Xenopus, mef2c and mef2d but not mef2a were recently shown to be expressed during cardiogenesis. We here investigated the function of Mef2c and Mef2d during Xenopus laevis cardiogenesis. Knocking down either gene by corresponding antisense morpholino oligonucleotides led to profound heart defects including morphological abnormalities, pericardial edema, and brachycardia. Marker gene expression analyses and rescue experiments revealed that (i) both genes are required for proper cardiac gene expression, (ii) Mef2d can compensate for the loss of Mef2c but not vice versa, and (iii) the γ domain of Mef2c is required for early cardiac development. Taken together, our data provide novel insights into the function of Mef2 during cardiogenesis, highlight evolutionary differences between species and might have an impact on attempts of direct reprogramming.The family of vertebrate Mef2 transcription factors is comprised of four members named Mef2a, Mef2b, Mef2c, and Mef2d. These transcription factors are regulators of the myogenic programs with crucial roles in development of skeletal, cardiac and smooth muscle cells. Mef2a and Mef2c are essential for cardiac development in mice. In  Isl1) and T-box factor 1 (Tbx1) cardiac progenitor cell (CPC) population.In the mouse, the heart arises from two populations of cells referred to as the first and second heart field, respectively Xenopus starts at the onset of gastrulation with the induction of two bilaterally located cardiac progenitor cell populations on either side of the Spemann Organizer Xenopus FHF already express genes indicating cardiac differentiation such as cardiac troponin (tnni3) and myosin heavy chain (myh6). Upon further development, these cells will later contribute to forming the single ventricle, the two atria and the inflow tract isl1 and tbx1 as well as bone morphogenetic protein 4 (bmp4). These cells have been shown to develop into the outflow tract Cardiac development in myocyte enhancer factor 2) proteins belong to the MADS  family of transcription factors. Members of the Mef2 family contain a common MADS-box and a Mef2-type domain at their N-terminus. The MADS-box serves as a minimal DNA-binding domain that requires the adjacent Mef2-type domain for dimerization and high-affinity DNA binding Mef2a, b, c and d- are expressed in precursors of the three muscle lineages as well as in neurons Mef2 gene gives rise to multiple isoforms through alternative splicing that are expressed in different embryonic and adult tissues Mef2 genes: the two mutually exclusive exons a1 and a2, and the short exons β and γ. The function of these isoforms depends on the exon composition after splicing. In particular the amino acids coded by the γ exon have been shown to function in gene repression Xenopus mef2c, three exons are alternatively spliced: two exons corresponding to the mammalian β and γ exons, as well as the so-called δ exon. No mef2d splice variants were detected in XenopusMef2  population at stage 20 whereas mef2d transcripts were strongly detected in CPCs  (mef2c is expressed in the myocardium (arrowhead) as well as in the endocardium (arrow) during tailbud stages , and Xenopus mef2d (xmef2d) were cloned into the pCS2+ expression vector in front of and in frame with the GFP gene. The RNAs of these constructs were bilaterally co-injected with either Control MO or Mef2c MO or Mef2d MO into two-cell stage embryos. GFP expression was monitored at stage 20  or due to introduced silent point mutations in the coding sequence (hMEF2D). Again, the efficiency was tested as described above. The expression of mMef2c-GFP or hMEF2D-GFP was not blocked by Mef2c MO. The Mef2d MO did not have an inhibitory effect on hMEF2D-GFP or mMef2c-GFP  Mef2c MO neither inhibits mMef2c nor hMEF2D translation and (III) the Mef2d MO does not interfere with mMef2c or hMEF2D expression. In conclusion, RNA coding for murine Mef2c and human MEF2D can be used as gene specific rescue constructs.For loss of function studies we relied on morpholino oligonucleotide (MO) based antisense strategies. To test the binding efficiency and functionality of the MOs used, the MO binding sites of stage 20 . Co-injeef2c-GFP . Taken tXenopus embryos to target cardiac