Pseudomonas aeruginosa and Staphylococcus aureus. Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice, and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether the genetic overexpression of acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa.Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including  Pseudomonas aeruginosa and Staphylococcus aureus. Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice, and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether the genetic overexpression of acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa. We demonstrate that the transgenic overexpression of acid ceramidase in CF mice corresponds to the overexpression of acid ceramidase in bronchial and tracheal epithelial cells and normalizes ceramide and sphingosine levels in bronchial and tracheal epithelial cells. In addition, the expression of β1-integrin, which is ectopically expressed on the luminal surface of airway epithelial cells in cystic fibrosis mice, an alteration that is very important for mediating pulmonary P. aeruginosa infections in cystic fibrosis, is normalized in cystic fibrosis airways upon the overexpression of acid ceramidase. Most importantly, the overexpression of acid ceramidase protects cystic fibrosis mice from pulmonary P. aeruginosa infections. Infection of CF mice or CF mice that inhaled sphingosine with P. aeruginosa or a P. aeruginosa mutant that is resistant to sphingosine indicates that sphingosine and not a metabolite kills P. aeruginosa upon pulmonary infection. These studies further support the use of acid ceramidase and its metabolite sphingosine as potential treatments of cystic fibrosis.Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including  CFTR; mouse, Cftr). Mutations of CFTR are relatively common, with a frequency of approximately 1 in 50 people in the European Union and the United States. Thus, cystic fibrosis is the most common autosomal recessive disorder, affecting 1 of every 2,500 children born in Western countries. Cystic fibrosis is characterized by several respiratory, reproductive, and gastrointestinal symptoms. However, at present, chronic pulmonary colonization with bacterial pathogens, in particular Pseudomonas aeruginosa, and the development of chronic pneumonia, chronic inflammation, and progressive pulmonary fibrosis are the primary causes of morbidity and mortality of these patients (Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator gene . We then determined alterations of lipids in these mice and, most importantly, the infection susceptibility of these mice in comparison to Cftr-deficient mice. Using a sphingosine-resistant P. aeruginosa mutant, we further analyzed whether sphingosine or a metabolite mediates the killing of P. aeruginosa.To analyze the role of acid ceramidase in cystic fibrosis, we generated mice that lack functional Cftr and overexpress acid ceramidase  mice with acid ceramidase transgenic mice in order to overexpress acid ceramidase in cystic fibrosis mice. This novel mouse strain was named CF-Asah1tg. The activity of acid ceramidase in the trachea of CF-Asah1tg mice was higher than that in wild-type mice but lower than the activity of the acid ceramidase in control Asah1 transgenic mice   . Ceramidr (COPD) , 25, andTherefore, collectively, these studies strongly support the notion that treatment of CF patients with acid ceramidase or sphingosine may be very beneficial and may protect patients from the development of pulmonary infections.CftrTgH(neoim)Hgu (CFMHH) congenic mice were generated as previously described (CftrTgH(neoim)Hgu mutant mice, which were generated by insertional mutagenesis in exon 10 of the Cftr gene, were inbred to create the congenic MHHCftr strain. These mice were then backcrossed for more than 20 generations to a C57BL/6 background. Because these mice still express low levels of Cftr, they can be fed a standard mouse diet. They exhibit normal development but also display pulmonary pathology typical of CF (–B6.129P2(CF/3)-escribed , 26, 27.al of CF –6, 28. SMHHCftr mice were crossed with mice transgenic for Asah1 to overexpress the acid ceramidase in mice lacking Cftr. Asah1 transgenic mice were also on a C57BL/6 background. The overexpression of the acid ceramidase was achieved by introducing an expression cassette in which Asah1 is expressed under the control of the CAG promoter into the mouse genome. Mice were cloned, and the integration of the transgene was tested by PCR. The overexpression of acid ceramidase was confirmed by measuring acid ceramidase activity in several tissues.Asah1 transgenic (Asah1tg0) mice served as controls.We used female and male mice at an age of at least 16 weeks and weighing between 25 and 35 g. Mice were divided into cages of equal sizes  by animal unit technical staff with no involvement in study design. Cages were randomly assigned to an experimental group. The investigators were blind to group allocation during the experiment and/or when assessing the outcome.https://felasa.eu/working-groups/guidelines), including S. aureus. The mice were free of all pathogens. Animal procedures were approved by the Bezirksregierung Duesseldorf, Duesseldorf, Germany, and the IACUC committee Cincinnati.Mice were housed and bred within isolated and ventilated cages in the mouse facility of the University Hospital, University of Duisburg-Essen, Germany. They were repeatedly tested for a panel of common murine pathogens according to the 2002 recommendations of the Federation of European Laboratory Animal Science Associations , Cy3-coupled anti-mouse IgM (for anti-sphingosine or anti-ceramide antibodies), or anti-rat IgG (for anti-β1-integrin antibodies) F(ab)2 fragments (Jackson Immunoresearch) in H/S plus 1% FCS for 30 min. Sections were washed three times as described above and once with PBS and embedded in Mowiol. Samples were evaluated by confocal microscopy on a Leica TCS-SL confocal microscope equipped with a 40× lens, and images were analyzed with Leica LCS software . All comparative samples were measured at identical settings. Control stainings were performed with Cy3-labeled secondary antibodies only or with irrelevant rabbit IgG, mouse IgM, or rat IgG, followed by staining with the corresponding Cy3-labeled secondary antibodies. These controls revealed very weak staining and confirmed the specificity of the antibody staining. Fluorescence intensities were quantified using ImageJ.Mouse trachea and lungs were stained for acid ceramidase, sphingosine, or ceramide as previously described –13. Mice–14C16]ceramide (55 mCi/mmol)  was pipetted onto the lumen of the trachea. Prior to the assay, [14C16]ceramide was dried, resuspended, and bath sonicated for 10 min. The assay was performed for 15 min, and the trachea was then extracted in H2O and CHCl3-CH3OH-HCl . The lower phase was dried, resuspended in CHCl3-CH3OH , separated by thin-layer chromatography (TLC) with CHCl3-CH3OH-NH4OH , and analyzed with a Fuji phosphorimager.Mice were sacrificed; the trachea was removed, opened, and placed on a 37°C prewarmed plastic plate; and 4 μl of a buffer consisting of 150 mM sodium acetate (pH 7.4), 0.05% octylglucopyranoside, and [32P]ATP for 15 min at 30°C. Controls were incubated with the same buffer without kinase or were left untreated. The kinase reaction was terminated by transferring the trachea into a solution containing CHCl3–CH3OH–1 N HCl , 170 μl of a buffered saline solution  and 30 μl of 100 mM EDTA were added, and lipids were extracted, separated on silica G60 TLC plates, and analyzed by liquid scintillation counting and a standard curve of C16- to C24-ceramides, as previously described  kinase  in a solution containing 4 μl of 150 mM sodium acetate (pH 7.4), 1 mM ATP, and 10 μCi ATP onto the luminal surface of the trachea. Controls were incubated with the same buffer without sphingosine kinase. The kinase reaction was terminated by the addition of the trachea to 100 μl H2O, followed by the addition of a solution containing 20 μl 1 N HCl, 800 μl CHCl3–CH3OH–1 N HCl , and 240 μl each of CHCl3 and 2 M KCl. Phases were separated, and the lower phase was collected, dried, dissolved in 20 μl CHCl3-CH3OH , and separated on silica G60 TLC plates with CHCl3-CH3OH-acetic acid-H2O  as a developing solvent. The TLC plates were analyzed with a phosphorimager. Surface sphingosine levels were determined with a standard curve of C18-sphingosine.Mice were sacrificed, and the trachea was removed, opened, and placed on a 30°C prewarmed plastic plate. We then added 4 μl of kinase buffer consisting of 150 mM sodium acetate (pH 7.4), 0.001 U of sphingosine kinase 1 , 1 μM ATP, and 10 μCi [γ-14C16]ceramide  (55 mCi/mmol) was added. To this end, [14C16]ceramide was dried for 10 min, resuspended in 0.1% octylglucopyranoside (OGP) in 150 mM sodium acetate (pH 4.5), and bath sonicated for 10 min. The samples were incubated at 37°C for 60 min. The reaction was terminated by extraction in H2O and CHCl3-CH3OH-HCl . The lower phase was dried, and samples were resuspended in CHCl3-CH3OH , separated by TLC with CHCl3-CH3OH-NH4OH , and analyzed with a Fuji imager.Tracheae were shock frozen, lysed in 1% Igepal in 150 mM sodium acetate (pH 4.5), and immediately sonicated with a tip sonicator to contain homogeneous lysates. Samples were diluted in 0.1% NP-40 in 150 mM sodium acetate (pH 4.5), and 0.3 μCi/sample ATP or 10 μl DAG kinase , 0.1 M imidazole-HCl (pH 6.6), 0.2 mM DTPA, 70 mM NaCl, 17 mM MgCl2, 1.4 mM ethylene glycol tetraacetic acid, 2 mM dithiothreitol, 1 μM ATP, and 5 μCi [γ-32P]ATP. Samples were incubated for 60 min at 37°C with shaking (350 rpm) and processed as described above.Tracheae or lungs were isolated, and epithelial cells were carefully removed from the surface and lysed in a solution containing 1% Igepal, 25 mM Tris-HCl (pH 7.4), 125 mM NaCl, 10 mM EDTA, 10 mM sodium pyrophosphate, and 10 μg/ml each aprotinin and leupeptin. Aliquots were extracted in CHCl3–CH3OH–1 N HCl . The lower phase was dried and resuspended in a detergent solution (7.5% [wt/vol] n-octyl glucopyranoside and 5 mM cardiolipin in 1 mM DTPA). The kinase reaction was initiated by the addition of a solution containing 10 μl DAG kinase , 0.1 M imidazole-HCl (pH 6.6), 0.2 mM DTPA, 70 mM NaCl, 17 mM MgCl2, 1.4 mM EGTA, 2 mM dithiothreitol, 1 μM ATP, and 5 μCi [γ-32P]ATP. Samples were incubated for 60 min at 37°C with shaking (350 rpm) and processed as described above.Tracheae or lungs were shock frozen; lysed in a solution containing 1% Igepal, 25 mM Tris-HCl (pH 7.4), 125 mM NaCl, 10 mM EDTA, 10 mM sodium pyrophosphate, and 10 μg/ml each aprotinin and leupeptin; and immediately sonicated with a tip sonicator to contain homogeneous lysates. Aliquots were extracted in CHClP. aeruginosa strain 762, ATCC 27858, a mucoid isolate from individuals with CF, P. aeruginosa strain 1242, or the sphingosine-resistant P. aeruginosa strain was grown overnight on tryptic soy agar (TSA) plates and resuspended in 40 ml prewarmed, sterile tryptic soy broth (TSB)  in Erlenmeyer flasks at an optical density (OD) of 0.200 to 0.250. Bacteria were then grown in TSB for 60 min at 37°C with shaking at 125 rpm until they reached the early logarithmic phase. After this, bacteria were centrifuged , the supernatant was removed, bacteria were washed with prewarmed RPMI 1640 plus 10 mM HEPES and resuspended in the same medium, and the concentration was determined by measurement of the OD and comparison to a calibration curve. Finally, the concentration of the bacteria was adjusted to 5 × 106 CFU per 20 μl in prewarmed RPMI 1640 medium plus 10 mM HEPES , a score of 1 for slightly affected (ruffled fur), a score