 h at RT, and then, incubated with primary antibodies: anti-MLKL antibody (1:300), anti-SIRT6 (1:500), anti-SIRT3 (1:300), anti-caspase-3 , and anti-PARP , before 1 h incubation at RT with secondary antibodies labelled with Alexa Fluor 633. For all slides, cellular architecture was marked by Phalloidin 488  and nuclei stained with 4,6-diamidino-2-phenylindole  for 7 min. Sections were quenched for autofluorescence using the Vector TrueVIEW Autofluorescence Quenching Kit  and mounted in Vectashield Mounting Medium . Slides were imaged using a Zeiss LSM 700 confocal microscope  with a plan apochromat ×63 (NA1.4) oil immersion objective and fluorescence intensity values, reported as arbitrary fluorescence units (AFU), evaluated by ImageJ 1.52n software .Cytokine (IFN-γ and TNF-α) levels in mouse extracts were determined by ELISA assays , according to the manufacturer’s instructions. Standards and samples (100 μL) were incubated overnight at 4 °C with gentle shaking to allow for cytokines binding to the wells, by the immobilised antibody, of precoated 96-well plate. The wells were washed 3 times and biotinylated antimouse IFN-γ and TNF-α antibodies (100 μL) were added and the plate was incubated at 37 °C for an additional hour. After washing away unbound antibody, HRP-conjugated streptavidin (100 μL) was added to each well and, after 45 min at 37 °C and 4 washes, TMB substrate solution (100 μL) was included for 30 min to develop colour, in proportion to the amount of cytokines. The addition of stop solution (100 μL) allowed a colour change from blue to yellow and the colorimetric intensity was measured at 450 nm using a microplate reader . The content of cytokines in the samples was determined by plotting the absorbance values against the concentrations of the individual standard curves.t-test. A p-value < 0.05 was considered significant.All data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 9 software . Comparisons between tissue samples from vehicle- and DMW-treated mice were performed using the two-tailed unpaired Student’s Patent n. 102021000024911; deposition date, 29 September 2021.


Gleevec (Imatinib Mesylate) is the first choice breakpoint cluster region (BCR)/ABL targeted oral therapy that produced a complete response almost in 71% to 80% of patients affected with CML. A complete blood count (CBC) of 37 patients was done during diagnosis, however only 21 showed abnormal CBC values which were selected for the study. Karyotyping study using bone marrow samples was performed on 21 CML patients for the conformation of 9;22, however, fluorescence in situ hybridisation was performed for the detection of the BCR–ABL fusion gene of 15 patients. Out of 21, 17 patients showed Ph-positive   translocation. Sixteen CML patients showed standard translocation however only CML patients showed a three-way variant/complex translocation with six additional chromosomes, 52XX, t ,+6,+8, i(9), +18,+19,+21 + der22 t). Here we report we report a novel case of six additional chromosomes with the three-way translocation of 52XX, t ,+6,+8, i(9), +18,+19,+21 + der22 t in blast phase. It is a result of a combination of two genes Breakpoint cluster Region (BCR) and Ableson (ABL) that form the fusion gene BCR–ABL that produces a chimeric protein, tyrosine kinase activity. This tyrosine kinase activity is accountable for regulating and keeping the adult tissues and helps in the signaling pathway and regulating cell division.Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disease, produced through the balance translocation between the long arms of chromosomes 9 and 22, so-called the Philadelphia chromosome.,3,4 However genetic additional abnormalities happen in <10% of chronic myeloid leukemia cases during the diagnosis stage and other 60% to 70% are detected in accelerated and blast crisis.,6 In CML trisomy 19 has usually happened as a secondary abnormality, non-common as trisomy 8, (17q) and extra chromosome Ph chromosome. Trisomy 19 has been observed only in about 15% of CML cases.–9The translocation 9 and 22 chromosome is established in up to 90% of CML-positive cases. On the other hand variant/complex translocations including one or more additional chromosomes instead of  were perceived only <8% of CML patients. This oral therapy is the first choice of treatment for almost 80% to 90% of CML patients of all phases. Imatinib Mesylate orally at 400 mg/daily dos indeciduate complete hematologic response in 95% of CML patients and complete cytogenetic responses in 61% to 90. After 18 months of follow-up, about 90% of CML patients are unable to transform to the accelerated or blastic phases. In conclusion, this drug has most effective for prolonged survival and better quality of life.,14Imatinib Mesylate (Gleevec) is an orally designed BCR/ABL protein tyrosine kinase inhibitor against BCR/ABL, platelet-derived growth factor receptor (PDGF-R), and c-kit.Here we reported a novel case of ACAs with three way translocation in CML patient with 6 additional chromosome 52XX, t ,+6,+8, i(9) , +18,+19, +21 + der22 t.The current study was conducted between July 2014 and September 2020 in different Hospitals in Quetta and Allied Hospital Faisalabad. In this study, a total of 21 patients with CML were enrolled of which 11 were male and 10 were female. All participants in the current study completed a written informed consent form. The study was approved by BUITEMS’s local ethical committee in Quetta, Pakistan.For complete blood cell count, we used a hematological analysis for the analysis of red blood cells, white blood cells (WBC), hemoglobin, mean corpuscular volume, platelet, and hematocrit.Karyotyping analysis was executed by the GTG-banding method. For this purpose, bone marrow samples were studied in direct or short-term (24-hour) cultures, and 22 metaphases were examined. In addition, karyotypes were expressed by the International System for Human Cytogenetic Nomenclature.The fluorescence in situ hybridisation test was executed by directly labeled dual color LSI/CEP probes provided by the supplier’s instructions (BCR/ABL– Oncor) for the recognition of BCR/ABL gene fusions. In this technique, 200 metaphase or interphase cells were counted.Table 3/µL, red blood cell 2.5 × 103/µL, Hbg 6.6 g/dL, PLT 287 × 103/µL, MCV 91/FL, MCH 26/pg, HCT (23%), MCHC 39.9 g/dL, neutrophils 52%, lymphocytes 01, eosinophils 03%, monocytes 05%, basophils 02%, promyelocytes 07%, myelocytes 15% meta-myelocytes 09% blast cells less then (5%). Cytogenetic bone marrow karyotyping result of the patient showed 46XX, t,)  . The patient was surviving in CP treated with Gleevec 400 mg/day.Table 3/µL, red blood cell 3.1 × 103/µL, hemoglobin 7.4 g/dL, PLTs 25 × 103/µL, MCV 109/FL, MCH 31.6/pg, HCT 25.5%, blast cells were 82% and the patient was migrating from first chronic to blast phase third phase of the disease. Cytogenetic karyotyping result of the patient showed 52XX, t  , +6, +8, i(9) , +18,+19,+21 + der22 t . All cells showed translocation among chromosomes 1q23, 9q34, and 22q11.2, +6, +8 an isochromosome 9, +18, +19, +21, and a derivative chromosome 22 generated by a translocation between chromosomes 9q34 and 22q11.2. Results of immunohistochemically stains on blast cells show TdT; diffuse positive, PAX-5; diffuse positive; CD34, positive, CD3; positive MPO; negative and CD117 also showed negative results. Before shifting to the second-generation drug Nilotinib (Tasigna) the patient died. After fluorescence in situ hybridisation analysis of 21, 11 CML patients shows BCR–ABL (+) gene fusion however, 04 showed BCR–ABL (–) and gene fusion.Because of regular pain, fever, massive splenomegaly, and weight loss doctor recommends bone marrow aspiration and cytogenetic tests for follow-up. The results of peripheral blood counts of bone marrow show that WBC was 67 × 10,15,16 Extra genetic discrepancies happen in <10% of CML cases during diagnosis. The most common chromosomal aberrations in addition to a Ph-chromosome are + 19, +8, and + Ph,), However, –Y, +21, +17, −7, and − 17 were also found in fewer CML patients.,17 Cytogenetic abnormalities like trisomy 8 (+8) and an extra copy of the Philadelphia chromosome are more common in CML cases.  is established in thereabouts 90% of CML patients, on the other hand, complex/variant chromosome translocation is observed only in nearly 5% to 8% of cases. In such cases, additional chromosomes other than 9;22 are tangled.,20In CML, overall, 41 cases of 52 chromosomes with additional 06 chromosomes have been reported. However, 52 chromosomes with 06 additional chromosomes and the three-way translocation at the same patient in CML have been reported twice earlier in the available literature.In this we investigate a new case of 52, XX, t, +6 + 8,, +18 + 19, +21 + der22 t  [20] in CML patient was cross checked at Mitel man Database of Chromosome Aberrations and Gene Fusions in Cancer.,21Nearly 30% of CML patients in accelerated and 71% to 80% and CML patients in BP have additional chromosomal abnormalities. Among the various additional chromosomal abnormalities, trisomy 08 and an additional copy of the Ph chromosome are usual in CML cases. Diverse additional chromosomal abnormalities have been exposed to be linked with diverse effects on drug response and survival. More or less ACAs are linked with disease advancement and drug resistance, while they may additionally just reflect the hereditary unpredictability prompted by incessant activation of BCR–ABL1.,22 In this study all the patients were treated with Imatinib Mesylate. Recently other protein tyrosine kinase inhibitors drugs approved by the FDA, and EMA like dasatinib, nilotinib, and bosutinib are used for the treatment of CML. Imatinib Mesylate is the first generation drug, however dasatinib, nilotinib and bosutinib are second generation tyrosine kinase inhibitors drugs.Imatinib Mesylate (IM) is the first-line therapy for CML. IM is a BCR–ABL protein tyrosine kinase inhibitor against c-Abl, BCR/ABL. IM exposed significant effects in all phases of CML. The prescribed dose 400 mg/day for CP CML and 600 mg/day for the accelerated phase is recommended.We are thankful to the ORIC BUITEMS, Quetta, and Faculty of Life Sciences BUITEMS, Quetta for providing funding for this research. Under (Ph.D. Registration ID no. 27934).