tissue GFP RNA was co-injected as a lineage tracer and to identify correctly injected embryos. Mef2c and Mef2d morphant embryos revealed abnormalities during cardiogenesis whereas Control MO-injected siblings showed a normal cardiac development  suggesting that their regulation by Mef2 factors is also conserved in Xenopus. In the case of myh6, a recent study identified a conserved Mef2 binding site in its promoter region Nkx2-5nkx2-5 expression in the CPC population at stage 20, but we have seen a down-regulation at the stage 28. This might suggest that nkx2-5 is a target gene of Mef2 proteins only at later stages of development or this observation might point to diverse functions of Mef2 proteins as discussed in the next section.Transcriptional targets of Mef2 factors that have been identified in different species include alpha cardiac myosin heavy chain, alpha cardiac actin and cardiac troponin I Nkx2-5 during early phases of cardiogenesis whereas this construct inhibits cardiogenesis at later time points Xenopus cardiogenesis may also be useful as another model suitable to gain further insights into the function of Mef2d during cardiac remodeling in mice, particularly in the reactivation of a fetal gene expression program.The γ domain of mMef2c has been shown to function as a transcriptional repressor Of note, our experiments have additional implications as Mef2c was used as one of three components in direct reprogramming experiments to generate cardiomyocytes out of cardiac fibroblasts Xenopus laevis embryos were obtained by in vitro fertilization, cultured and staged according to Nieuwkoop and Faber mMef2c and hMEF2D constructs were purchased from ImaGenes GmbH and the open reading frames were subcloned into pCS2+ hMEF2D by mutagenesis using the QuickChange II Site-Directed Mutagenesis Kit (Stratagene). All constructs were verified by sequencing. The mMef2cγ- deletion construct was generated by inverse PCR using the mMef2cγ+/pCS2+ rescue construct as template and the proof reading Phusion DNA polymerase (Finnzyme) followed by re-ligation. The primers used for the inverse PCR were: for: 5′-GAC CGT ACC ACC ACC CCT TCG A-3′; rev: 5′-GCT GAG GCT TTG AGT AGA AGG CAG G-3′. For cloning full length mef2c from heart enriched explants the following primers were used: Mef2c cloning for: 5′-GTT GGA GCA GAG GGG AAA AT-3′, Mef2c cloning rev: 5′-GGT ATA AGC ACA CAC ACA CTG CA-3′.The 2O and stored as aliquots at −20°C. Mef2cMO: 5′-CCA TAG TCC CCG TTT TTC TGT CTT C-3′; Mef2dMO: ′-AAT CTG GAT CTT TTT TCT GCC CAT G-3′5. For knock down approaches, we injected the MOs (10 ng) into both dorso-vegetal blastomeres of eight-cell embryos to target the presumptive heart region GFP RNA. Only correctly injected embryos were considered for the experiments. For control injection experiments, the standard Control MO of Gene Tools was used. Control MO: 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′ For rescue experiments, mRNA (0.5 ng) was injected together with MO. The binding specificity of MOs was tested in vivo were cloned in frame with and in front of the GFP open reading frame in pCS2+. The indicated RNA and MO were co-injected bilaterally into two-cell stage embryos and GFP translation was monitored at stage 20 using a fluorescence microscope.All MOs were purchased from Gene Tools, LLC, OR USA, resuspended in DEPC-Hmef2c and mef2d were used as previously described GFP RNA. The uninjected side served as internal control. Wild type or injected embryos were fixed at +4°C in MEMFA  at indicated stages. WMISH experiments were performed according to a standard protocol as previously described 2O2. For sections, stained embryos were embedded in gelatine/albumine overnight at +4°C, sectioned using a vibratome with a thickness of 25 μm, coverslipped, and imaged with an Olympus BX60 microscope.Probes for Xenopus embryos at different stages with peqGOLD RNAPure (peqLab) according to the manufacturer's protocol. To analyze mef2c variants in Xenopus cardiac tissue, the anterior-ventral part of stage 24, 28, and 32 of wild type embryos (heart-enriched regions) posterior to cement gland were dissected and total RNA was isolated using peqGOLD RNAPure (peqLab). cDNA was synthesized using random