of 2 for moderately affected , and a score of 3 for severely affected . Bacterial numbers were then determined in the mouse lungs 4 h after infection. To this end, mice were sacrificed, and the lungs were removed, homogenized, and lysed in 5 mg/ml saponin to release intracellular bacteria. Bacteria were then pelleted at 2,240 × g, washed once in sterile PBS, diluted, plated, and grown in duplicate on TSA plates for 12 h. Bacterial numbers were counted. Infection experiments were approved by the Bezirksregierung Duesseldorf, Duesseldorf, Germany, under permission numbers 84-02.04.2013.A348 and 81-02.04.2019.A134 and by the IACUC committee Cincinnati. The actual care and treatment of the animals were performed and/or overseen by veterinarians of the Central Animal Facility of the University Hospital Essen, Essen, Germany.mM HEPES , and themM HEPES . If indiescribed . The micP. aeruginosa strains in vitro, we incubated 1 × 103 bacteria in H/S with increasing amounts of sphingosine . To this end, a sphingosine stock solution of 10 mM in 0.9% NaCl was prepared by sonication and stored at −20°C. The stock solution was bath sonicated directly before use, diluted in H/S, and sonicated again for 10 min. Bacteria were incubated with the indicated sphingosine concentrations for 1 h at 37°C on a horizontal shaker at 125 rpm. After this time, an aliquot of the bacteria was plated on Luria broth (LB) agar plates and incubated overnight to determine bacterial survival.To test the antibacterial efficacy of sphingosine in killing different P. aeruginosa strain ATCC 27853 was prepared as described above, and 24 aliquots of 1,000 CFU each were incubated for 60 min with 40 μM sphingosine in 500 μl as described above. We then added 500 μl TSB, and cultures were grown overnight. Resistant cultures were plated on TSB agar plates, and clones were picked after growth overnight. Clones were treated for 5 additional rounds with 40 μM sphingosine and then used for experiments.All clinical samples were used after performing conventional microbiological diagnostics. The study did not include patients’ details and did not result in additional constraints for the patients. All data were anonymously analyzed without the need for patient consent due to the retrospective nature of the study. All procedures and methods were carried out in accordance with approved guidelines.Pseudomonas aeruginosa strains were included  for more than two groups followed by post hoc Student’s t test for all pairwise comparisons applying Bonferroni correction for multiple testing. The P values for the pairwise comparisons were calculated after Bonferroni correction. All values were normally distributed. The statistical details  are given in the figure legends. The sample size planning for the continuous variables in in vivo infection experiments was based on two-sided Wilcoxon-Mann-Whitney tests . Investigators were blind for histology experiments and animal identity as described above. All data were quantified using ImageJ and are expressed as arbitrary units. In each photo, 20 randomly chosen areas corresponding to the luminal membrane of 20 different cells were quantified and averaged with the values obtained in the other photos from the fluorescence microscopy studies. G*Power version 3.1.7 of the University of Duesseldorf, Germany, and ImageJ are public programs available online without charge.Data are expressed as arithmetic means ± standard deviations (SD). For the comparison of continuous variables from independent groups, we used Student’s


This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR–Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10 African swine fever (ASF) is a highly contagious, fatal infectious disease of domestic and wild pigs caused by an Asfarviridae family DNA virus called the African swine fever virus (ASFV) . It can At present, the detection of ASFV can be divided into two categories according to different diagnostic tests: one is detecting antibodies; the other is the detection of viral DNA. Conventional detection methods are serological enzyme-linked immunosorbent assay (ELISA)  and DNA-Recently, CRISPR–Cas12/Cas13/Cas14 systems have been exploited in the analytical method field and have been found suitable for detecting various targets, including bacteria, drug resistance genes, human DNA genotypes, and cancer mutations ,16,17,18Here, we present a CRISPR/Cas–LFB assay, an ultrasensitive and specific lateral flow biosensor (LFB) that can respond to cleaved ssDNA reporter from crRNA-guided Cas12a cleavage of ASFV DNA target. The principle is that ASFV-containing samples were pre-amplified by PCR to enhance sensitivity. The amplified product was then added to the Cas12a detection system . The Cas12a and crRNA complex recognized and cleaved the target DNA and subsequently triggered the biotinylated ssDNA reporter trans-cleavage, which cannot hybridize to its complementary DNA immobilized at the LFB test line . The CRIAll oligonucleotides, including the ASFV specific target sequence, primers, crRNAs (designed using benchling), reporters, and lateral flow capture probes were synthesized by Sangon Biotech  . Biotin-4.3H2O), trisodium citrate, and streptavidin (SA) were purchased from Sigma–Aldrich . Absorbent and fiberglass papers and nitrocellulose membranes were purchased from Sartorius AG . A dispenser to immobilize the biotin-labeled goat anti-mouse IgG and probe DNA on the LFB and a nitrocellulose membrane cutter were purchased from Shanghai Kinbio . A portable strip reader was from Goldbio Technology Co. . Genome and plasmid extraction kits were purchased from Qiagen  and Tiangen Biotech , respectively. All buffers used in this study were prepared in our laboratory. Other chemicals were purchased from standard commercial sources and were of pure analytical grade.Gold trichloride acid  a 270 bp ASFV specific sequence targeting conserved regions of VP73 genes of 7 different types of ASFV  synthesi2O to final reaction volume. The reaction mixture was incubated in a thermocycler using 3 steps PCR protocol for 45 min: 95 °C, 5 min for denaturation; 95 °C, 10 s for each cycle; 55 °C, 10 s for annealing; 72 °C, 30 s for elongation; 35 cycles. PCR products were analyzed using 2.5% agarose gel electrophoresis.For PCR amplification, 25 µL PCR premix was composed of 1 µL forward and reverse primers (0.3 µM), 12.5 µL 1 × rTaq Premix, 2 µL of extracted genomic DNA (200 ng), and ddH2, 1% glycerol, 0.5 mM DTT, 20 nM HEPES (pH 7.5)) or 3 µL NEB buffer 3.1, 3 µL crRNA (300 nM), 1 µL LbaCas12a (36 nM), and 5 µL activator (40 nM) incubated at 37 °C for 10 min. Then, an LbaCas12a detection was prepared using 21 µL LbaCas12a-crRNA-activator complex, 2 µL biotinylated ssDNA reporter (20 nM), and 2 µL extracted target DNAs. Finally, the reaction was incubated at 37 °C for 30 min and analyzed using LFB after adding ddH2O up to 60 µL. The reporter trans-cleavage was monitored by the naked eye on the LFB within 10 min, and an LFB reader provided peak area values of the test and control lines’ signal intensities. The test lines’ peak area intensities were plotted after LFB photo-analysis using Image J , which provided similar intensities between each analyzed triplicate.For CRISPR/Cas-LFB assay analysis of trans-cleaved reporters, the cleavage assay was carried out according to  with sli2 LB 943 . The fluorescence from reporter sequence cleavage was measured every 5 min from 0 to 1 h with detection at λ excitation = 495 and λ emission = 520.For fluorescence analysis of trans-cleavage reaction, 50 µL reaction set-up was prepared as mentioned above with 5′FAM and 3′TAMRA labeled ssDNA reporter instead of biotinylated ssDNA reporter. The reaction was incubated and proceeded at 37 °C in a 96-wells microplate reader Mithras3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose, and 0.1% NaN3). The solution was gently shaken for 3 h at 4 °C, centrifuged , and rinsed three times with suspension buffer to remove unbound SA. The red pellet was resuspended in 100 µL of suspension buffer before being dispensed on the glass fiber conjugate pad. (3) The nitrocellulose membrane consisted of the capture probe (complementary to ssDNA reporters) and biotin-labeled goat anti-mouse IgG dispensed on the LFB nitrocellulose membrane strip (25 mm in width) to form test and control zones, respectively. The LFB parts were dried at room temperature for 12 h and stored in a desiccant container. All pads were sequentially assembled along the adhesive part of the nitrocellulose membrane with an overlap of 2 mm and then cut into 0.4 cm-wide strips.The LFB was composed of 3 main parts: (1) a sample pad (16 mm in width) prepared using immersion buffer , dried and stored in a desiccant container. (2) a conjugate pad containing AuNPs prepared according to the previous method ,23,24, wTo confirm our approach’s applicability, we used ASFV-negative and ASFV-positive whole blood samples collected and preserved at 4 °C in EDTA tubes in Prof. Liangxue Lai’s swine blood collection in Guangzhou Institutes of Biomedicine and Health. ASFV genomic DNA was extracted using Qiagen Blood Kit without pretreatment from 200 µL of each clinical sample. Two microliters of each extracted DNA were subjected to PCR and then applied to Cas12a cleavage assay.Each result presented is the mean ± standard error of the mean (SEM) of the LFB test line’s peak area readings from the triplicates.2O and shorter reporter sequences were applied (As illustrated in  applied a. The te applied a. In theThe system was first studied using pUC57-ASFV recombinant plasmid DNA target a. We sel−8–0.5 × 10−15 M) were tested using both PCR and CRISPR/Cas–LFB. PCR alone achieved >25 × 10−15 M sensitivities (−15 M (~1554.46 copies/µL) was detected. Our biosensor’s qualitative activity was also confirmed by the previously reported fluorescence-based CRISPR diagnostics assays [To evaluate the sensitivity, different concentrations of the serially diluted recombinant plasmid , viruses , and co-incubation in the presence of ASFV. Except for the absence of ASFV, which showed similar detectable test lines, the distinctive cleavage was only observed in the presence of ASFV (The selectivity assay consisted of various bacteria strains ( of ASFV . The co- of ASFV , confirmA total of 6 available swine blood samples with 4 ASFV-positive and 2 ASFV-negative were confirmed by PCR  and examASFV is one of the most detrimental viruses occurring in swine, with high morbidity and mortality, causing severe socio-economic concerns. The early detection of ASFV is crucial for early control, monitoring, and treatment. Traditional detection methods such as ELISA and PCR-based methods have been utilized, but they are still laborious, costly, and non-deployable. Nucleic acids isothermal amplification methods are currently considered promising in the rapid detection of ASFV. Nevertheless, carry-over cross-contamination, tedious gel electrophoresis analysis, and toxic dyes still pose difficulties. The currently developed CRISPR–Cas-based detection systems promise potential diagnosis of broad-range nucleic acids ,18,19,20Most importantly, our designed probe-based biosensor is universal for any singleplex CRISPR–Cas detection. Although PCR is a mature and commonly used method, its on-site availability in resource-limited areas is limited; thus, isothermal amplification methods such as loop-mediated isothermal amplification and recombinase polymerase could be integrated with our CRISPR/Cas–LFB. In addition, a multiplexing biosensor can also be designed to detect other pathogens simultaneously by using their specific reaction components.