The late endosome/lysosome (LE/Lys) lipid bis(monoacylglycero)phosphate (BMP) plays major roles in cargo sorting and degradation, regulation of cholesterol and intercellular communication and has been linked to viral infection and neurodegeneration. Although BMP was initially described over fifty years ago, the enzymes regulating its synthesis remain unknown. The first step in the BMP biosynthetic pathway is the conversion of phosphatidylglycerol (PG) into lysophosphatidylglycerol (LPG) by a phospholipase A2 (PLA2) enzyme. Here we report that this enzyme is lysosomal PLA2 (LPLA2). We show that LPLA2 is sufficient to convert PG into LPG in vitro. We show that modulating LPLA2 levels regulates BMP levels in HeLa cells, and affects downstream pathways such as LE/Lys morphology and cholesterol levels. Finally, we show that in a model of Niemann-Pick disease type C, overexpressing LPLA2 alleviates the LE/Lys cholesterol accumulation phenotype. Altogether, we shed new light on BMP biosynthesis and contribute tools to regulate BMP-dependent pathways. Lysosomal phospholipase A2 (LPLA2) is the PG to LPG converting enzyme in the BMP biosynthetic pathway, and changes in LPLA2 levels in cells affect cholesterol levels and late endosome/lysosome morphology. LE/Lys are a sorting crossroad for both proteins and lipids that will be targeted towards retrograde transport, degradation or extracellular export. Indeed, in addition to their essential intracellular sorting function, LE/Lys can also fuse with the plasma membrane and release ILVs as nanovesicles in the extracellular milieu, known as exosomes.Select lipids have emerged as key regulators of intracellular trafficking and consequently, as essential components of cellular homeostasis. Amongst them, bis(monoacylglycero)phosphate  is uniquely localized to late endosomes/lysosomes (LE/Lys), where it is further concentrated in intralumenal vesicles (ILVs)3. BMP also affects the correct sorting and degradation of proteins and lipids. For example, it plays a key role in the retrograde transport of mannose-6-phosphate receptor to the trans-Golgi network1 and facilitates the correct degradation of sphingolipids4. BMP also promotes cell survival through the stabilization of lysosomes5.As BMP presents with the intrinsic capacity to induce the formation of multivesicular liposomes in vitro, BMP and its binding partner Alix are considered to be key players in the formation of ILVs7. The majority of cellular cholesterol originates from extracellular cholesteryl ester-rich low-density lipoprotein (LDL) particles that are internalized by the LDL receptor and trafficked to the lumen of LE/Lys. Cholesteryl esters are then hydrolyzed by lysosomal acid lipase into free cholesterol. Free cholesterol can then be incorporated into BMP-rich ILVs membranes before being redistributed to the LE/Lys limiting membrane and then to the rest of the cell7. It was recently proposed that Niemann Pick type C 2 (NPC2) plays a key role in bringing together cholesterol and BMP-rich ILVs membranes with the limiting membrane of LE/Lys, as it can bind both BMP and proteins on the LE/Lys limiting membrane 8.Importantly, BMP controls cholesterol storage capacity in LE/Lys and subsequently, intracellular cholesterol levels12. Moreover, most neurodegenerative diseases show alterations in BMP-regulated pathways and BMP levels are profoundly altered in patients with neurodegenerative disorders of childhood and aging, as well as in model systems of these diseases; e.g., lysosomal storage disorders16, Alzheimer’s disease18, Down Syndrome17 and dementia with Lewy bodies19. Finally, the presence and abundance of BMP regulate the intracellular delivery of certain candidate drugs, such as cationic cell-penetrating peptides20 and antisense oligonucleotides21.Unsurprisingly, given the number of essential homeostasis pathways it regulates, BMP has also been implicated in a vast number of diseases. BMP is required for the initial entry step of specific viruses—e.g., vesicular stomatitis virus, dengue virus, Lassa virus—through the back-fusion of ILVs with the limiting membrane of LE/Lys and release of the virus nucleocapsid within the cell cytoplasm22, and despite the fact that its levels and localization can be probed by a specific antibody1, its exact biosynthetic enzymes remain unknown. Here we propose a major advance in answering this question lingering for over 55 years.Modulating endogenous BMP levels would thus be of crucial interest not only to understand the molecular mechanisms involved in the pathogenesis of these disorders, but also potentially as a therapeutic tool. These studies are limited by the fact that although BMP has been described as early as 196723. In the currently prevailing model of BMP biosynthesis24, the first and limiting step involves the conversion of sn-3:sn-1’ PG to sn-3:sn-1’ LPG by an enzyme with phospholipase A2 (PLA2) activity. The sn-3:sn-1’ LPG intermediate then goes through two transacylation steps to give the final product, sn-1:sn-1’ BMP. Of all sixteen classes of PLA2 enzymes, only lysosomal PLA2 (LPLA2 or Group XV LPLA2), coded by PLA2G15, localizes to the LE/Lys25. We thus tested whether LPLA2 could be the PLA2 enzyme regulating the first and limiting step of the BMP biosynthetic pathway. LPLA2 was initially identified in Madin-Darby Canine Kidney cells26 and then purified from bovine brain27. The human and mouse isoforms contain 412 amino acids, with a signal peptide cleavage site, a lipase AXSXG motif, and four N-linked glycosylation sites28. A serine, aspartic acid, and histidine catalytic triad is essential for enzymatic activity29. LPLA2 displays 1-O-acylceramide synthase activity as well as PLA1 and PLA2 activities, is Ca2+-independent and has an acidic pH optimum30. Its known substrates include phosphatidylcholine (PC), phosphatidylethanolamine (PE), PG, and phosphatidylserine (PS) but not phosphatidylinositol (PI) or sphingomyelin (SM)31.It has been established that phosphatidylglycerol (PG) is the precursor in the de novo synthesis of BMPIn this study, we first confirm that LPLA2 is sufficient to catalyze the conversion of PG to LPG in an in vitro liposomes assay. We further show that over-expressing or down-regulating LPLA2 leads to increased or decreased BMP levels in human cell lines, respectively. In addition, we show that decreasing LPLA2 levels also affect BMP-dependent pathways, such as LE/Lys homeostasis and cholesterol regulation. Finally, we demonstrate that in a cellular model of Niemann-Pick disease type C, a lysosomal storage disorder in which cholesterol accumulates in LE/Lys, overexpressing LPLA2 alleviates the cholesterol accumulation phenotype. Taken together, our results strongly support that LPLA2 plays a key functional role in the BMP biosynthetic pathway and provide tools to regulate BMP levels and BMP-dependent pathways in control and diseased model systems.31. Some LPG 18:1 was also detected in the presence of LPLA2 , suggesting that LPLA2 may have a stronger PLA2 than PLA1 activity towards PG.We first tested whether the PLA2 activity of LPLA2 was sufficient to catalyze the conversion of PG to LPG, the first step in the biosynthesis of BMP. To finely dissect the activity of LPLA2, we used an in vitro system where purified recombinant LPLA2 was incubated with liposomes containing PC and PG 16:0/18:1. After incubation at 37 °C for 1 h, the lipid content of the liposomes was extracted and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) Fig. . While nLA2 Fig. , indicatp = 0.907, unpaired Student’s t test, Fig. p < 0.05, unpaired Student’s t test, Fig. We also tested the pH-sensitivity of the PLA2 activity of LPLA2. Indeed, while endoplasmic reticulum (ER) has a neutral pH (7.4), LE exhibit an acidic pH (5-5.5) that is tightly linked to their function. In order to gain insight into the functional localization of LPLA2, we tested whether the capacity of LPLA2 to convert PG into LPG is pH-dependent by incubating recombinant LPLA2 with PC:PG liposomes at a pH of 5.5 or 7.4. LPG 16:0 was detected in both pH conditions in comparable amounts  reporter (Lpla2-GFP) or with the control empty GFP vector were processed by immunocytochemistry with antibodies targeting endogenous BMP Fig. . Quantifvs. 