hexamers and the SuperScript II reverse transcriptase (Invitrogen). PCR was performed with the Phire Hot Start II DNA Polymerase (Thermo scientific). Primers for amplification were: gapdh_for: 5′-GCC GTG TAT GTG GAA TCT-3′; gapdh rev: 5′-AAG TTG TCG TTG ATG ACC TTT GC-3′; H4 for: 5′-CGG GAT AAC ATT CAG GGT ATC ACT-3′; H4 rev: 5′-ATC CAT GGC GGT AAC TGT CTT CCT-3′; Xenopus mef2c for: 5′-AGA GCG CAC GGA CTA CTG AT-3′; Xenopus_mef2c_rev: 5′-TCA CCT GTC GGT TAC GTT CA-3′; Xenopus mef2d for: 5′-GCA GCT TTA AAT TCC GCA AG-3; Xenopus rev: 5′-CGG TGT CAC TTG GCC TTT AT-3′; Quantitative PCR was performed on RNA isolated from heart-enriched regions using SYBR Green Master Mix (Fermentas) and a Roche Light Cycler 1.5. gapdh was used as housekeeping gene. Each sample was analyzed in triplicate. Primer pairs used were: Xenopus gapdh for: 5′-GCC GTG TAT GTG GAA TCT-3′; Xenopus gapdh rev: 5′-AAG TTG TCG TTG ATG ACC TTT GC-3′; Xenopus mef2cγ+ for: 5′-GCACAATATGCCTCATTCAGCC-3′; Xenopus mef2cγ+ rev: 5′-GGAGGAGAAACAGGTTCTGACTTG-3′; Xenopus mef2cγ- for: 5′-TGGCTCAGTTACTGGCTGGCAGC-3′; Xenopus mef2cγ-rev: 5′-TAGTACGGTCTCCCAGCTGGCTGAG-3′; Xenopus mef2d for: 5′AGA CCT GGC ATC CCT CTC TA-3′; Xenopus mef2d rev: 5′TTG CGG TTG GTT ATG TTG TT-3′.Total RNA was isolated from whole 30 embryos injected with RNA were homogenized in 300 µl lysis buffer  and incubated on ice for 5 min. Protein samples were cleared at 13,000 rpm at 4°C for 5 min. For removal of lipids, the supernatant was mixed with 50 µl Freon followed by centrifugation at 13,000 rpm at 4°C for 5 min. The upper protein containing phase was collected. Concentration of protein samples was determined by Bradford assay with BSA as standard. Western blotting was performed according to standard procedures and proteins were visualized using a Li-COR ODYSSEY Imager. Primary antibodies were purchased from Abcam , Santa Cruz  and Serotec . Secondary antibodies used were IRDye conjugates from Li-COR.Mef2a, b, c and d in human, mouse and X. tropicalis were compared using NCBI and Xenbase G Browse.For synteny analysis, genomic structure and chromosomal organization of p value of ≤0.05 was considered to be significant.Data were obtained from at least three independent experiments and analyzed with statistical program GraphPad Prism. The number of embryos (N) and the number of independent experiments (n) performed for each experiment is indicated in the corresponding figures. For rescue experiments, embryos of the same batch were evaluated upon injection either with either MO or MO along with the RNA of interest. The nonparametric Mann-Whitney rank sum test was used to determine statistical differences. A Figure S1Spatio-temporal expression of mef2cand mef2d in Xenopus.A. Temporal expression of mef2c. mef2c is maternally supplied. Mef2c embryonic expression starts at stage 9 and increases until stage 40. gapdh was used as loading control. –RT serves as negative control. B. Temporal expression of mef2d. mef2d is maternally supplied. Mef2d embryonic expression starts at stage 12. H4 was used as loading control. –RT serves as negative control. C–j. Spatial expression of mef2c. C. Anterior view with the dorsal side to the top. c. Sagittal section. D, F, H, J. Lateral views with anterior to the right. E, G, I, K. Ventral views with anterior to the top. Black arrowheads indicate the expression in the FHF, the red arrowhead highlights mef2c transcripts at the lateral sides of the SHF. C. Parasagittal section. d, f, h, j. Transverse sections. Black arrowheads indicate cardiac expression; the arrowhead in h shows mef2c expression in the endocardium, the black arrow in the myocardium. L–s. Spatial expression of mef2d. L. Anterior view with the dorsal side to the top. l. Sagittal section. M, O, Q, S. Lateral views with anterior to the right. N, P, R, T. Ventral views with anterior to the top. m, o, q, s. Transverse sections. White arrows indicate mef2d expression in cardiac progenitor cells. The white arrowhead indicates cardiac cells with low mef2d expression. Black arrows indicate mef2d expression in the myocardium, black arrowheads show mef2c expression in the first heart field (FHF). St: stage(TIF)Click here for additional data file.Figure S2In vivo MO