Gallus gallus; TBCs) are an indigenous breed found in the Qinghai-Tibet Plateau that are well-adapted to a hypoxic environment. As of now, energy metabolism of the TBCs embryonic brain has been little examined. This study investigated changes in energy metabolism in TBCs during hypoxia, and compared energy metabolism in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed, to explore underlying mechanisms of hypoxia adaptation. We found TBCs exhibited decreased oxygen consumption rates (OCR) and ATP levels as well as an increased extracellular acidification rate (ECAR) during hypoxia. Nevertheless, OCR/ECAR ratios indicated aerobic metabolism still dominated under hypoxia. Most important, our results revealed significant differences in TBCs brain cellular metabolism compared to DLCs under hypoxia. Compared to DLCs, TBCs had higher OCR and TCA cycle activities during hypoxia. Also, TBCs had more mitochondrial content, increased mitochondrial aspect ratio and MFN1, MFN2, and OPA1 proteins which have previously been reported to control mitochondrial fusion were expressed at higher levels in TBCs compared to DLCs, suggesting that TBCs may regulate energy metabolism by increasing the level of mitochondrial fusion. In summary, TBCs can reduce aerobic metabolism and increase glycolysis to enable adaptation to hypoxia. Regulation of mitochondrial fusion via MFN1, MFN2, and OPA1 potentially enhances the ability of TBCs to survive on the Qinghai-Tibet Plateau.The Tibetan chickens ( Gallus gallus; TBCs) are widely distributed at altitudes of 2,200–4,100 m in the Qinghai-Tibet Plateau. This unique breed has been present in the region for at least 1,000 years. Through a long period of natural and artificial selection, the appearance and physiology of TBCs have changed to adapt to conditions of extremely high altitude, low oxygen, and cold. Compared with lowland chickens, TBCs are smaller, have an enhanced blood oxygen transport capacity due to an increased number of red blood cells, lower arterial oxygen partial pressure, lower venous blood pH, and higher hemoglobin concentration  and hypoxia (13% O2) incubators. One hundred eggs were incubated from each breed per condition. Temperature was maintained at 37.8°C with a relative humidity of 60%.The altitude of TBCs distribution is a range from 2,200 to 4,100 m, we simulated the 13% O−ΔΔCT method.Six eggs were collected from each breed and oxygen condition after 8, 12, and 18 days of incubation. Total RNA was extracted from each sample using the TRIzol reagent . cDNA was synthesized using the Fast Quant RT Kit (with gDNase) . Beta-actin was used as an endogenous control for normalization. qRT-PCR amplification was performed using primer pairs specific for target genes . The cycBrains from chicken embryos obtained on days 8, 12, and 18 of incubation were dissected in cold PBS (Gibco). Brains were homogenized in RIPA lysis buffer on ice for 30 min with a protease inhibitor cocktail (Thermo Fisher™), and centrifuged at 12,000 g for 0.5 h. Samples were boiled and denatured in loading buffer containing 5% 2-mercaptoethanol (Invitrogen) and subjected to electrophoresis through a 10% SDS polyacrylamide gel. The separated proteins were transferred to a PVDF membrane (Bio-Rad) and probed with primary antibodies for MFN1 , MFN2, OPA1 , DRP1 , α-Tubulin  and the appropriate secondary antibody conjugated with horseradish peroxidase . Bound proteins were detected using a Pierce ECL Western Blotting Substrate kit (Thermo Scientific™). The results were visualized by scanning and then analyzed using Image J software (NIH).6 cells/plate. After cultured 3 h, the inoculation fluid was replaced with serum-free medium . FBS, neurobasal™ media, B27, Penicillin-Streptomycin, and glutamine were all purchased from Gibco .Primary brain cells were isolated from chicken embryos on days 8, 12, and 18 of incubation. Intact brains were removed under sterile conditions and cleaned in pre-cooled Hanks solution . Brains were cut into pieces and digested with 0.125% trypsin (Gibco) for 30 min at 37°C. The digestion was terminated by addition of Fetal Bovine Serum (FBS). The mixture was fully suspended with inoculation fluid  and filtered through a 300-mesh sterile stainless steel sieve (BD Biosciences) into a 15 ml centrifuge tube (Corning) and then centrifuged for 8 min at 800 rpm. After removing the supernatant, cells were re-suspended and plated onto Poly-lysine–coated (100 μg/ml) Seahorse XF24 cell culture microplates (Seahorse Bioscience) at 102, 5% CO2) and hypoxia  for immunofluorescence assay. Cells were fixed for 10 min in 4% paraformaldehyde at room temperature then 0.1% TritonX-100 for 10 min. After blocked for 1 h using 1% BSA, cells were labeled with primary antibodies  for overnight at 4°C. Next day, cells were washed 3 times with 1xPBS and then stained with respective Alexa flour 488 secondary antibody (1/250) for 1 h at room temperature. Cells were then washed and 10 mg/ml Hoechst was added for 15 min. For EdU assay, primary brain cells were cultured for 24 and 48 h under normoxia  and hypoxia . Cells were cultured in fresh growth medium which contained 10 mM EdU for 24 h. Then, the cells were fixed, permeabilized, and stained following the manufacturer's instruction  and the EdU-stained cells were observed using the fluorescent microscope.Primary brain cells were cultured for 48 h under normoxia  and hypoxia  for metabolic flux analysis. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience). The medium was supplemented with 10 mM glucose, 1 μM oligomycin, 100 mM 2-deoxyglucose (2-DG), 1 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), 1 μM antimycin A, and 1 μM rotenone . OCR and ECAR values were normalized to the total protein per well (BCA assay).Primary brain cells were cultured for 48 h under normoxia (21% OThe activities of α-Ketoglutarate dehydrogenase (α-KGDH) and citrate synthase (CS) were measured using commercial assay kit from Nanjing Jiancheng Bioengineering Institute and Solarbio (China).13C6 (50 μg/mL) and evaporated to dryness under a nitrogen stream. The residue was reconstituted in 30 μL of 20 mg/mL methoxyamine hydrochloride in pyridine, and the resulting mixture was incubated at 37°C for 90 min. A 30 μL aliquot of BSTFA (with 1% TMCS) was added to the mixture. The reaction was conducted at 70°C for 60 min prior to GC-MS metabolomics analysis.After grinding and homogenizing the whole brain in liquid nitrogen, each 40 mg sample was cooled in an ice bath and homogenized for 3 min at 30 Hz in 800 μL chloroform/methanol/water solvent (v/v/v = 2:5:2), using a Tissue Lyser  with zirconia beads. Following centrifugation at 14,000 × g at 4°C for 15 min, 160 μL of supernatant was mixed with 10 μL deionized water containing glucose-GC-MS analysis was performed on an Agilent 7890A gas chromatography system coupled to an Agilent 5975C inert MSD system . An OPTIMA® 5 MS Accent fused-silica capillary column  was utilized to separate the derivatives. Helium (>99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min through the column. The injection volume was 1 μL and the solvent delay time was 6 min. Oven temperature was initially 70°C for 2 min, ramped to 160°C at a rate of 6°C/min, to 240°C at a rate of 10°C/min, to 300°C at a rate of 20°C/min, and finally held at 300°C for 6 min. The temperatures of injector, transfer line, and electron impact ion source were set to 260, 250, and 230°C, respectively. The impact energy was 70 eV, and data was collected in both full scan and SIM mode (m/z 50–600).Live-cell imaging used Nikon A1HD25 confocal microscope equipped with a 60x1.3NA oil immersion objective. Primary brain cells were stained with 20 nM MitoTracker Green  for 15 min and 10 mg/ml Hoechst for 20 min at 37°C. Fluorescence was excited with a 405 nm laser diode (Hoechst) and a 488 nm Argon laser.Mitochondrial aspect ratio (the ratio of length/width) and content  were quantified using Image J (NIH) according to previously described . Differences were considered to be statistically significant for p < 0.05. Scale bars show the SEM of at least three separate experiments.Significance was analyzed using one-way analysis of variance (ANOVA) to test homogeneity of variances via Levene's test, followed by Student's Primary brain cells were first isolated from chicken embryos on days 8, 12, and 18 of incubation and cultured. To determine what are exactly the cells isolated from the embryonic brains, we performed the immunofluorescence assay. Cells were labeled with microtubule-associated protein 2 (MAP2), a neuron-specific cytoskeletal protein expressed in the soma and dendrites of mature neurons Matus,  and Hoec−1μg−1 protein. ECAR results are presented in units of mpH min−1μg−1 protein. We compared OCR and ECAR in TBCs primary brain cells at different developmental stages under normoxia and hypoxia . The DLCs are a dwarf laying hen found in Beijing . The results were shown in Surprisingly, we identified differences in energy metabolism between TBCs and DLCs under hypoxia . TBCs di.g−1 tissue compared to 3.00 μg.g−1 tissue), but TBCs have higher levels of pyruvate (75.97 μg.g−1 tissue) and lactate (679.67 μg.g−1 tissue) (HK-1) mRNA levels show that HK-1 expression was significantly higher in TBCs  . Citric  tissue) . We incl tissue) . The res tissue) . Isocitr tissue) . Despite tissue) . Unexpec tissue) , which s in TBCs . These rHaving observed a higher energy metabolism level in TBCs than DLCs under hypoxia, we sought to determine whether the increase is related to mitochondrial. The results demonstrated no difference in mitochondrial including mitochondrial content and mitochondrial aspect ratio between TBCs and DLCs under normoxia . Then weHaving observed a higher mitochondrial quality in TBCs than DLCs under hypoxia, we sought to determine whether it is related to mitochondrial fusion which is regulated by MFN1, MFN2, and OPA1. We performed western blotting and quantitative qRT–PCR assays to detect MFN1, MFN2, and OPA1 expression. The results suggested that mitochondrial fusion was promoted in DLCs compared to TBCs on day 8 of incubation under normoxia . SurprisMFN1, MFN2, and OPA1 decline significantly during hypoxia  to produce G6P, which can be processed by glycolysis, the pentose phosphate pathway (PPP), and glycogenesis (Bélanger et al., HK-1 mRNA level was significantly different in TBCs and DLCs (Compared to DLCs, TBCs embryonic brain also exhibited a higher level of glycolysis with rising OCR on days 8 and 12, which was inconsistent with the ECAR results under normoxia . Higher and DLCs . G6P mayand DLCs , but thiMFN1, MFN2, and OPA1 (Chan, MFN2 as demonstrated by Western blot assay on day 8 (MFN genes or OPA1 decreases cellular respiration in mouse fibroblasts and myoblasts (Bach et al., Low oxygen reduces oxidative phosphorylation and Krebs cycle rates, and both processes occur within mitochondria (Raimundo et al., A1 Chan, . A signion day 8 . Surprison day 8 , 8, whicon day 8  and the on day 8 . These ron day 8 . MFN2 pron day 8  and OPA1on day 8 . DeletioBesides, we also investigated HIF-1a, which was found to modulate the expression of many metabolic enzymes as well as the expression/activity of mitochondrial fusion/fission-controlling factors. More HIF-1a transferred to nuclear in TBCs under hypoxia , which iWe conclude that TBCs embryonic brain can reduce aerobic metabolism and increase glycolysis to enable adaptation to hypoxia. Compared to DLCs, TBCs embryonic brain have a higher energy metabolism level, which may be associated with regulation of mitochondrial fusion via MFN1, MFN2, and OPA1, to enhance the ability of TBCs to survive on the Qinghai-Tibet Plateau. We have a hypothesis that this is an important mechanism via regulating mitochondrial fusion to change metabolic status for hypoxia adaptation in TBCs.The original contributions generated for