100 ± 7% in cells treated with a non-targeting siRNA)  in HeLa cells, a technique that does not require the use of lipid-based transfection agents. We fixed the siRNA-transfected cells and processed them for immunocytochemistry with antibodies directed towards endogenous BMP and LPLA2 Fig. . ControlNA) Fig. . We alsoNA) Fig. . BMP levNA) Fig. . We alsoNA) Fig. . DecreasWe then reasoned that if LPLA2 levels were indeed regulating BMP levels, LPLA2 levels would also impact structural and functional cellular outcomes associated with BMP levels, such as LE/Lys morphology and cholesterol levels.To test the impact of LPLA2 levels on LE/Lys, protein levels of LAMP1, a LE/Lys marker, were analyzed using western blot Fig. . We foun7. We fixed siRNA-treated cells and processed them for staining with filipin, a fluorescent antibiotic that recognizes free cholesterol. We found that in cells showing reduced levels of LPLA2, where BMP levels were also reduced  disease, a fatal neurodegenerative lysosomal storage disorder associated with accumulation of cholesterol and other lipids in the endolysosomal system32. While levels of BMP and cholesterol shift in parallel in non-diseased cells  or phospholipases D (conversion of phosphatidylcholine to PA). Of note, diacylglycerol kinase α activity has been linked to the formation of mature MVBs and the secretion of exosomes in T lymphocytes35, suggesting a possible functional connection between levels of BMP, DG, and PA.We also showed that overexpression of Lpla2 leads to increased levels of BMP, as demonstrated both by immunofluorescence and by LC-MS/MS. In addition to increased BMP levels, lipidomic analysis revealed that overexpression of Lpla2-GFP also resulted in an increase of diacylglycerol (DG) and phosphatidic acid (PA) and a decrease in plasmogen lysophosphatidylethanolamine (LPEp) and N-Acyl Serine (NSer), compared to overexpression of GFP only. Although small in effect size, the fact that both DG and PA were increased is interesting, as these two lipids are metabolically linked. Indeed, DG can be converted to PA by the action of diacylglycerol kinases, while PA can be converted to DG through lipin activity7. Similarly, the observed increase in levels of SM and dhSM corroborates the proposed involvement of BMP in the degradation of sphingolipids4.Conversely, we observed that decreasing LPLA2 levels resulted in decreased levels of BMP, as demonstrated both by immunofluorescence and by LC-MS/MS. As expected, LC-MS/MS results displayed more variability than imaging results did. This is likely due to the fact that while imaging focuses on one population of cells (with decreased LPLA2), LC-MS/MS results correspond to the average of multiple populations of cells (with varying levels of LPLA2). In addition to decreased BMP 36:4 levels, lipidomic analysis of LPLA2-depleted cells revealed multiple changes compared to controls, including decreased levels of free cholesterol (FC) and phosphatidylethanolamine (PE) and increased levels of alkyl phosphatidylcholine (PCe), sphingomyelin (SM), Acyl Carnitine (AC) and dihydrosphingomyelin (dhSM). This decrease in free cholesterol was confirmed by filipin staining and is in good accordance with the proposed role of BMP in the control of cellular cholesterol levels1. This suggests that decreasing BMP levels or sterically hindering its accessibility with an antibody interferes with its function in the proper formation of ILVs, either by disrupting its interaction with Alix3, by inhibiting the formation of BMP clusters proposed to play an essential role in the release of ILVs from the limiting membrane36 or both. However, we cannot fully rule out the alternative possibility that decreasing BMP levels leads to the formation of clusters of aggregated individual LE/Lys.Levels of BMP also profoundly impact the morphology of LE/Lys. Indeed, we observed modifications in the number and morphology of LAMP1-positive LE/Lys. The aberrant LAMP1 membranes observed after decreasing LPLA2 and BMP levels are reminiscent of the inner membrane disorganization that was reported after internalization of an antibody directed against BMP37, but not in LE/Lys. As proposed by others39, the two most likely sources of PG are autophagy/mitophagy and inter-organelles contact sites between LE/Lys and a PG-containing organelle. This will require further investigation.Altogether, our results strongly support that LPLA2 plays a central role in the biosynthesis of BMP, and consequently on BMP-dependent pathways. An important issue that remains unsolved is the source of the PG used to synthesize BMP. Indeed, PG is mostly found in mitochondria40, suggesting that there could be a positive feedback loop at play that enables both the continual synthesis of BMP and the increased catabolism of other lipids by LPLA2 and other lipases in the LE/Lys. Indeed, the unique sn-1:sn-1’ configuration of BMP makes it a poor substrate for phospholipases39 while its negative charge at acidic pH makes it a docking site for lipid binding and lipid degrading enzymes, thus facilitating lipid catabolism4.Interestingly, it has been reported that anionic phospholipids, and in particular BMP, can stimulate PLA2 enzymatic activityPla2g15−/− mice have been reported in the literature41. In these mice, exon 5 of Pla2g15—which codes for the lipase motif—was deleted, leading to an absence of PLA2 or 1-O-acylceramide synthase activity. The lifespan and fertility of Pla2g15−/− mice were unaffected, yet they showed splenomegaly, as well as an increased number of enlarged lung alveolar macrophages that accumulated phospholipids41. This model was also used to propose a role of LPLA2 in host immunity to respiratory pathogenic bacteria42. Another Pla2g15−/− mouse model supported that LPLA2 could also play a role in atherosclerosis43. To our knowledge, BMP levels have not been investigated in Pla2g15−/− mice. However, it is interesting to note that levels of its precursor PG were found to be increased in one of these models41, in good accordance with our hypothesis.Of note, 44. Interestingly, while BMP typically accounts for only 1–2% of total phospholipids, it is highly enriched in alveolar macrophages, representing up to 17% of all phospholipids45, drawing parallels between LPLA2 activity and BMP levels and supporting our hypothesis that LPLA2 plays a key role in BMP synthesis.While LPLA2 is ubiquitously expressed, it is highly enriched in alveolar macrophages and has a 50-fold higher activity in these cells than in any other cell type46. Although the function of LPLA2 was not explored further in this report, these results are in good accordance with our findings.Very recently, LPLA2 was a hit in two genome-wide CRISPR screens designed to identify genes that, when knocked out, alter levels of cholesterol or BMP47. It is also well established that the LXR/RXR pathway acts as a cholesterol sensor and regulates intracellular cholesterol metabolism48. This suggests that there could be a transcriptional component to the cholesterol/BMP relationship, through control of LPLA2 expression.Moreover, the expression of LPLA2 has been proposed to be regulated by an RXR-dependent pathway33, and highlighting the potential of LPLA2 as a therapeutic target for NPC. As BMP and BMP-dependent pathways are involved in a vast array of disorders, we anticipate that modulation of LPLA2 levels, activity, or both, could be of therapeutic relevance in a number of these diseases.We also provide evidence that expressing Lpla2 in a cellular model of NPC disease is sufficient to alleviate the LE/Lys cholesterol accumulation phenotype that is typical of this disorder. We observe that the filipin-positive puncta first decrease in size and then in number, supporting the known beneficial effect of BMP in NPC models43 as well as LPLA2-selective inhibitors31 already exist, yet it is likely that additional models and compounds will be developed in the near future. Based on our findings, we expect that present and future models and compounds will provide genetic and pharmacological tools to assess the contribution of LPLA2 and BMP to pathogenic mechanisms of viral infection and neurodegeneration, and pave the way for potential therapeutic avenues for these disorders.Our findings, taken together, provide strong evidence that LPLA2 is one of the elusive enzymes regulating the first step of BMP biosynthesis first alluded to several decades ago. We thus believe that we are strongly contributing to a long-standing question in the field of cell biology with implications for intracellular trafficking, lysosomal degradation, intercellular communication, and associated disorders stemming from the dysfunction of these pathways. Mouse models in which LPLA2 is selectively targetedLipids were obtained from Avanti Polar Lipids. HEPES, KCl, MES, dimethyl sulfoxide (DMSO), filipin, sucrose, bovine serum albumin (BSA) were from Sigma-Aldrich. Saponin was from Acros. Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Media (MEM), Hank’s balanced salt solution (HBSS), and Phosphate buffered saline (PBS) were from Gibco.Recombinant active human lysosomal phospholipase A2 was obtained from Echelon Biosciences. The antibodies were obtained from the following sources: rabbit antibodies to LAMP1 , 1/500 for western blots (WB)), LPLA2 ; a rat monoclonal antibody (mAb) to LPLA2  and mouse mAbs to BMP  and GAPDH . Peroxidase-conjugated secondary antibodies were from Biorad and Alexa-conjugated fluorescent secondary antibodies were from Life Technologies.g. Lipids were then dried using a SpeedVac vacuum concentrator. The resulting dried lipids were resuspended in 100 μL of buffer A  or buffer B  and sonicated in a water bath sonicator for 10 min to help with the formation of multilamellar vesicles/liposomes (crude liposomes). The resulting solutions were stored overnight at 4 °C. The next day, liposomes were sonicated for 10 min and placed on ice. They were then diluted 1/100 in buffer A or B to prepare the working solution. For t0 conditions, lipids were extracted right away. For t60 conditions, lipids were extracted after incubation for 1 h at 37 °C with shaking. For t60 + LPLA2 conditions, lipids were extracted after incubation for 1 h at 37 °C with shaking in the presence of recombinant active human lysosomal phospholipase A2 . All buffers and tubes used for lipid extraction were pre-chilled at 4 °C. Lipids were extracted from each initial 50 μL reaction volume using 900 μL chloroform:methanol (5:4). The resulting solution was vortexed for 10 sec and supplemented with 200 μL KCl 1 M. The resulting solution was vortexed for 30 s and incubated on ice for 1 min. This step was repeated twice. The solution was then centrifuged for 2 min at 16,100 × g. The organic layer was then transferred to a new tube, dried using a SpeedVac vacuum concentrator and stored at −80 °C.Lipids used were PC 16:0/18:0 and PG 16:0/18:1 PG at 10 mg/mL in chloroform (Avanti Polar Lipids). All experiments were performed protected from light. On day one, PC was either used alone or mixed with PG in a 90/10 ratio for a final volume of 50 μL. The resulting lipid mixes were vortexed for 10 s and centrifuged for 1 min at 16,100 × r2 > 0.9 in QC dilution series. Quantification data was extracted using Agilent MassHunter Quantitative Analysis (QQQ) software version B.08. The data were manually curated to ensure that the software integrated the right peaks. Areas under curve (AUC) of the extracted ion chromatograms peaks for each MRM transition were extracted to Microsoft Excel.Dried lipid extracts were re-suspended in 50 μL of chloroform/methanol (C/M) 2/1 v/v. 10 µL of these solutions were transferred to mass spectrometry vials and diluted 1/1 v/v with C/M 2/1 containing 10 µg/mL PC 14:0/14:0, 10 µg/mL LPC 17:0, 2 µg/mL PG 14:0/14:0, 2 µg/mL LPG 14:0, 2 µg/mL PS 14:0/14:0, and 2 µg/mL LPS 17:0. 2 µL of each sample were pooled together to make a quality control (QC) sample for instrument response stability monitoring. These solutions were used directly for LC-MS/MS analysis, without further dilution. In addition, the QC samples were also diluted 2×, 4×, 8×, and 16× with C/M 2/1 v/v for response linearity monitoring. Chromatographic separation was achieved on an Agilent 1290 Infinity Binary Pump, using hydrophilic interaction liquid chromatography (HILIC) on a Kinetex HILIC column . Gradient elution used solvents A (95% Acetonitrile/5% 25 mM ammonium acetate pH 4.6) and B (50% Acetonitrile/50% 25 mM ammonium acetate pH 4.6) as follows: from 1% to 25% B in 6 min, from 25% to 90% B in 1 min, back to 1% B in 0.1 min, keep at 1% B for 3 min (runtime: 10.1 min). The flow rate was 0.5 mL/min and the column temperature 30 °C. Quantification of PC, LPC, PG, and LPG was accomplished on an Agilent 6490 triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) transitions under both positive (headgroup-specific transitions) and negative (fatty acyl-specific transitions) electrospray ionization (ESI) with the following parameters: gas temperature, 200 °C; gas flow, 12 L/min; capillary voltage, 3500 V. 10 QC samples were first analyzed to stabilise instrument response. When instrument stability was satisfactory, the analytical sequence was started. Blank and QC were injected every 11 sample injections to monitor the stability of the instrument response. QC dilution series was used to check linearity of response. To be quantified in the samples, an MRM transition must pass the following criteria: average signal in blank lower than 10% average signal in pooled QC, RSD < 25% over all the QC, Pearson 2 atmosphere in DMEM with Glutamax supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin . Human NPC1-deficient fibroblasts (GM18453) were obtained from the Coriell Institute and maintained at 37 °C in a humidified 5% CO2 atmosphere in MEM supplemented with 15% fetal bovine serum and 1% penicillin/streptomycin . Cells were negative for mycoplasma contamination.HeLa cells were maintained at 37 °C in a humidified 5% COLpla2-GFP and the corresponding empty GFP vector were obtained from Origene (MG225901 and PS100010). HeLa cells and NPC fibroblasts were transiently transfected using Lipofectamine LTX (Life Technologies) and protein expression was assessed after 24 h  or after 48 h (NPC fibroblasts). For KD experiments, Accell Human PLA2G15 siRNA SMARTpool and Accell Non-targeting Pool were obtained from Horizon Discovery. HeLa cells were treated with Accell Human siRNA using Accell siRNA delivery media (Horizon) as per the manufacturer’s instructions and LPLA2 knock-down was assessed at 120 h.50. Briefly, lipid extracts were prepared from cell lysates spiked with appropriate internal standards using a modified Bligh and Dyer method, and analyzed on a platform comprising Agilent 1260 Infinity HPLC integrated to Agilent 6490A QQQ mass spectrometer controlled by Masshunter v 7.0 (Agilent Technologies). Glycerophospholipids and sphingolipids were separated with normal-phase HPLC as described before18, with a few modifications. An Agilent Zorbax Rx-Sil column  maintained at 25 °C was used under the following conditions: mobile phase A  and mobile phase B ; 95% A for 2 min, decreased linearly to 30% A over 18 min and further decreased to 25% A over 3 min, before returning to 95% over 2 min and held for 6 min. Separation of sterols and glycerolipids was carried out on a reverse phase Agilent Zorbax Eclipse XDB-C18 column  using an isocratic mobile phase, chloroform, methanol, 0.1 M ammonium acetate (25:25:1) at a flow rate of 300 μL/min. Quantification of lipid species was accomplished using multiple reaction monitoring (MRM) transitions52 under both positive and negative ionization modes in conjunction with referencing of appropriate internal standards: PA 14:0/14:0, PC 14:0/14:0, PE 14:0/14:0, PG 15:0/15:0, PI 17:0/20:4, PS 14:0/14:0, BMP 14:0/14:0, APG 14:0/14:0, LPC 17:0, LPE 14:0, LPI 13:0, Cer d18:1/17:0, SM d18:1/12:0, dhSM d18:0/12:0, GalCer d18:1/12:0, GluCer d18:1/12:0, LacCer d18:1/12:0, D7-cholesterol, CE 17:0, MG 17:0, 4ME 16:0 diether DG, D5-TG 16:0/18:0/16:0 (Avanti Polar Lipids). Lipid