specificity test. Two-cell stage embryos were bilaterally injected and GFP fluorescence was monitored at stage 20. MO binding sites of Xenopus, mouse and human are indicated. Red letters indicate different bases in the MO binding sites, green letters indicate the ATG start codon. Upper panels show the light view, lower panels provide the fluorescent view. A. GFP fluorescence was observed upon injection of mef2c-GFP together with Control MO but not with Mef2c MO. Neither mMef2c-GFP nor hMEF2D-GFP were targeted by Mef2c MO. B. GFP expression was observed after the injection of Control MO. Co-injection of xmef2d-GFP and Mef2d MO led to an inhibition of GFP expression. Neither the expression of hMEF2D-GFP nor mMef2c-GFP was influenced by Mef2d MO.(TIF)Click here for additional data file.Table S1Gene abbreviations used in (PDF)Click here for additional data file.


Celecoxib (CXB) is a widely prescribed COX-2 inhibitor used clinically to treat pain and inflammation. Recently, COX-2 independent mechanisms have been described to be the targets of CXB. For instance, ion channels such as the voltage-gated sodium channel, L-type calcium channel, Kv2.1, Kv1.5, Kv4.3 and HERG potassium channel were all reported to be inhibited by CXB. Our recent study revealed that CXB is a potent activator of Kv7/M channels. M currents expressed in dorsal root ganglia play an important role in nociception. Our study was aimed at establishing the role of COX-2 independent M current activation in the analgesic action of CXB.We compared the effects of CXB and its two structural analogues, unmethylated CXB (UMC) and 2,5-dimethyl-CXB (DMC), on Kv7/M currents and pain behavior in animal models. UMC is a more potent inhibitor of COX-2 than CXB while DMC has no COX-2 inhibiting activity. We found that CXB, UMC and DMC concentration-dependently activated Kv7.2/7.3 channels expressed in HEK293 cells and the M-type current in dorsal root ganglia neurons, negatively shifted I–V curve of Kv7.2/7.3 channels, with a potency and efficiency inverse to their COX-2 inhibitory potential. Furthermore, CXB, UMC and DMC greatly reduced inflammatory pain behavior induced by bradykinin, mechanical pain behavior induced by stimulation with von Frey filaments and thermal pain behavior in the Hargreaves test. CXB and DMC also significantly attenuated hyperalgesia in chronic constriction injury neuropathic pain.+ channels with effects independent of COX-2 inhibition. The analgesic effects of CXBs on pain behaviors, especially those of DMC, suggest that activation of Kv7/M K+ channels may play an important role in the analgesic action of CXB. This study strengthens the notion that Kv7/M K+ channels are a potential target for pain treatment.CXB, DMC and UMC are openers of Kv7/M K In clinical practice, non-steroid anti-inflammatory drugs (NSAIDs) are the most frequently used pain relief drugs. It is believed that NSAIDs mainly relieve pain by suppressing the activity of cyclooxygenase (COX) COX-1 is a ubiquitous constitutive form of the enzyme that is involved in the regulation of various physiological processes such as platelet aggregation, and gastrointestinal tract and kidney homeostasis. COX-2 is an inducible isozyme mainly observed during pathological processes such as inflammation and cancer Recently, COX-2-independent mechanisms have been described to be the targets of CXB. For instance, several non-COX-2 components of the cell, such as sarcoplasmic/endoplasmic reticulum (ER) calcium ATPase (SERCA) Certain ion channels were also recently described as additional targets of CXB. For example, the voltage-gated sodium channel in rat retinal neurons + channels may also contribute to the analgesic action of CXB. It is well established that Kv7.2 and Kv7.3  constitute the molecular basis of the neuronal M-type potassium channel + currents 2+-activated Cl− currents in DRG neurons The strong modulation of Kv7.2/7.3 by CXB found in our previous work led us to think that activation of KIn light of the findings mentioned above, we propose that, in addition to reducing the generation of PGs by inhibiting COX-2, activation of M currents in DRG neurons may also be involved in the NSAID analgesic action of CXB. In the present study, we