the study are included in the article/The animal study was reviewed and approved by Experimental Animal Ethics Committee of China Agricultural University.QT, CD, and MF conceived the study design. QT and CD conducted the experiments. QT wrote the paper. QiaX and KW helped prepare the materials. YB and QinX took part in performing the experiments. QT and MF refined the manuscript. All the authors discussed the results and improved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zea mays) in Spencer County, Indiana was observed with heavily stunted plants, and from the affected roots a large number of cysts were recovered. Soil samples were submitted to one of us (JF), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and the recovered second-stage juveniles (J2) that were examined morphologically conformed to the measurements of Vittatidera zeaphila, the goose cyst nematode originally described from Tennessee, USA in 2010. The molecular analysis of J2 showed the sample from Spencer County matched exactly with V. zeaphila according to ribosomal DNA markers ITS, 28S, and 18S, and with mitochondrial cytochrome oxidase I (COI). The nuclear marker heat shock protein 90 (Hsp90) was also analyzed for the first time from the Indiana population of V. zeaphila. Similarities to existing cyst nematode sequences are reported herein. Geographically, although the county is across the Ohio River from Kentucky, the previously reported Hickman County, Kentucky location and Indiana detection are approximately 200 miles apart. To the best of our knowledge, this is the first report of V. zeaphila in Indiana.In the summer of 2016, a field of corn ( Vittatidera zeaphila (V. zeaphila were published by Zea mays) in Spencer County, Indiana was observed with heavily stunted plants, and from the affected roots a large number of cysts were recovered. Soil samples were submitted to one of us (JF), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and second-stage juveniles (J2) conformed morphologically and morphometrically to V. zeaphila. We report here the first occurrence of this species in Indiana, thus representing the third state after Tennessee, and Kentucky in the United States.Globally, cyst nematodes species cause serious damage to a wide variety of economically important crops. The need for information on cyst-forming nematode species has been instrumental in stimulating growth of nematology worldwide. The cyst nematode group currently contains eight genera, with a total of 121 valid species . The genzeaphila , the gooCysts, white females, and J2 were obtained from soil and roots associated with corn plants from Spencer County, Indiana. Juveniles were separated from soil by sieving and Baermann funnel extraction or were collected from cysts removed from fresh roots and kept in water in watch glasses. Juveniles were fixed in 3% formaldehyde and processed to glycerin with a formalin glycerin method . FemalesPhotomicrographs of cyst vulval cones, and J2 were made with an automatic 35-mm camera attached to a compound microscope having an interference contrast system. Roots and whole cysts were photographed under a dissecting microscope Nikon SMZ18, and light microscopic images of fixed nematodes were taken on a Nikon Eclipse Ni compound microscope using a Nikon DS-Ri2 camera. Measurements were made with an ocular micrometer on a Leica WILD MPS48 Leitz DMRB compound microscope. All measurements are in micrometers unless otherwise stated.2, 10 mM dithiothreitol (DTT), 4.5% Tween 20 and 0.1% gelatin) , 15 mM MgClgelatin)  and stor2, 0.2 mM dNTPs, 1U Platinum Taq DNA polymerase , 3 μl nematode DNA extract, and supplied enzyme reaction buffer in a total volume of 25 μl. Cycling included one step of 95°C for 2 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 90 sec, finished with one cycle at 72°C for 5 min  and AB28 , 1.5 mM MgClor 5 min .28S: Amplification of the 28S large ribosomal subunit (LSU) D2-D3 expansion segment included the primers D2A [5′-ACAAGTACCGTGAGGGAAAGTT-3′] and D3B [5′-TCGGAAGGAACCAGCTACTA-3′] and were amplified as previously described .2, and 2.5 μl PCR buffer in a total volume of 25 μl. PCR cycling conditions were 95°C for 3 min, 35X , 72°C for 5 min, 4°C until finish.18S: The 18S  sequence was amplified with primers in one fragment with forward primer 18S-CL-F3: [5′-CTTGTCTCAAAGATTAAGCCATGCAT-3′]  and reveCOI: Mitochondrial cytochrome oxidase I (COI) was amplified with primers Het-coxiF [5′-TAGTTGATCGTAATTTTAATGG-3′] and Het-coxiR [5′-CCTAAAACATAATGAAAATGWGC-3′]. Amplifications were performed in 25 μl reactions with 1x PicoMaxx (Agilent) buffer, 0.2 mM dNTPs, 0.3 μM each primer, 0.125 μl Dream Taq DNA Polymerase (Thermo Fisher), 0.5 μl PicoMaxx Taq, and 3 μl DNA extract. Cycling conditions were as described previously .2, 0.3 μM each primer, 1.25 U PicoMaxx Taq, 1 U Platinum Taq, and 3 μl nematode DNA extract. PCR cycling conditions were 94°C 2 min, followed by 45 cycles of , ending with 1 cycle of 68°C for 15 min.Hsp90: Heat shock protein 90 (Hsp90) fragments were amplified with degenerate primers U288 [5′-GAYACVGGVATYGGNATGACYAA-3′] and L1110 [5′-TCRCARTTVTCCATGATRAAVAC-3′] . CyclingV. zeaphila (MK580824-MK580829), Heterodera avenae , and the outgroup Helicotylenchus digonicus (MK580830).PCR products were analyzed by electrophoresis on 2% agarose with 1X SB (sodium borate-EDTA) buffer. Gels were stained with ethidium bromide and visualized using the U:Genius gel documentation system . Hsp90 fragments were cloned using the Strataclone PCR Cloning Kit  according to manufacturer’s instructions. Plasmid clone DNA was prepared with the QiaPrep Spin Miniprep Kit  and digested with Eco RI to verify the presence of insert. Sequencing was performed by Genewiz, Inc. Direct sequencing of PCR amplicons was used to obtain the 28S sequences (assigned GenBank accession numbers MK121965-MK121968), the ITS 1&2 rDNA sequence (MK121952), the 18S rDNA sequence (MK182465), and COI sequences (MK253554-MK253558). Accession numbers were assigned for new sequences from cloned Hsp90 of www.geneious.com) with built-in parameters or the MAFFT plug-in, with auto-selection of best algorithm depending on data. Alignments of Hsp90 intron regions were adjusted further using Geneious Alignment with free end gaps and identity (1.0/0.0) cost matrix and inspected visually to ensure preservation of exon boundaries and reading frames. Phylogenetic analysis using Bayesian inference . Multiple DNA sequence alignments were created using Geneious Prime 2019.0.3  included length of body . Lip region rounded, slightly set off with three to four annules, stylet short, well developed  with rounded knobs, lateral field with four distinct lines, tail elongate conoid with narrowly rounded terminus , and hyaline tail terminus . Shapes of the tail, tail terminus, and stylet knobs were consistent with V. zeaphila. The cysts were lemon-shaped, dark to reddish light brown in color; vulval cone was not protuberant, with large egg masses (n = 5) included body length (L) including neck ; body width (W) ; neck length  and width ; fenestra length  and width . The cysts had light to wavy line type of cyst wall cuticular pattern  collected from the cysts were examined morphologically and molecularly for species identification. Observations of morphological characters critical for identification C-J indicV. zeaphila  showed that the 18S sequences were identical. For Hsp90, six sequences were obtained from cloning the amplicons from a single J2. Sequence lengths were 1412 bp with four introns. Excluding the degenerate primer regions at each end, the sequences varied from 4 to 11 bp among the clones, mostly within introns or at third codon positions. No Hsp90 sequence was available from the TN population for comparison.Three sequences of ITS 1&2 rDNA obtained from separate J2 were assembled into a 936 bp alignment of identical sequences that overlapped with a 540 bp region from JF741961 previously described from the TN population of zeaphila . Except Vittatidera zeaphila sequences had two introns (273 bp and 200 bp) that were significantly longer than the corresponding introns from other cyst nematodes in the alignment. Vittatidera zeaphila formed a sister group to the Globodera clade, although support was moderate (0.82). Consistent with previously published 18S, 28S, and ITS trees . To assess the effect of additional taxa on topologies, another tree was constructed from a shorter, 594 bp genomic alignment of 50 sequences; this tree resulted in weaker support for the position of V. zeaphila relative to the Globodera clade and it did not further resolve the other clades (not shown). This is most likely due to the shorter alignment containing fewer informative characters. Moreover, a third alignment that included only coding regions with introns removed contained essentially the same topology and branch support as seen in V. zeaphila was not resolved in relation to the rest of the Punctoderinae . BlastN showed that the next closest sequence matches had maximum similarity of only 82% to H. glycines and other Heterodera species. In total, 46 selected cyst nematode COI fragments were assembled in a trimmed 373 bp alignment representing 33 taxa, with the resulting IQ-TREE shown in V. zeaphila appeared as a sister taxon to the Cyperi group of Heteroderinae, with 97% support. Taxa within the Avenae and Schachtii groups were also strongly supported and agreed with trees based upon ITS rDNA (H. filipjevi and H. latipons in unresolved positions relative to the rest of the Avenae group, which disagreed with previous phylogenies (Globodera spp. were not conclusively resolved by COI, possibly due to gene duplications and variability within the multipartite mitochondrial genomes in those species (V. zeaphila relative to Cactodera spp. and Globodera spp. was best supported in the tree shown in H. medicaginis relative to populations of H. glycines, a COI tree placed V. zeaphila in a weakly supported clade with Meloidodera spp., but its relationship relative to Punctodera spp. and Cactodera spp. was not well resolved (V. zeaphila in a clade with a single C. betulae sequence, but could not resolve its relationship to the clade containing Globodera spp. and Heterodera spp. (V. zeaphila as a sister taxon to Heterodera, Cactodera, Punctodera, Globodera, and Dolichodera. The relationship of V. zeaphila to the other circumfenestrate nematodes remains fluid and will be strengthened as additional sequences become available.COI sequences obtained from five J2 were assembled into a 456 bp alignment. All sequences were identical to each other and were 100% match to ITS rDNA . A numbelogenies . Relatio species . The relresolved . An 18S era spp. . The 28SV. zeaphila was previously established (Although evidence of damage to host plants from ablished , there r