levels for each sample were calculated by summing the total number of moles of all lipid species measured by all three LC-MS methodologies, and then normalizing the total to mol %. The final data are presented as mean mol %.Lipidomics profiling was performed using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS)Lipid abbreviations are as follows: (FC) Free Cholesterol, (CE) Cholesterol Ester, (AC) Acyl Carnitine, (MG) Monoacylglycerol, (DG) Diacylglycerol, (TG) Triacylglycerol, (Cer) Ceramide, (dhCer) Dihydroceramide, (SM) Sphingomyelin, (dhSM) Dihydrosphingomyelin, (Sulf) Sulfatide, (MHCer) Monohexosylceramide , (LacCer) Lactosylceramide, (GM3) Monosialodihexosylganglioside, (GB3) Globotriaosylceramide, (PA) Phosphatidic acid, (PC) Phosphatidylcholine, (PCe) Alkyl phosphatidylcholine, (PE) Phosphatidylethanolamine, (PEp) Plasmalogen phosphatidylethanolamine, (PS) Phosphatidylserine, (PI) Phosphatidylinositol, (PG) Phosphatidylglycerol, (BMP) Bis(monoacylglycero)phosphate, (AcylPG) Acyl Phosphatidylglycerol, (LPC) Lysophosphatidylcholine, (LPCe) Alkyl lysophosphatidylcholine, (LPE) Lysophosphatidylethanolamine, (LPEp) Plasmogen lysophosphatidylethanolamine, (LPI) Lysophosphatidylinositol, (LPS) Lysophosphatidylserine, (NAPE) N-Acyl Phosphatidylethanolamine, (NAPS) N-Acyl Phosphatidylserine, (NSer) N-Acyl Serine.53. Briefly, cells grown on glass coverslips were washed once with HBSS and fixed with a solution of 4% paraformaldehyde (Electron Microscopy Sciences) and 4% sucrose (Sigma) in HBSS for 20 min at room temperature. Cells were then washed twice in HBSS and incubated with NH4Cl (50 mM in HBSS) for 10 min. Cells were then washed twice in PBS and permeabilized with solution A  for at least 45 min at 37 °C. They were then incubated with primary antibodies diluted in solution A overnight at 4 °C. The next day, cells were washed three times in solution A and incubated with fluorescent secondary antibodies diluted in solution A for 1 h at room temperature. When appropriate, filipin (0.5 mg/mL) was added to the secondary antibodies solution. Cells were then washed again three times in solution A. Cells were finally washed once with PBS and coverslips were mounted in Vectashield mounting medium (Vectorlabs).Immunofluorescence experiments were performed as in ref. 54, as in55.Z-stacks were acquired by confocal laser scanning microscopy (Zeiss LSM 800). Fluorescence was collected with a 63× plan apochromat immersion oil objective (NA 1.4). When appropriate, the AiryScan module of the confocal microscope was used to generate higher-resolution z-stacks. Extraction of single z-frame and maximum intensity projections were performed with ImageJ software. Integrated densities, i.e., cell area × [(mean cell intensities summed on the whole z-stack)-(mean background intensities summed on the whole z-stack)], were measured with Image J software. The number, size, and intensity of puncta were quantified with ICY softwareg at 4 °C. Pellets were resuspended in RIPA buffer (Pierce) complemented with cOmplete protease inhibitor cocktail (Roche) and proteins were extracted on a wheel at 4 °C for at least 30 min. Samples were then centrifuged for 15 min at 16,000 × g and proteins in the supernatant were processed for protein dosage . SDS–PAGE was performed on samples  loaded in NuPAGE 4–12% Bis-Tris gels (Life technologies). Wet transfer was performed at 80 V for 1 h 45 min at 4 °C (Bio-Rad). Primary and secondary antibodies were incubated overnight at 4 °C and 1h at room temperature, respectively. Revelation was performed with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) and the chemiluminescent signal was imaged with ImageQuant LAS4000 mini . Quantification was performed with ImageJ. Uncropped blots are shown in Supplementary Fig. Cells were washed with ice-cold HBSS, scraped, and centrifuged twice for 10 min at 16,000 × t-test was performed. If a clear hypothesis was postulated that made it valid, then we used a one-tailed t-test. When variances were not comparable, Welch’s correction was applied. When the distribution could not be assumed to be Gaussian , we used a non parametric Mann–Whitney test. When more samples were compared and Bartlett’s test showed that variances could be compared, we used one-way ANOVA with Dunnett’s post-test. If variances could not be compared , then we used multiple t-tests. Outliers, defined as values that were superior to (mean+3 standard deviations) or inferior to (mean−3 standard deviations) were excluded.Statistical calculations were performed using GraphPad Prism software (version 9.2.0). All the data are given as mean ± s.e.m. In most cases, when comparing two samples, two-tailed Student’s Further information on research design is available in the Supplemental FiguresReporting Summary


They are common globally in arid ecosystems and have recently been identified in agroecosystems. However, unlike natural ecosystem biocrusts, agroecosystem biocrusts receive regular fertilizer and irrigation inputs. These inputs could influence seasonal biocrust N2-fixation and their relationship with soil nutrients in perennial agroecosystems, which is of particular interest given crop management requirements. In this study, biocrust and adjacent bare soil N2-fixation activity was measured in the field during the summer, fall, spring, and winter seasons in a Florida citrus orchard and vineyard using both acetylene reduction assays and 15N2 incubations. Samples were analyzed for microbial and extractable carbon , nitrogen , and phosphorus . In both agroecosystems, biocrusts had greater microbial biomass and extractable nutrients compared to bare soil. The citrus and grape biocrusts were both actively fixing N2, despite crop fertilization, with rates similar to those found in natural arid and mesic systems, from 0.1 to 142 nmol of C2H4 g–1 of biocrust dry weight h–1 . Lower soil temperatures and higher EC:EN ratios were associated with higher N2-fixation rates in citrus biocrusts, while higher soil moisture and higher EP were associated with higher N2-fixation rates in grape biocrusts. The N2-fixation activity of these agroecosystem biocrusts indicates the possibility of biocrusts to enhance N cycling in perennial agroecosystems, with potential benefits for crop production.Biocrusts are communities of microorganisms within the top centimeter of soil, often dominated by phototrophic dinitrogen-fixing (N Biological soil crusts (biocrusts) are communities of organisms on soil surfaces and often include diazotrophic organisms such as lichens , cyanoba2-fixation) is one of the key biocrust processes, and it is estimated that biocrusts contribute approximately half of the total N fixed in arid lands  fertilization applications. In particular, fertilizer N could inhibit biocrust N2-fixation, as shown both in field-collected biocrusts and multi-species laboratory cultures. For example, N additions mimicking atmospheric deposition significantly reduced N2-fixation rates in arid light and dark biocrusts , spring (16–26°C), and summer (25–29°C) than in the cooler winter (10–22°C) due to their narrow temperature range.1While Nosystems  and seasosystems , 2014, a to 30°C , higher 2-fixation activities have not been examined in perennial agroecosystems, and the influence of consistent N and P fertilization and irrigation on agroecosystem biocrust N2-fixation activities is unknown. Therefore, we conducted a year-long field study of agroecosystem biocrusts and bare soil controls to quantify seasonal N2-fixation rates, compare biocrust and bare soil nutrient concentrations, and identify the relationships between biocrust N2-fixation activity, soil nutrients, and environmental variables. We chose two agroecosystems with biocrusts (a vineyard and a citrus orchard) that had similar climatic conditions but differed in fertilization and irrigation management. We hypothesized that due to limited seasonal temperature change and consistent water input through irrigation, N2-fixation rates of these agroecosystem biocrusts would not have a seasonal pattern. However, based on differences in crop management, we hypothesized that