Citrobacter species often occur in sewage, food, soil, wastewater, and in the intestinal tract of animals and humans. Citrobacter spp. cause urinary tract infections (UTIs) and infantile meningitis in humans. Due to the presence of plasmid-encoded resistance genes, Citrobacter spp. are often resistant to many antibiotics. In this study, Citrobacter virus HCF1, a novel virulent bacteriophage capable of killing Citrobacter amalonaticus and Citrobacter freundii, was isolated from the sewage water. The isolated bacteriophage was characterized with respect to transmission electron microscopy, one-step growth curve, host range, in vitro efficacy, storage stability, and environmental stress tolerance. The one-step growth curve analysis revealed that the latent period of HCF1 was 30 min and the estimated burst size was 121 plaque-forming units (PFU) per bacterial cell. Host range testing indicated that the HCF1 was specific to the Citrobacter genus. In vitro efficacy assay in the effluent of an anaerobic biodigester showed that the HCF1 completely eliminated the host within 4 and 5 h at MOI:100 and MOI:10, respectively, thereby indicating its potential for combating C. amalonaticus infections. The isolated bacteriophage is considerably stable and tolerant to environmental stress. Furthermore, the complete genome of HCF1 was sequenced using Oxford Nanopore sequencing and the data were subjected to detailed bioinformatic analyses. NCBI-BLASTn analysis revealed that the HCF1 genome had a query coverage of 15–21% and a maximum similarity of 77.27–78.49% with 11 bacteriophages of the Drexlerviridae family. Detailed bioinformatic analysis of the genome profile suggests that HCF1 is a novel T1svirus belonging to the Tempevirinae subfamily of the Drexlerviridae family. Citrobacter is a genus of aerobic, Gram-negative bacteria belonging to the Enterobacteriaceae family. Citrobacter spp. have been reported from sewage   . In a reisolates . Similarisolates  conducte A study  reportinC. amalonaticus infection reported in Marseille, France, suggested that the bacterial isolates associated with various cases exhibited resistance to different antibiotics such as amoxicillin, ticarcillin, cotrimoxazole, amoxicillin/clavulanate, and ticarcillin/clavulanate. Although Citrobacter spp. are regarded as rare human pathogens, this genus contains plasmid-encoded resistance genes which confer considerable resistance to many antibiotics. Therefore, Citrobacter spp. pose a significant public health threat to hospital patients as well as the general public. In a study of 36 patients with C. freundii bacteremia in a tertiary medical center in northern Taiwan between 2009 and 2014, a high degree of antibiotic resistance was reported for various first- and second-generation antibiotics including cefuroxime (66.7%), cefoxitin (97.2%), and cefazolin (100%). Furthermore, polymerase chain reaction (PCR)-based detection of resistance genes in these bacterial isolates suggested the presence of β-lactamase genes , a mediator of carbapenem resistance. A recent study from a 22-bed digestive rehabilitation center in Buenos Aires (Argentina) reporting an episode of a sudden 2-week outbreak of C. amalonaticus infection revealed the presence of blaNDM-1 strain . By whole-genome sequencing, a total of 16 resistance genes were detected, including blaNDM-1 and mcr-1.5  is reported  according to standard protocols.The sources of all bacterial strains used in this study are detailed in Citrobacter amalonaticus virus HCF1 was isolated from sewage water collected from the Defence Research and Development Establishment (DRDE) campus , Gwalior, India. The sewage sample was centrifuged at 4000 × g for 10 min to eliminate possible solid impurities. The supernatant was then filtered using a 0.22-μm syringe filter  to remove bacterial debris. The bacteriophage was isolated using the enrichment method described by C. amalonaticus NAIMCC 1364 culture  was inoculated into 90 mL of the sewage filtrate and incubated in a shaking incubator at 100 r/min for 24 h at 37°C. Following incubation, the mixture was centrifuged at 4000 × g for 10 min and filtered using 0.22-μm syringe filter (Millipex GP). Bacteriophage purification and propagation was carried out using double agar plating assay as described by 11 PFU mL–1) was deposited on a 300-mesh copper grid and stained with 2% phosphotungstic acid (pH 4.5) for 30 s. After air-drying, grid was observed with a TEM (JEOL JEM-1400plus) at an accelerated voltage of 80 kV. Three virions were measured to determine the size of head and tail. Accordingly, the values have been represented in results as average ± standard deviation (SD).To prepare bacteriophage sample for the morphological examination using transmission electron microscope (TEM), the purified bacteriophage solution . This mixture was plated onto LB agar plate . Subsequently, 10 μL of bacteriophage suspension (∼1010 PFU mL–1) was spotted onto the LB agar medium and allowed to incubate overnight at 37°C. Each experiment was performed in triplicate. The relative EOP with respect to various susceptible bacterial hosts was determined using the double agar plating assay as described previously , as well as at −20 and −80°C in SM buffer supplemented with 15% (v/v) glycerol in multiple pre-sterilized glass vials (10 mL volume). Samples were removed from the glass vials at a 15-day (D) interval and the bacteriophage titer was determined using the double agar plating assay as described earlier . Acidity and alkalinity in solutions were produced by, respectively, using HCl (6 M) and NaOH (6 M) as described by 8 PFU mL–1) was added to 90 mL of autoclaved SM buffer with varying pH and salt concentrations and incubated at 37°C in conical flask (250 mL volume) made of borosilicate glass. Samples were drawn from the flasks 30, 60, 120, 240, and 360 min after incubation, and the bacteriophage titer was determined using the double agar plating assay as described previously , pH , and NaCl concentrations  and in an anaerobic biodigester effluent at 37°C. Effluent was obtained from a local anaerobic biodigester that treats human excreta. Briefly, 10 mL of bacterial culture (109 CFU mL–1) was added to 89 mL of autoclaved saline buffer/effluent in a conical flask  which resulted in 108 CFU mL–1 of bacterial suspension. One milliliter each of bacteriophage suspension having 1010, 1011, and 1012 PFU mL–1 was used to make three different MOIs , and the flasks were incubated at 37°C. Next, 100 μL of the sample was drawn at 1 h intervals up to 6 and at 24 h. CFU counts were performed using the Miles and Mishra drop-plate method (e method . In the –1 = Average number of colonies for a dilution × 50 × dilution factor.CFU mL9 PFU mL–1) was incubated with DNase (2 mg mL–1) and RNase (5 mg mL–1) for 1 h at 37°C. To this, bacteriophage lysis buffer  was added, mixed well, and incubated at 50°C for 30 min. For protein precipitation, 3.5 M ammonium acetate was added to this mixture at the rate of 60 μL mL–1, incubated on ice for 30 min, and centrifuged at 13,000 × g for 10 min. Thereafter, the supernatant was taken into a fresh tube, where an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added, and the sample was centrifuged at 10,000 × g for 10 min. This step was repeated once. The aqueous upper phase was then transferred into a fresh tube, and an equal volume of chloroform was added to it before the sample was centrifuged at 10,000 × g for 10 min. The aqueous upper phase was again transferred to a new tube and an equal volume of isopropanol was added and the sample was allowed to precipitate at 20°C for 2 h. The precipitated nucleic acid was centrifuged at 12,000 × g for 5 min at 4°C, and the pelleted nucleic acid was washed with 70% (v/v) ethanol. The purified bacteriophage nucleic acid was visualized on a 0.9% (w/v) agarose gel. The nucleic acid was distinguished as being DNA or RNA following treatment with DNAse I and RNAse A (Fermentas) separately as per the manufacturer’s instructions.Bacteriophage HCF1 nucleic acid extraction was performed as described by fast5” format) were basecalled (“fastq5” format) and demultiplexed using Albacore v2.3.1. Basecalled reads were error-corrected and assembled using “Canu” assembler v1.8, for sequence polishing.End-repairing of bacteriophage HCF1 DNA samples was done using NEBnext ultra II end repair kit . Clean-up of end-repaired samples was carried out with 1x AmPure beads . NEB blunt/TA ligase (New England Biolabs) using NBD103 (ONT) was used for Native barcode ligation. Following ligation, clean-up process was repeated with 1x AmPure beads (Beckmann Coulter). The Qubit-quantified barcode ligated DNA sample was then pooled at equimolar concentrations to obtain 500 ng pooled sample. BAM Adapter ligation was performed for 15 min using NEBnext Quick Ligation Module (New England Biolabs). The library mix was cleaned up using 0.4X AmPure beads (Beckmann Coulter) and, finally, the sequencing library was eluted in 15 μL of elution buffer and used for Nanopore sequencing. Sequencing was performed on a GridION X5  using a SpotON flow cell (R9.4) in a 48 h sequencing protocol on MinKNOW 2.1 v18.05.5. In order to eliminate probable errors in long-read assemblies, all the Nanopore raw reads  was carried out using the GGDC web server  with theequences . Furtherequences . In addiequences .C. amalonaticus virus HCF1 has been deposited in NCBI GenBank under accession number MN545971. In addition, fastq file pertaining to raw sequence data has been submitted to NCBI Sequence Read Archive (SRA) database under the identifier number SRP267319.The complete genome sequence of post hoc test. A p-value ≤ 0.05 was considered to be statistically significant. Accordingly, significant differences have been marked in tables and figures as relevant.Statistical analysis for the empirical data was performed using SPSS Statistics v17.0 . Significant differences among the different conditions for each tested parameter were assessed by one-way analysis of variance (ANOVA) followed by Tukey’s HSD 12 PFU mL–1 by following the classical method for purification and propagation as suggested by n = 3; ± SD) and a tail of 233 ± 4 nm dia  as analyzed using TEM  in bacterial counts within 1 h of bacteriophage treatment even at an MOI:1 compared to the control (p ≤ 0.05 as per Tukey’s HSD test). Data recorded after 1 h suggested that an increase in the MOI resulted in a significant decrease in bacterial growth. HCF1 completely eliminated the host within 4 and 5 h at MOI:100 and MOI:10, respectively. Even at MOI:1, there was 6.05 log reduction in bacterial counts within 6 h compared to the control  at 4°C. Even at 75 and 90 D, bacteriophage survival was as high as 8.80 and 7.43 log PFU mL–1. In contrast, when stored at −20 and −80°C, bacteriophage viability decreased significantly over time from 15 D onward (p < 0.05 as per Tukey’s HSD test) and, at 90 D, only 2.77 and 2.34 log PFU mL–1 survival was recorded, respectively. There was no statistically significant decrease in bacteriophage survival following storage at 25°C, for up to 30 D (p > 0.05 as per Tukey’s HSD test). However, after 30 D, bacteriophage titer decreased significantly over time with the survival of only 2.14 log PFU mL–1 at 90 D  on viability of bacteriophage HCF1. The results revealed that bacteriophage viability did not decrease significantly for up to 60 D . Bacteriophage HCF1 tolerated pH 11.0 for up to 30 min without any significant decrease in viability. At pH 9.0, bacteriophage HCF1 demonstrated increased tolerance toward alkaline conditions as shown by the considerable phage survival (6.81 log) for up to 360 min. However, bacteriophage HCF1 showed no statistically significant decrease in viability during the tested period at pH 7.0 for up to 360 min  in its viability over the studied time compared to the control. In contrast, we observed moderate sensitivity of bacteriophage HCF1 to 10 g NaCl L–1 . However, further increase in salinity to 15 and 20 g NaCl L–1 resulted in decreased viability with survival rates of 4.53 and 3.24 log, respectively . The bacteriophage survival rate at 50°C after 360 min was 7.50 log  and putative transcriptional regulator (encoded by orf51) with 68 and 46% sequence similarity to antirepressors of Salmonella spp. phage 36 and putative transcriptional regulator of Escherichia spp. phage vB_EcoS_W011D, respectively, were identified . TMHMM2.0 analysis could not provide useful information regarding orf59. Hence, Pfam analysis of orf59 was carried out which suggested the presence of a transmembrane domain with a signal peptide  in the bacteriophage lysozyme, indicating a likely function of O-glycosyl hydrolases (EC 3.2.1.17). Phyre2 server analysis suggested that orf59 corresponding to the HCF1 lysozyme had a maximum identity to coliphage 21 lysozyme  and gp41 (orf34), and were found to encode for neck protein Type 1 (Ne1) and Tail