the availability of N and P fertilizer would regulate biocrust N2-fixation patterns and relationships with biocrust nutrient concentrations more strongly than soil temperature and soil moisture.Biocrust N1 Agroecosystem biocrusts were assessed in two perennial crops: 2-month-old Vitis rotundifolia (muscadine grape) located at the University of Florida Plant Science Research and Education Unit in Citra, Florida  and a 2-year-old Citrus sinensis (orange) orchard located at the University of Florida Citrus Research and Education Center in Lake Alfred, Florida . Soils of both sites were classified as excessively drained Entisols of the Candler series, sandy soil formed from eolian and loamy marine deposits with 6.0–6.5 pH as measured by –1 10–10–10 NPK granulated fertilizer applied in June 2019, July 2019, and March 2020, while Citrus was fertigated (5–0–7 NPK or 7–2–7 NPK) weekly and received 29 N kg ha–1 12–4–8 NPK controlled release fertilizer in July 2019 , November 2019 , January 2020 (Winter), and May 2020 (Spring); and at Grape in August 2019 (Summer), October 2019 , January 2020 (Winter), and May 2020 (Spring). Samples were collected from six plots at each location. Each plot was randomly located on either side of the crop trunk or vine and contained an intact biocrust and adjacent bare soil (no more than 10 cm away from the biocrust) located within the crop row . There w2 up to 6427 cm2. Plots were at least 2 m apart from each other within a site. After each sampling point, the sample collection plot locations were shifted to the nearest intact biocrusts  because not enough material remained for repeated sampling.Biocrusts and bare soils were randomly sampled at each plot and ranged in area from 1045 cm2-fixation rates from both sites  were examined for the presence of cyanobacteria and algae using an inverted microscope, Nikon Eclipse Ti2 .Biocrusts were identified by field observations and referencing the visual development scale . The barn = 6) when samples were collected. Temperatures were measured at the surface without plant shading using a thermocouple attached to a DIGI-SENSE 20250-02 temperature meter . Light intensity measurements were recorded using a LI-250A light meter . Additional data about precipitation and solar irradiance were obtained from the Florida Automated Weather Network1  and during the fall season in Citrus (biocrusts: n = 2). Bare soil samples from Citrus were not collected for 15N2 enrichment incubations.Three subreplicates each of biocrust and adjacent bare soil were collected intact from each plot to 0.5 cm depth using a 3 cm diameter corer . Cores wBoth sites were irrigated daily in the morning, including the morning before sample collection. However, at the January and May 2020 sample collections, the Grape soil was very dry, therefore, on these dates, the soil was saturated with deionized water before soil core collection. No deionized water was added during other collection times.2-fixation was measured under field conditions immediately after collection using an adapted version of the acetylene reduction assay (ARA) (2-fixing phototrophic organisms (Biocrust and bare soil Nay (ARA) . Measurerganisms . The lid2 fixed, separate incubations with 15N2 gas were conducted simultaneously with ARA on two biocrust replicates from Citrus in the fall, three biocrust replicates from Grape in the summer, and three bare soil replicates from Grape in the summer season following the method of 15N2 was injected into the jar headspace of samples and into three empty jars. Gas samples were collected from these jars after a 2-h incubation to determine the headspace 15N2 concentration. The jars of 15N2 enriched samples were then opened and aerated for 10 min before being placed on ice to stop the incubation. These samples were used for N isotopic determination. Acetylated samples from the same plots where 15N2-incubated samples were collected served as unenriched controls. 15N2-enriched biocrust subreplicates from 15N2-enriched jars and non-enriched control biocrust subreplicates from acetylated jars were separated from loose soil particles with a 0.5 mm sieve. The three sieved enriched biocrust, non-enriched biocrusts, enriched bare soil, and unenriched bare soil subreplicates from each plot were pooled, homogenized, and dried at 70°C.To identify the conversion ratio from acetylene reduction to Nn = 6). The soil moisture content of biocrusts and bare soils was determined gravimetrically after drying in the laboratory oven for 72 h at 70°C to avoid additional mass loss due to organic matter volatilization and to allow for subsequent N analyses (n = 12). However, only biocrust soil moistures were averaged together for principal component analysis (PCA).Biocrust samples from acetylated jars were separated from loose soil particles with a 0.5 mm sieve. The three sieved biocrust and bare soil subreplicates from each plot were pooled, homogenized, and then subsampled for microbial biomass, extractable nutrients, and moisture determination , N (MBN), and P (MBP) were measured using the fumigation extraction approach . BrieflyMicrobial biomass C, MBN, and MBP were determined by calculating the differences between fumigated and non-fumigated sample pairs with 0.37 adjustment factor (extraction efficiency) for MBC, 0.54 for MBN, and no adjustment factor for MBP following 2H4 gas  was used for calibration, and results were reported as micromoles of C2H4 per square meter of soil core surface area per hour (μmol m–2h–1).Gas samples from ARA measurements were stored at 25°C and analyzed for ethylene within 2 weeks of collection using a Shimadzu GC-8A gas chromatograph equipped with a flame ionization detector (FID) and HayeSep N column (2 m). The operating temperatures for the column and injection ports were 80 and 110°C, respectively. A 100 ppm standard C15N2-enriched biocrust subreplicates from 15N2-enriched jars and non-enriched control biocrust subreplicates from acetylated jars were analyzed for isotopic N, and total N. Atom% 15N was measured using a Thermo Finnigan Delta Plus XL isotope ratio mass spectrometer with a ConFlo III preparation system at the UF/IFAS Soil and Water Sciences Elemental Analysis Laboratory, Gainesville, FL, United States. Total N was simultaneously determined using a Costech ECS 4010 CHNS-O elemental analyzer. Atom% 15N of headspace N2 was calculated by subtracting 15N/14N atom% of air from 15N/14N atom% of gas blanks. Atom% excess biomass was divided by atom% excess in headspace to calculate the fraction of N2-fixation derived N according to the equation adapted from Inglett (2013).2-fixation rate in nmols of N-N2 g–1 DW h–1. The conversion factor from acetylene reduction to N2-fixation was calculated by dividing the average biocrust acetylene reduction rate by the average enriched biocrust N2-fixation rate. The conversion factor was obtained separately for the summer season Grape and the fall season Citrus samples. Analogous estimates were also made using the theoretical conversion factor of 3 . Each site was analyzed separately and only measurements from biocrusts were included. Prior to analysis, the data were preprocessed by filtering out zeros , testing for multivariate normality using the mvn function from the MVN package . At the Grape site, biocrust and bare soil temperature ranged from 27.0 to 41.9°C, with the highest average temperature in spring and the lowest average in winter, while at the Citrus site, it ranged from 23.1 to 41.8°C, with the highest average in the summer and lowest average in winter  for acetylene reduction rates . In Grap–2 h–1 with the highest average rate in summer (260 μmol m–2 h–1) and the lowest average rate in spring (4.19 μmol m–2 h–1) (–2 h–1 with the highest rate in winter (78 μmol m–2 h–1) and the lowest in summer (13 μmol m–2 h–1).Grape biocrusts had higher average acetylene reduction rates than Citrus biocrusts. Grape biocrust acetylene reduction rates ranged from undetectable to 401 μmol mm–2 h–1) . AcetyleAverage Grape biocrust acetylene reduction rates decreased from summer to spring, while average Citrus biocrust acetylene reduction rates increased from summer to spring. Grape biocrust acetylene reduction rates were significantly greater during the summer when compared to Grape biocrust acetylene reduction rates during fall, winter, and spring . While n2H4:N2 conversion factor was lower in Citrus  than in Grape (2.69 ± 0.92), leading to higher average annual N input estimates from N2-fixation in Citrus. Estimates of biocrust N2-fixation rates using both the experimental and theoretical 3:1 ratios are shown in 2H4:N2 conversion factor and using experimental ratios, the N2-fixation rates in Grape ranged from 1.87 × 10–6 to 4.18 × 10–3 g N m–2 h–1, while in Citrus it ranged from 2.05 × 10–6 to 7.49 × 10–3 g N m–2 h–1. Based on the assumption that phototrophic diazotrophs were engaged in N2-fixation for 10 light hours, we estimated the daily N2-fixation rate of these agroecosystem biocrusts to be 6–32 mg of N per day. Averaging N2-fixation inputs separately for each season and site, and then adding for an annual estimate with the assumption that biocrust agroecosystems had 12.5% biocrust soil surface area coverage, Citrus biocrusts were estimated to provide 8.1 kg N ha–1 year–1, while Grape could provide 4.9 kg N ha–1 year–1.The C–1 with the highest concentration in fall (2563 mg kg–1) and lowest in spring (1122 mg kg–1). Citrus biocrust MBC ranged from 232 to 3998 mg kg–1 with the highest concentration in fall (2149 mg kg–1) and lowest in summer (1578 mg kg–1)  .–1 with the highest concentration in winter (121 mg kg–1) and lowest in spring (36 mg kg–1). Citrus biocrust MBN ranged from 16 to 480 mg kg–1 with the highest concentration in summer (189 mg kg–1) and lowest in fall (120 mg kg–1). There was also a significant interaction between site and season for biocrust MBN  and lowest in winter (10 mg kg–1). Citrus biocrust MBP ranged from undetectable to 68 mg kg–1 with the highest concentration in spring (29.5 mg kg–1) and lowest in winter (3 mg kg–1) (Grape biocrust MBP ranged from undetectable to 83 mg kgmg kg–1) . Due to There were different seasonal patterns in MBC:MBN ratios in Grape and Citrus. Mass-based MBN:MBP and MBC:MBP ratios were excluded because 30% of MBP values were below the detection limit. Grape biocrust MBC:MBN ranged from 6 to 44, while Citrus biocrust MBC:MBN ranged from 7 to 30. The highest biocrust MBC:MBN in Grape was during the summer (18.1 ± 5.52), while in Citrus it was during the fall (18.2 ± 2.53). The lowest biocrust MBC:MBN in Grape was in fall (9.2 ± 1.08), while in Citrus it was during the summer (9.82 ± 2.54) .–1 with a high in fall (512 mg kg–1) and a low in spring (292 mg kg–1). Citrus biocrust EC ranged from 83 to 1107 mg kg–1 with a high in spring (419 mg kg–1) and a low in summer (174 mg kg–1).In general, EC, EN, and EP were higher in biocrusts than in bare soils in Grape and Citrus. EC was significantly greater in biocrusts than in bare soils during the summer (Grape), fall (Grape and Citrus), and spring (Citrus) . Grape b–1 with a high in spring (78 mg kg–1) and a low in winter (28 mg kg–1). Citrus biocrust EN ranged from 9 to 132 mg kg–1 with a high in spring (58 mg kg–1) and a low in winter (32 mg kg–1).EN was significantly greater in biocrusts than in bare soils during the summer (Citrus), fall (Grape), winter (Citrus), and spring (Citrus). Grape biocrust EN ranged from 7 to 255 mg kg–1 with a high in summer (156 mg kg–1) and a low in spring (80 mg kg–1). Citrus biocrust EP ranged from 12 to 67 mg kg–1 with a high in summer (48 mg kg–1) and a low in winter (26 mg kg–1).There was a significant interaction between site and sample type for EP . EP tendNo strong seasonal patterns or significant differences between sites, biocrusts and bare soils, or seasons were detected in extractable nutrient ratios. Due to undetected EN and EP, certain ratios were excluded from the following comparisons. Grape biocrust EC:EN ranged from 3 to 18, while Grape bare soil EC:EN ranged from 7 to 15. Citrus biocrust EC:EN ranged from 3 to 13, while Citrus bare soil EC:EN ranged from 1 to 10. Grape biocrust EN:EP ranged from 0.05 to 3, while Grape bare soil EN:EP ranged from 0.2 to 0.6. Citrus biocrust EN:EP ranged from 0.2 to 4, while Citrus bare soil EN:EP ranged from 0.2 to 2. Highest average Grape biocrust EC:EN was in winter (11.2 ± 1.89), while the lowest Grape biocrust EC:EN was in summer (4.9 ± 1.07). Highest average Citrus biocrust EC:EN was in fall (9.1 ± 1.98), while the lowest Citrus biocrust EC:EN was in summer (4.86 ± 1.07). Highest average Grape biocrust EN:EP was in spring (0.9 ± 1.12), while the lowest Grape biocrust EN:EP was in summer (0.4 ± 0.22). Highest average Citrus biocrust EN:EP was in spring (1.8 ± 1.23), while the lowest Citrus biocrust EN:EP was in summer (0.8 ± 0.33) .2-fixing biocrusts were included in the PCA. At Grape, MBC:MBN, extractable nutrients, extractable nutrient ratios, and soil moisture were significant drivers of variation, as indicated by the significance of loadings , nutrient variables , acetylene reduction rates, and environmental variables  measured in Nloadings . Based oariation . Based o2-fixing biocrust samples in Grape (46%) and Citrus (29%) were significantly separated based on the season   . It shou–1 year–1, while Grape biocrusts could provide 39 kg N ha–1 year–1 per hectare of biocrust. However, while in natural ecosystems up to 70% of soil surface can be covered with biocrusts  which occurred 1 month after application of a controlled release fertilizer containing ammonium and nitrate   . Thereforust N:P . Such a rust N:P , suggest2-fixation rates between Grape and Citrus biocrusts. Grape and citrus have different disturbance histories from planting, which could explain the differences in their biocrust morphologies. Using the qualitative indicators of potentially different organism compositions in biocrusts , suggesting that different communities of organisms were involved in N2-fixation at each site. Due to the variety of diazotroph nitrogenases, the conversion factor can differ between not only phyla but also species of cyanobacteria, as it did between Anabaena culture (4–5) and Nostoc culture (0.1–0.5)  . Additio2-fixers . Therefo2-fixation activity within ranges of natural biocrusts. These agroecosystem biocrusts have the potential to supplement available N for the crops through N2-fixation activity. While biocrusts could contribute less than 1% of the daily fertigation requirement of 1–3-year-old citrus, their continued N2-fixation activity during favorable conditions could contribute 7-14% of yearly N requirements for perennial crops such as grape and citrus.Despite regular N fertilizer inputs, biocrusts in Florida citrus and grape agroecosystems maintained N2-fixation rates and patterns for better estimates of N inputs from N2-fixation on a crop field yearly scale, studies across more time and spatial scales are necessary. Additional diel N2-fixation rate measurements would show if there are contributions from non-phototrophic diazotrophs (dark), or more N2-fixation activity during other times of the day that this study did not consider. This study used 3 cm diameter cores for N2-fixation rate measurements, but there is a need to scale up to the whole field area for more accurate N input estimates, which could be done with more and larger core collections, biocrust percent cover measurements, and potentially paired with remote sensing technology as was done for biocrusts in natural ecosystems  and stimulation (P) of biocrust N2-fixation rates. Ultimately, further agroecosystem biocrust studies should aid in determining whether biocrust N inputs make any substantial improvements to crop productivity, or at least help satisfy N crop requirements more sustainably.Detailed analysis of biocrust microbial communities could help identify agroecosystem biocrusts NThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PI, SS, and KS designed the experiment. KS conducted the experiment, processed the samples, analyzed the data, and wrote the manuscript. SS and PI revised the manuscript. All authors contributed to the article and approved the submitted version.