completion protein Type 1 (Tc1), respectively . Out of these, orf53 was the largest ORF  encoding a protein similar to the ATP-dependent DNA helicase of Citrobacter virus Stevie sharing 59.97% identity (accession number YP_009148736.1). The predicted proteins of orf10, orf47, orf48, orf49, orf50, orf54, and orf63 displayed 74.01, 61.67, 82.22, 81.13, 57.30, and 97.46% identity to polynucleotide kinase, putative exodeoxyribonuclease VIII, putative recombination protein, Ssb, DNA primase/helicase, putative endonuclease, and helicase with Escherichia phage vB_EcoS_PHB17, Salmonella phage YSP2, Salmonella phage 36, Escherichia virus TLS, Citrobacter phage CF1 DK-2017, Salmonella phage GJL01, and Salmonella phage 36, respectively (orf47) in HCF1 genome may confer it stability against DNA damage.BLASTp analysis suggests that the DNA metabolism module consists of 10 ORFs  . VRR-Nuc is known to be member of the primordial restriction endonuclease-like superfamily possessing a mixed fold of α/β fold of αβββαβ topology. VRR-Nuc-containing proteins usually occur as a single-domain nuclease in several bacteria and viruses. FANCD2/FANCI-associated nuclease 1 (FAN1), a structure-specific nuclease, is the only example of multi-domain eukaryotic protein containing a VRR-Nuc domain. FAN1 is considered to play an important role in the repair of inter-strand DNA crosslinks like the ERCC1-XPF nuclease  contained an N-terminal signal sequence which lacked any transmembrane domains. Pfam analysis suggests that the protein corresponding to orf50 in bacteriophage HCF1 consists of a zinc-binding domain , a transmembrane domain, a signal peptide, and a coiled-coil. In addition, the protein corresponding to orf53 in HCF1 contains a helicase conserved C-terminal domain restricted to DEAD/H helicases , a transmembrane domain, and a signal peptide. Furthermore, the predicted protein of orf55 of HCF1 exhibited 64.23% identity with DNA adenine methyltransferase  of Salmonella phage vB_SenS_PHB07. DAM is linked with several biological functions in bacteria . In addition, DAM is known to induce DNA methylation in T1 bacteriophages  was from the Indian sub-continent  with each other at protein level. Taken together the data pertaining to nucleotide and protein profiles, we therefore propose that Citrobacter spp. virus HCF1 is a novel bacteriophage under the Tempevirinae subfamily of the Drexlerviridae family.BLASTn analysis of the whole-genome sequence of the newly isolated bacteriophage HCF1 suggested that only 11 bacteriophages had an E-value of 0.0. These 11 bacteriophages were taken forward for detailed comparison with HCF1. Maximum score, query cover, and percent identity of these bacteriophages with HCF1 ranged from 974 to 1037, 15 to 21%, and 77.27 to 78.49%, respectively. All these 11 bacteriophages belonged to the us genus . Phylogeus genus . Phylogeus genus . The perus genus . To our us genus , Phage Cus genus , and Phaus genus  belonginontinent . Howeveronaticus . The disonaticus . Moreoveonaticus . It has onaticus , 2009. AC. amalonaticus as well as C. freundii. The detailed analysis of its whole-genome properties and putative protein profiling as elucidated using various bioinformatic tools has allowed us to propose it as a novel species of bacteriophage belonging to the genus T1svirus under Tempevirinae subfamily within the Drexlerviridae family. The novel bacteriophage HCF1 exhibits no significant loss of viability up to 75 D when stored at 4°C. In addition, it tolerates high temperature (50°C) and alkaline pH as high as 11 without significant loss of the viability. Furthermore, in vitro efficacy assay indicated its potential for combating infections caused by C. amalonaticus and C. freundii. The predicted presence of DAM (orf55) in HCF1 suggests that it is able to evade the host R-M system. This study may further be extended for devising suitable strategies for the field applications of bacteriophage for validating its use as antimicrobial agent against Citrobacter spp.In conclusion, we have isolated a novel virulent bacteriophage capable of killing the bacterium The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.MM and DK conceived and designed the study. PK and MM performed the experiments, analyzed the data, and prepared the initial draft of the manuscript. All authors checked and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thus, this study contributes to understand the complicated relationship between fat ingest and CRC.Etiology of colorectal cancer (CRC) is related, at least in part, with nutritional profile and epidemiological data indicating a key role of dietary fat on CRC pathogenesis. Moreover, inflammation and eicosanoids produced from arachidonic acid might have a pivotal role in CRC development. However, the effect of specific fatty acids (FAs) on intestinal epithelial cell growth is not completely studied now. By this reason, the aim of this work is to unravel the effect of different saturated and unsaturated long-chain fatty acids (LCFA) and some LCFA metabolites on CRC cell line growth and their possible mechanisms of action. Our results demonstrated that oleic acid is a potent mitogenic factor to Caco-2 cells, at least in part, through 10-hydroxy-8-octadecenoic synthesized by lipoxigenase pathway, whereas polyunsaturated FAs such as eicosapentaenoic (EPA) acid has a dual behavior effect depending on its concentration. A high concentration, EPA induced apoptosis through intrinsic pathway, whereas at low concentration induced cell proliferation that could be related to the synthesis of eicosanoids such as prostaglandin E Cancer causes around 7 million of deaths annually, becoming 12.5% in the entire world and colorectal cancer (CRC) is the third leading cause of cancer-related death in developed countries . AccordiCRC is linked with environmental factors, being lifestyle and nutritional profile the major but controllable implicated factors. In fact, around 90% of the CRC cases appear to be related to lifestyle, with the highest incidence in economically developed countries . Refinedtrans fats and unsaturated fats can act in opposite ways to influence human health, including promotion of cancer. Thus, the data indicated that not only the amount of dietary fat but also fat diet composition could be determinant in the pathogenesis of different neoplasms as CRC . However, not all fats are the same and it is now well established that saturated, s as CRC . Some exs as CRC . Howevers as CRC , others s as CRC , while ss as CRC . In the s as CRC  but othes as CRC .Eicosanoids produced from arachidonic acid (AA) might have a pivotal role in these events. AA biosynthesized from linoleic acid is the substrate of cyclooxygenase (COX) and lipoxygenase (LOX) for the production of eicosanoids such as 2-series prostaglandins (PGs), 4-series leukotrienes (LTs) and HETEs that may facilitate CRC progression by stimulating cell proliferation and survival, tumor cell invasiveness, and angiogenesis . PUFAs sConsidering all together, the effect of FAs on intestinal epithelial cell growth is not completely understood now. By this reason, the aim of this study is to determine the effect of representative saturated and unsaturated long-chain FAs (LCFA) and some fatty acid metabolites on CRC cell line growth and their possible mechanisms of action.2, PGE3, leukotriene (LT) B4, LTB5, 12-S-HETE, 12-S-HEPE, ketoprofen, baicalein, MK571, MK886 LY171883, zileuton, U75302, and the PPARγ ligand screening assay were supplied by Cayman Chemical Co. . Cell proliferation ELISA–BrdU (Colorimetric) Kit was from Roche . LY255283 was from Tocris Biosc. . AH23848 and ONO-329 were kindly provided by Glaxo-Wellcome  and Ono Pharmaceutical Co. Ltd. , respectively. The MebStain Apoptosis Kit was supplied by MBL International  and the Multi Kinase Array (MKA) ElA by Symansis .Dulbecco’s modified Eagle’s medium (DMEM), trypsin/EDTA, penicillin, and streptomycin were supplied by GIBCO . Acridine orange, bovine serum albumin (BSA) de-fatty BSA, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), Dulbecco’s PBS, ethidium bromide, fetal bovine serum (FBS), nonessential amino acids, paraformaldehyde extra pure, propidium iodide, ribonuclease A from bovine pancreas, SC19220, tetramethylrhodamine ethyl ester perclorate (TMRE), and Triton X-100 were supplied by Sigma-Aldrich . 10-hydroxy-8-octadecenoic acid (10-HODE) was from Parchem . Tissue culture supplies and sterile material were obtained from Corning, Nirco S.L., NORM-JECT and Biosigma S.R.L. . The BioRad Protein Assay was obtained from Bio-Rad Laboratories, Inc. Myristoleic, palmitoleic, stearic, oleic, elaidic, linoleic, α-linolenic, γ-linolenic, mead, arachidonic (AA), EPA, erucic and DHA acids, prostaglandin (PG) E2 plastic flasks at a density of 1 × 104 cells/cm2 and cultured in DMEM with 4.5 g/L D-glucose and 2 mM L-glutamine, and supplemented with 1% (v/v) nonessential amino acids, 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated at 37°C under a humidified atmosphere of 5% CO2 in air. Cells grown to ~80% confluence were released by trypsinization and subcultured at a density of 1.5–2 × 104 cells/cm2 in 12 mm diameter plastic clusters and of 1 × 104 cells/cm2 in 60 mm diameter plastic dishes. Growth medium was replaced twice per week. Although cancerous in origin, Caco-2 cells undergo a gradual differentiation process that takes place spontaneously once confluence has been reached and that is completed after 21–25 days in culture (Caco-2 cells were derived from a moderately well-differentiated primary colon adenocarcinoma and were provided by American Type Culture Collection (HTB-37) . The cells were routinely grown in 25 or 75 cm culture . Conside3 cells/cm2). Cells were cultured for 96 h in DMEM supplemented with 10% FBS. Then, cells were incubated for 48 h in the presence of treatments. Finally, cells were washed, trypsinized, and counted under a microscope using ethidium bromide/acridine orange staining to count viable cells.The effect of the treatments was assessed on Caco-2 cells clusters in 24-well plates . Caco-2 cells were cultured at 1,000–1,500 cell/well in 96 well plates for 96 h in DMEM supplemented with 10% FBS. Then, cells were incubated for 48 h in the presence of compounds. Thereafter, cells were treated following the manufacturer instructions. Absorbance was measured at 450 nm in a plate reader .Degradation of chromosomal DNA was evaluated with TUNEL method using a MebStain apoptosis kit (MBL Int.). After 96 h in culture, Caco-2 cells were cultivated in media containing treatments for 48 h. Next, cells were fixed with 4% paraformaldehyde and permeabilized with 70% ethanol. Thereafter, 3′-OH DNA ends generated by DNA fragmentation were labeled with fluorescein-dUTP, mediated by terminal deoxynucleotidyl transferase, and were analyzed on an Epics XL flow cytometer .2 and PGE3 concentrations were determined in the Caco-2 culture medium. Cells were overnight with FBS or FBS plus EPA (10 μM). Two hundred fifty μl aliquots of the culture medium were acidified with 1 ml of formic acid and PGs were extracted in ethyl acetate. After discarding the aqueous phase, the organic phases were evaporated under a stream of N2 and PGE2 or PGE3 were determined with a monoclonal enzyme immunoassay kit  according to the manufacturer’s protocol.PGE4 cells/cm2). After 4 days, the cultures were incubated with the treatments in absence of FBS. Maximal phosphorylation was observed after 5 min incubation for ERK1/2, Akt, and p38, and after 15 min for GSKβ and CREB. To measure the kinase activation with total cellular lysates, Caco-2 cells were lysed using a denaturing cell lysis buffer containing 6 M urea and protease  and phosphatase  inhibitors. The resulting solutions containing 80–100 µg of proteins were then added to a kinase ELISA plate and the assay was performed following the manufacturer’s recommendations (Symansis). Finally, optical density was measured at 450 nm. Thus, we studied the effect of PGE2/PGE3 on the phosphorylation of AKT1 (pS473), AKT2 (pS474), ERK1/2 , GSK3β (pS9), p38α (pT180/Y182), and CREB (pS133).Cells were seeded in 60 mm plastic clusters . This assay is based on the competition of free ligand in the samples or standards for the affinity binding site of PPARγ occupied by a probe conjugated to fluorescein. Finally, the polarization was quantified as milli-polarization units (mP). This assay was adapted to be performed in a microcuvette with a luminescence spectrometer .Mitochondrial membrane depolarization (MMP) was measured through tetramethylrhodamine ethyl ester perclorate (TMRE) incorporation into active undifferentiated Caco-2 cell mitochondria. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control for MMP. Cell cultures were treated with experimental compounds for 20 min and, after tripsinization, cell suspension was labeled with TMRE for 30 min. Finally, samples were analyzed in FC500 flow cytometer (Coulter Corporation) to measure mitochondrial depolarization or hyperpolarization.The results are expressed as mean ± SEM. All data were compared by one-way ANOVA and the Student’s t-test, using SPSS software . Significance was taken as p < 0.05.When undifferentiated Caco-2 cells were incubated with different LCFA in absence of FBS we observed that palmitoleic (10–100 µM), oleic (1–100 µM), myristoleic (100 µM), and elaidic (100 µM) acids as well as the lowest EPA and DHA concentration (10 µM) induced cell proliferation , and LY255283 (BLT1 and BLT2 antagonist) (4 antagonist), SC19220 (EP1 antagonist) also reverted the mitogenic effect induced by EPA  whereas PGE3 was not detected. Interestingly, EPA (10 μM) supplementation of Caco-2 cell culture medium increases PGE3 synthesis (1.02 ± 0.13 ng/ml), whereas we observed the impairment of PGE2 (1.02 ± 0.09 ng/ml). In 3 progressively increased Caco-2 cell growth and DNA synthesis up to 10 nM in a similar way to PGE2. 3 was totally inhibited by an EP1 antagonist (SC19220) and by an EP4 antagonist (AH23838). However, an EP3 antagonist  did not have any effect. Therefore, these results indicate that PGE3 acts through EP1 and EP4 receptors, but not through the EP3 receptor.EPA can be presented into cell membrane phospholipids , it is r1 antagonist (U75302), and by a BLT1 and BLT2 antagonist (LY255283)  induced significant cell growth and DNA synthesis in Caco-2 cell cultures in the absence of growth factors, in a similar way to 12-S-HETE (100 nM). This mitogenic action of 12-S-HEPE was blocked by a COX inhibitor (ketoprofen), by a BLT4 has a mitogenic effect on Caco-2 cells (5 derived from EPA did not induce proliferation in the range of 1–100 nM (Previously we observed that LTB-2 cells . However3 (10 nM) and 12-S-HEPE (100 nM) to phosphorylate pivotal elements in the cell signaling pathways implicated in the regulation of cell growth. Our findings show that PGE3 presents a similar pattern to PGE2. Thus, PGE3 could increase the phosphorylation of ERK 1/2, CREB, GSKβ, and p38α , but not by BLT1 and BLT2 antagonist (LY255283) nor by MK571, a cysteinyl leukotriene antagonist  such as acetate, propionate, and butyrate. SCFAs and specially butyrate induces cellular differentiation and decrease proliferation of CRC-derived cell lines . These e pathway . Thus, S pathway . However18:1trans, elaidic acid. These findings agree with min/+ mice feed with oleic acid present a high number of intestinal tumors, and recently, this mitogenic effect of oleic acid has also been observed in prostate cancer cell growth . Furthermore, we demonstrated that this PGE3 proliferative effect was a consequence of interaction with the PGE2 receptors EP1 and EP4, in agreement with their affinities  or LCFA metabolites on Caco-2 cell growth through bind to GPR40 and the subsequent cell signaling activation involved in cell proliferation , by Generalitat de Catalunya (2009SGR438) and by CIBEROBN .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constituted as a potential conflict of interest.


Raman spectral data acquisition was carried out using a non-contact fiber optic probe on the surface of aqueous samples in 50–3398 cm−1. ICP-AES was used as a reference method for the determination of the mineral contents in aqueous INF samples. Results showed that the best performing partial least squares regression (PLSR) models developed for the prediction of minerals using all samples for calibration achieved R2CV values of 0.51–0.95 with RMSECVs of 0.13–2.96 ppm. The PLSR models developed and validated using separate calibration (n = 42) and validation (n = 41) samples achieved R2CVs of 0.93, 0.94, 0.91, 0.90, 0.97, and 0.94, R2Ps of 0.75, 0.77, 0.31, 0.60, 0.84, and 0.80 with RMSEPs of 3.17, 0.29, 3.45, 1.51, 0.30, and 0.25 ppm for the prediction of Ca, Mg, K, Na, Fe, and Zn respectively. This study demonstrated that RS equipped with a non-contact fiber optic probe and combined with chemometrics has the potential for timely quantification of the mineral content of aqueous INF during manufacturing.This study investigated the use of Raman spectroscopy (RS) and chemometrics for the determination of eight mineral elements  in aqueous infant formula (INF). The samples were prepared using infant formula powder reconstituted to concentrations of 3%–13%  Infant formula (INF) is intended for infants and young children and can be a complete or partial substitute for human milk . Dairy-bMulti-element determinations at the ultra-trace level can be achieved using atomic absorption spectroscopy (AAS), inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and ICP-mass spectroscopy (ICP-MS). These techniques have been well investigated for the quantification of trace elements in milk and INF ,7,8,9,10In the dairy industry, powdered INF is usually manufactured using either a dry blending process or a wet mixing-spray drying process . The wet2CV—0.99), protein (R2CVs—0.98–0.99), albumin, blood urea nitrogen (R2CV—0.82), lactose (R2CVs—0.86–0.92), glucose, and somatic cell count (R2CV—0.85) [2CV—0.29–0.97) and poor accuracy on the content of major minerals (R2CV—0.41–0.48) [2CV—0.42–0.71) [2CV—0.59–1) [2CV value of 0.90 with RMSECV of 680 mg/kg (ppm) for the prediction of Ca; R2CVs of 0.83 with RMSECVs of 16.1 mg/kg (ppm) and 226.4 mg/kg (ppm) for the prediction of Cu and Fe, respectively. To date, almost all the published studies using process analytical technology for quantification of mineral elements in infant formula were carried out on dry powder samples. To the authors’ knowledge, no information is currently available for quantifying mineral contents of aqueous INF using RS.Previously, process analytical methods have been widely investigated to verify nutrient levels of milk and INF. Near-infrared spectroscopy (NIR) has been successfully applied to predict various constituents in milk, including fat (RCV—0.85) ,14,15. M41–0.48) . Other s42–0.71)  and high—0.59–1) . Raman s—0.59–1) ,20,21,22—0.59–1)  and mela—0.59–1) ,25,26. A—0.59–1) . Laser-i—0.59–1) ,29 and y2CV of 0.775 with RMSECV of 0.33%) [2CV of 0.954 with RMSECV of 0.490 mg/g) [As a non-invasive technique, RS has considerable advantages for the analysis of aqueous samples compared to other methods because water has weak Raman scattering properties. Unlike infrared spectroscopy, this technique is not limited by water interference. For trace element determination, RS can provide sophisticated spectral information of chemical composition with high resolution . It has f 0.33%)  and to p90 mg/g) . Therefon = 14) were produced using a pilot-scale research facility at Teagasc food research center  and stored in sealed bags at ~15 °C for three months. Moreover, five commercial INFPs  were purchased from local supermarkets in Ireland. Altogether, 19 INFP samples were included in this study. The moisture content of each powder was measured using a halogen rapid moisture analyzer  at 105 °C until a constant weight (<1 mg) was attained in 140 s. For aqueous sample preparation, powder samples were weighed and dissolved in 200 mL of deionized water, considering moisture content to obtain solutions at five concentrations, i.e., 3%, 5%, 8%, 10%, and 13% w/w. During homogenization, an overhead 4-blade impeller of a high shear mixer  was positioned at the center of the deionized water and operated at 400 rpm; the weighed powder was added and mixed at ambient temperature (∼15 °C) for 20 min. In total, eighty-three aqueous samples were prepared for this study including 75 aqueous samples prepared from 15 powder samples  at concentrations of 3%, 5%, 8%, 10% and 13% w/w; 7 aqueous samples prepared from 7 powder samples  at a concentration of 13 % w/w, and one aqueous sample prepared from one powder sample at a concentration of 8% w/w due to the limited amount of these particular INFP samples. After sample preparation, all aqueous samples were kept overnight at 4 °C before measurements.Infant formula powder (INFP) samples . Each sample was scanned twice at different locations of the aqueous surface. The averaged spectrum of the duplicate was used in the subsequent chemometric analysis.Raman spectral data were collected using a non-contact fiber optic probe on the surface of aqueous INF samples. A fiber optic probe and the connected coaxial cable were fixed on a probe stand to avoid effects caused by subtle changes of instrument configuration. Each sample (10 mL) was transferred into an aluminum cylindrical container  and placed on a variable height platform under the probe. Samples were raised so that their surface was 2 mm away from the quartz screen of the probe head. A DXR SmartRaman spectrometer  was used for spectral data acquisition. The system was equipped with a diode laser operating at 780 nm and a charge-coupled device (CCD) detector. The sample surface was exposed under the aperture (50 μm slit) of the fiber optical probe accessory. For each scan, a 120 mW laser power was used; spectra of each sample were automatically accumulated until the maximum signal to noise ratio of the averaged spectrum was obtained. All aqueous samples were scanned in random order at ca. 20 °C. Raman intensity counts per second (cps) were recorded in the wavelength range of 50−3380 cmv/v detergent  diluted with tap water followed by rinsing, air-drying and soaking in 10% v/v HNO3 prepared from 65% v/v HNO3 . Before use, glassware was rinsed three times with deionized water and dried in a thermal cabinet. For ICP-AES instrumental calibration, standard solutions were prepared using the ICP multi-element standard stock solution of 1000 ppm  diluted to 1.25, 2.5, 5, 12.5, 25 and 50 ppm respectively using 5% v/v HNO3 prepared from 69% v/v HNO3 . Standard solutions were stored overnight in ash-free polypropylene containers at ~4 °C before ICP-AES measurements. Deionized water (resistivity−18.2 MΩ-cm) was produced using a Barnstead™ Smart2Pure™ water purification system  for the preparation of reagents and standard solutions for ICP-AES analysis. To avoid mineral contamination, all glassware sets used in this study were washed using 2% Each aqueous INF sample (10 mL) was transferred into a porcelain crucible (25 mL) and heated in a chamber furnace  at 650 °C for three hours until white ashes were obtained.v/v HNO3. The acid digestion solution of each sample was prepared by dissolving ash residue in 25 mL of 5% v/v HNO3 for analysis of INFP minerals at low concentrations (<140 ppm), e.g., Mg, Cu, Fe, Zn, Mn, etc. [v/v using 5% v/v HNO3. All solutions were kept in the polypropylene containers at 4 °C before ICP-AES analysis.Ashed samples were digested using 5% Mn, etc. . For anav/v HNO3) and multi-element standards of 12.5, 25, and 50 ppm for elements present at higher concentrations and the blank and multi-element standards of 1.25, 2.5, and 5 ppm for