Citrus aurantium L.) plants developed from somatic embryos or haploid tissues. Sour orange anthers were cultured in N6 and MS media to induce calli and somatic embryos. N6 liquid medium supplemented with 1 mg·L−1 gibberellic acid and 200 µM spermidine resulted in a 10% increase in callus and embryo induction rates. Regenerated plants were validated using simple sequence repeat markers. Out of the 109 regenerated plants, ploidy analysis identified 99 diploids, two haploids, and eight putative aneuploids; out of the 99 diploid plants, 33 were haploid-derived homozygous diploids. The chromosomal analysis confirmed most plants as diploids, whereas some were identified as aneuploids (19–21 chromosomes). Furthermore, phylogenetic analysis confirmed that the resultant homozygous or heterozygous plants were haploid-derived. This is the first report of haploid-derived homozygous diploid and aneuploid sour orange plants obtained through anther culture. Moreover, the anther cultivation technique described herein can be applied to other citrus varieties.Citrus plants are important fruit tree species; however, the breeding of high-quality varieties of citrus species is a time-consuming process. Using haploid-derived plants from anther culture may reduce the time required for obtaining purebred lines. This study aimed to genetically verify whether anther culture-derived sour orange ( Citrus aurantium L.) propagates through seeds, it is mainly cultivated using rootstocks [Citrus plants belong to the family Rutaceae, which includes some of the most economically important and globally widespread fruit tree crops ,2,3,4. Iotstocks ,7. Recenotstocks ,9. Howevotstocks ,10. Furtotstocks . Despiteotstocks .Generating superior lines of citrus cultivars using conventional breeding methods, such as crossing, is time-consuming and labor-intensive owing to their high heterozygosity, long juvenility, self-incompatibility, and polyploidy ,15,16,17Poncirus trifoliata (L.) Raf. (trifoliate orange), Citrus clementina Hort. ex Tan. (clementine), Citrus sinensis (L.) Osbeck (sweet orange), Citrus sinensis (L.) Osbeck , Citrus limon (L.) Burm. f. (lemon), and Clausena excavata [Haploid plants cannot undergo normal meiosis, which hinders generational progress ,19. In cexcavata ,30,31,32Aneuploidy is a type of chromosomal anomaly that involves an increase or decrease in the number of chromosomes  and is cIn this study, an optimum medium for anther culture of sour orange was developed. This medium was used to culture citrus embryos to determine the efficiency of anther culture of sour orange plants. Furthermore, the ploidy status of haploid-derived plants was verified and the associated genetic changes were investigated. Additionally, chromosomal analysis was used to verify the presence of homodiploid and aneuploid plants, and phylogenetic analysis was used to confirm whether homozygous or heterozygous plants were haploid-derived.−1 GA3 and 200 µM spermidine was added to the anther culture medium, the rates of callus induction and embryo formation increased by approximately 10%; the original callus induction rate was 25% and that of embryo formation was approximately 2% without the addition of the liquid medium  markers  out of eight combinations of heterozygous SSR markers were used. Out of >1000 plants obtained through anther culture, 271 were randomly selected for analysis using P1 and P2 markers . Of the The SSR marker CiSSR-43 showed two amplification bands, similar to that shown by CiSSR-P1 in the control b. PutatiThe SSR markers CiSSR-254 and CiSSR-260 showed two amplification bands, which was similar to the pattern exhibited by the CiSSR-P1 marker in the control e,f. ThreUnlike CiSSR-P1, the SSR markers CiSSR-P2 a and CiSPolyploidy was investigated in 109 plants obtained through anther culture. These included 41 out of 43 (two specimens were lost due to contamination) genetically selected plants and 68 genetically validated plants .Verification of ploidy in the 109 plants identified about 91% of plants as diploids  chromosomal regions were analyzed to determine the genetic relationship between the donor and regenerated plantlets and to establish whether the anther culture-derived plants were somatically mutated or haploid-derived. The results indicated a genetic difference in the chromosomal ITS region between plants identified using SSR markers and the control plants . SubsequCitrus plants are characterized by long juvenility [Citrus species [Citrus species, sour orange varieties are widely used as rootstocks for industrial use and tissue culture. The rootstocks also serve as useful models for anther culture [This study investigated embryo formation and callus induction in sour oranges using anther culture to obtain haploid and double-haploid plants. The production of haploid- derived plants through anther culture is more challenging in citrus cultivars than in herbaceous species  because venility . Successvenility ,53,54,55 species ,16,17,56 species ,57. Amon culture . However culture .3) and spermidine are indicated in callus induction or stimulate somatic embryogenesis [3, when added to the solid medium during culturing of sour orange anthers, caused swelling of the anthers and exerted a greater effect on embryo and callus formation , 33 of the remaining plants were haploid-derived homozygous diploids, two plants were haploids, and eight were identified as putative aneuploids . Hidaka 3 (1 mg·L−1) and spermidine (200 µM) was added—except in the control treatment—to two types of culture media [Approximately 100 sour orange (seed-forming variety) flower buds were randomly sampled from approximately 15-year-old C. aurantium L. trees obtained from the greenhouse at Citrus Research Center, RDA, Korea, in April 2020 a,b. The re media . The cul−1) and malt extract (500 mg·L−1). The following growth regulators were added to the two media: 0.2 mg·L−1 2,4-dichlorophenoxy acetic acid, 0.2 mg·L−1 α-naphthalene acetic acid, 1.0 mg·L−1 kinetin, 0.8 mg·L−1 6-benzyladenine, 0.43 mg·L−1 zeatin, and 0.44 mg·L−1 thidiazuron. The pH of the media was adjusted to 5.8 using 1 N KOH and 0.1 N HCl; 0.8% (w/v) agar was used for solid media. Callus and embryo induction rates were compared between anther cultures with and without N6 liquid medium supplemented with GA3 (1 mg·L−1) and spermidine (200 µM). The anthers were cultured in the dark at 4 °C for the first 14 d and then cultured under a 16 h light-dark photoperiod at 25 ± 2 °C.Callus cells and embryos were induced in an anther culture medium comprising N6 medium  suppleme−1 malt extract and 164 mM lactose) according to the method described by Jin et al. [−1), GA3 (1 mg·L−1), malt extract (500 mg·L−1), sucrose (50 g·L−1), and Gelrite® (0.2%) to induce germination. The embryos showing shoot development were transferred and cultured in a Magenta box  containing MS medium with GA3 (0.5 mg·L−1), malt extract (500 mg·L−1), sucrose (50 g·L−1), and agar (8 g·L−1). The roots were induced following a 6-week culture period. Each of the newly formed shoots was grafted onto the cultivated citron root. Subsequently, these plants were transferred outdoors from the greenhouse for acclimation to field conditions.Callus cells induced in the anther culture medium were transferred to a somatic embryo induction medium , comprising 250 μM dNTPs, 1.5 mM MgCl2, 1 U Taq DNA polymerase, 10 mM Tris-HCl (pH 9.0), and 40 mM KCl. To this mixture, 20 ng of 0.5 μM primer was added, and the reaction volume was adjusted to 20 µL. The PCR cycle conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 40 s, followed by final extension at 72 °C for 5 min. The PCR products were then analyzed electrophoretically using the QiAxcel Advanced System .To verify whether the plants obtained from the anther culture were haploid-derived, two SSR marker primers  specific to the heterozygote were used . Primers® filter (Sysmex Partec GmbH) directly into the sample test tube. This test tube was loaded into the machine for analysis. A total of 109 regenerant lines were analyzed.To verify the ploidy of the plants derived from anther culture, leaf samples were collected from each plant obtained through in vitro grafting. The samples were prepared according to Chiancone et al. . The plo−1 4,6-diamidino-2-phenylindole , mounted in VECTASHIELD® , and examined under an Olympus BX53 fluorescence microscope (100× magnification).To confirm ploidy, the samples were pretreated and prepared according to the method described by Ha et al. . Young r® Gel purification kit . The purified amplification products were cloned using the pLUG-Prime® TA-Cloning Vector kit  [The genetic relationship between the donor plant (control plants) and anther culture-derived regenerated plants was analyzed as described previously ,66. The , Korea)  and usedIn this study, anther culture of sour orange plants was performed and haploid-derived homozygous diploid and aneuploid plants were obtained. The findings of this study can be effectively applied to mutant breeding of citrus cultivars through anther culture. More importantly, the results would be helpful in the study of anther cultures of other citrus varieties since haploid-derived homozygous diploid plants were obtained using anther culture in this study. Future research should focus on chromosomal analysis of pollination-derived individuals and incorporate microspore culture for the development of marketable, seedless varieties.