the elements at lower concentrations. For sample analysis, the optimal wavelength for monitoring each mineral element was selected from three relevant wavelengths suggested by the supplied software of the ICP-AES system. The optimal wavelength for each specific element was decided based on the highest linearity (R2~1) of the developed calibration curves. Each sample was measured in triplicate; the mean value of each mineral element was acquired, and the repeatability value for each element detection was also calculated [A Vista RL  CCD simultaneous ICP-AES  spectrometer powered by a 40 MHz radiofrequency generator at 1.2 kW was used for the determination of all minerals. This equipment employed an echelle polychromator with a cross dispersion prism to form a two-dimensional diffraction pattern across its focal plane. A charge-coupled device detector was equipped to detect electromagnetic signals at a wavelength range of 167–784 nm. Argon gas was used as a plasma source at a flow rate of 15 L/min, and the auxiliary gas flow rate was 1.5 L/min. Before the operation, instrumental calibration was carried out to develop standard curves with < 10% of maximum error and >0.995 correlation coefficient values. During the analysis, the prepared standard solution or acid digestion solution was introduced into the system via a SeaSpray concentric nebulizer  operated with argon aerosol gas (200 kPa) at a 0.75 L/min flow rate. Sample aspiration was forced using an Alitea v-10R peristaltic pump at 15 rpm with a 1.4 mL/min sample delivery rate. To avoid nebulizer contamination and clogging, a sample uptake delay (15 s) and rinse time (10 s) with deionized water were applied between each measurement. The signals were obtained at an observation height of 13 mm above the load coil and were corrected using the embedded background correction method. Calibration curves were developed using a blank solution . After dry matter analysis, the certified reference material (1 g) was transferred into a porcelain crucible (100 mL) and heated at 400 °C for 20 min using a hot plate  until no further smoke was observed. Then the crucible was placed into a furnace  to heat the material at 650 °C for 3.5 h until white ashes were obtained. For ICP-AES analysis, dry digestion and ICP-AES measurements were carried out following the procedures described in 2, MgCl2, KCl, NaCl, MnCl2, iron(III)chloride hexahydrate (FeCl3 + 6 H2O), CuCl2, and ZnCl2 crystals  dissolved in deionized water. These chloride salt solutions were separately kept in polypropylene containers. Before Raman measurements, each solution was well shaken using a vortex mixer  for 1 min to ensure complete dissolution. During the experiment, 10 mL of each solution was transferred into a 50 mL quartz beaker with aluminum foil smoothly covering the outer surface and then placed on an elevating frame under the fiber optic probe stand of the Raman spectrometer . The sample was raised to ensure that its surface was 2 mm away from the quartz screen of the probe head. Detailed measurement procedures and instrumental settings are described in 1M chloride salt solutions were prepared using CaCl−1 and 50–1800 cm−1 frequency ranges based on observed spectral signal intensities. The ICP-AES measured values of each mineral element for all INF aqueous samples (n = 83) were used as individual Y variable. For further investigations, PLSR models were also developed using calibration samples (n = 42) and validated using the rest samples (n = 41); quasi-random selection was used to split the whole sample group into calibration and validation sample sets. Venetian blind cross-validation was performed to evaluate the performance of PLSR models. Parameters such as root mean square error of calibration (RMSEC) and cross-validation (RMSECV), the coefficient of determination on calibration (R2C), cross-validation (R2CV) and prediction (R2P) were calculated. The bias of cross-validation and prediction was also determined. The satisfactory prediction results from a PLSR model were expected to have R2 values close to 1, RMSE values, and bias close to 0. Robust PLSR models were developed using a small number of latent variables (PLS loadings) [Raw Raman spectra of all the measurements were imported into Matlab 2015a . The mean spectrum of each sample was calculated. Baseline correction on raw data was carried out using asymmetric least squares correction (AsLs), adaptive iteratively reweighted penalized least squares (airPLS) , Savitzkples n = ; quasi-rples n =  and valioadings) . Chemometrics was also employed for the specification of mineral-related Raman spectral variables. Based on PLSR modeling, the Raman spectral frequencies of significant regression coefficient intensities for each mineral element prediction were used to compare with the pre-processed Raman spectral signals of the chloride salt solution  in order to verify their common locations of Raman spectral signals. −1) of aqueous INF samples (n = 83) are shown in −1 region is shown in −1 are assigned to the vibrational mode of the glycosidic bond of α or β lactose [−1 may also be related to δ (C-C-C) and τ (C-O) bonds [−1 are also relevant to δ (C-C-O) or δ (C-C-H) bonds of tryptophan [−1 has been assigned to δ (C-O-C), δ (C-O-H) and ν (C-O) bonds; 1065–1082 and 1121cm−1 has also been assigned to δ (C-O-H), ν (C-O) and ν (C-C) of aspartic and glutamic acid [−1 have been strongly related to the ring-breathing structure of phenylalanine reported in many previous studies [−1 are assigned to γ (CH2), τ (CH2), δ (CH2) and ν (C=O) bonds of aliphatic chains in lipids and amino acid residues, respectively [−1 are related to δ (N-H) and ν (C=N) of Amide II, and ν (C=O) of Amide I, respectively [−1, 2900–2927 cm−1, and around 3005 cm−1 may be attributed to symmetric ν (CH2), asymmetric ν (CH3), and the symmetric γ (CH2) vibrational mode of aliphatic chain and aromatic structures of lipids [−1 has been assigned to ν (N-H) of amino groups [Raw Raman spectra  bonds . 645, 76yptophan ,46; 950 mic acid . Raman b studies ,47,48. Pf lipids . The Ramo groups . n = 83) are summarized in Statistics of the ICP-AES results on the eight selected mineral elements determined from all the aqueous INF samples  based on the preparation and analysis procedures mentioned in the current work with the reference values of the certified product . For theVIP informative variable selection was demonstrated to be the most effective method to select the most relevant Raman spectral variables to enhance the robustness of PLSR models developed for each mineral element prediction. Therefore, results from other informative spectral variable selection algorithms  are not discussed in this paper.−1) and data pre-treated respectively using AsLs, air-PLS, BOC, and S.G. first derivatives baseline correction methods are shown in n = 83) for the prediction of Ca, Mg, Na, Cu, Fe, Zn. One sample with an unusual ICP-AES measured value of K was eliminated from the PLSR modeling for K prediction. Generally, four or six latent variables were required to attain: R2CV values of 0.95 for Ca, Mg and Fe prediction with RMSECV values of 1.44, 0.13 and 0.16 ppm, respectively; an R2CV value of 0.94 with an RMSECV value of 0.12 ppm for Zn prediction; R2CV values of 0.82, 0.88 and 0.92 with RMSECV values of 0.04, 1.28 and 0.66 ppm for Cu, K, and Na prediction  modes of lipids and >3300 cm−1, which is consistent with ν (N-H) and ν (O-H) modes of protein and lactose [Minerals and trace elements exist in cows’ milk as ions, salts, or bind with proteins, peptides, carbohydrates, fats, and small molecules to form complexes . In the peptides ,47. Minepeptides . Raman se and Zn . Other sproteins . Fe and proteins . For the lactose . −1 and 50–1800 cm−1 spectral regions. Models developed based on AsLs baseline-corrected Raman spectra performed best for mineral element prediction. Models developed using VIP selected spectral variables in the Raman frequency range (50–1800 cm−1) had similar prediction performance to those developed using spectral variables selected in 50–3398 cm−1. A summary of model performances is shown in 2Cs of 0.98, 0.98, 0.99 and 1; R2CVs of 0.93, 0.94, 0.97 and 0.94; R2Ps of 0.76, 0.77, 0.84 and 0.80 with RMSEPs of 3.05 ppm, 0.29 ppm, 0.30 ppm and 0.25 ppm for Ca, Mg, Fe and Zn prediction, respectively. While for K and Na prediction, R2CV values of 0.91 and 0.93 were achieved with RMSECVs of 1.15 ppm and 0.74 ppm and R2P values of 0.31 and 0.57 with RMSEPs of 3.45 ppm and 1.51 ppm, respectively. The R2P of Cu prediction is 0.08, confirming that the PLSR models failed to predict Cu content in the validation samples. As PLSR models were developed on collinearity of both Raman spectral data (X-variables) and ICP-AES results of each element for each sample (Y- variables), the prediction performances could also reflect the accuracy (R%) of ICP-AES measured values shown in w/w). RS with chemometrics did demonstrate high sensitivity for the determination of mineral elements. Theoretically, PLSR model prediction performances could be improved using even more accurate chemical reference values (Y- variable) in future studies.To further exploit the potential of RS for mineral content prediction, PLSR models were also developed using two separate calibration and validation sample sets over 50–3398 cm−1 from PLSR modeling for each mineral element are shown in −1 to ca. 1800–1900 cm−1. Patterns of the significant regression coefficient intensities for the prediction of each element are different. However, some significant regression intensities exist at the same Raman shift for different elements’ prediction, e.g., 1003 cm−1 assigned to phenylalanine for the prediction of K, Na, Mn, Cu, and Zn. It is assumed that these mineral elements bind to phenylalanine in infant formula samples. Nevertheless, previous publications have rarely mentioned the specific Raman shifts assigned to neutral or ionized minerals. Some previously published studies reported Raman spectral peaks of calcite (CaCO3) shown around 709, 1085, 1434, and 1758 cm−1 [The regression coefficient intensity of each wavelength was derived from statistics on the regression results of PLSR modeling. Information on regression coefficients from PLSR modeling is useful for the specification of Raman shifts that related to mineral content. Higher absolute regression coefficient intensities of the wavelengths indicate that these Raman wavelengths are more related to the anions or organic functional groups, which have been bonded with certain mineral ions in studied INF. The intensities of regression coefficients in the spectral range 50–3398 cm758 cm−1 ,31; whil2, CuCl2, FeCl3, KCl, MgCl2, MnCl2, and ZnCl2) were also collected to observe Raman spectral variations caused by mineral element content. The AsLs baseline-corrected Raman spectra of these chloride salt solutions are illustrated in −1, especially in the range of 50–465 cm−1. However, the Raman signal noise level was found to increase gradually in the range of 1800–3398 cm−1, and the spectral variance of different chloride salt solutions cannot be clearly observed in 1800–3398 cm−1. Thus, the Raman signal in this frequency range cannot be used to compare with the corresponding Raman shifts of high regression coefficient intensity in the same frequency range. Correspondingly, significant regression coefficient intensities of the minerals determined  during the wet mixing process of the INF manufacture. With more engineering innovations the fiber optic probe setting of the Raman spectrometer could allow online monitoring. However, to develop an established process analytical tool for online application, more studies are needed. Future research should investigate a more sophisticated experimental design and modeling using RS and chemometrics for accurate quantification of multiple mineral elements in INF.