compared the effects of CXB and its two structural analogues, unmethylated CXB (UMC) and 2,5-dimethyl-CXB (DMC), on Kv7/M currents and pain behavior in animal models. As UMC is a more potent inhibitor of COX-2 than CXB and DMC has no COX-2-inhibiting activity, the role of COX-2-independent M current activation in the analgesic action of CXB can be assessed by comparing the effects of these three CXB analogues.UMC and DMC are structural analogues of CXB . With reThe Kv7.2/7.3 currents were recorded using the protocol shown in the right panel of 50 for the Kv7.2/7.3 current activation were 22.7 ± 3.0 µM, 4.5 ± 0.7 µM, and 2.5 ± 0.2 µM for UMC, CXB and DMC, respectively. Thus, the order of both efficacy and potency of these CXBs were UMC < CXB < DMC, which is opposite to the order of their COX-2 inhibition activity (UMC > CXB > DMC). These results strongly suggest that the modulation of Kv7.2/7.3 currents by CXB analogues does not depend on their COX-inhibitory activity.Next, we examined the concentration-response relationships for the UMC, CXB and DMC activation of Kv7.2/7.3 channels. 1/2 from the fitting of Boltzmann function (see Methods) showed that CXB, DMC and UMC shifted voltage dependent activation of Kv7.2/7.3 to more negative potentials to different degrees  neurons + currents in DRG neurons are an important modulator of nociception. In this study, we explored the effects of UMC, CXB and DMC on M-type K+ currents from rat DRG neurons.Our previous work showed that CXB and DMC not only activated Kv7.2/7.3 currents expressed in cell lines but also activated the native M-type K+ currents were recorded from small-diameter DRG neurons by amphotericin B perforated patch clamp using the voltage protocol depicted in + currents from DRG neurons  following the hind paw injection of 50 µl of saline containing the relevant compounds. Intraplantar injection of BK (200 µM) into the hind paw produced strong nocifensive behavior  were injected into the plantar of the rat hind paw and the response to the mechanical stimuli was measured 8 min later. As demonstrated in We used the Hargreaves test The neuropathic pain model of chronic constriction injury (CCI) to the sciatic nerve was used in this part of the study. Nocifensive response changes to mechanical or thermal stimuli after surgery were monitored using von Frey filaments or the Hargreaves test as discussed above. As shown in On the first day after surgery, the mechanical pain threshold and thermal pain threshold were significantly decreased. From the first day after the surgery, the rats were divided into four groups with each group receiving intragastric administration of either CXB, DMC or RTG  or solvent in a volume of 1 ml, twice a day (for details see Methods).th day after surgery, the mechanical pain threshold in the solvent group was further reduced to 4.3 ± 0.3 g, and all three drug treatments significantly increased the threshold compared with the solvent control . On the other hand, drug treatment did not affect the reduced thermal pain thresholds .On the 5th day after surgery, the mechanical pain thresholds in the drug groups were significantly increased compared with the solvent group . For the thermal pain threshold, only CXB treatment significantly increased the threshold .On the 10th day after surgery, only CXB treatment significantly increased mechanical threthold compared with the solvent treatment . For the thermal pain threshold, none of the treatments significantly affected the thermal latencies: control, 16.1 ± 0.9 s; CXB, 19.8 ± 1.3 s; DMC, 19.8 ± 1.2 s; RTG, 19.1 ± 0.7 s).On the 14+ conductance plays a role in the analgesic action of CXB. We found that all three CXB analogues concentration-dependently activate Kv7/M channels, with both potency and efficacy of the stimulatory effects inversely related to their COX-2 inhibitory activity: DMC showed the greatest effect while UMC showed the weakest effect in activating Kv7/M channels. Furthermore, CXB analogues showed similar order of potency on negatively shifting I–V curve of Kv7 channel. These results support our previous study showing that CXB modulates Kv7/M channel in a COX-2 independent manner + currents from DRG neurons were also observed, except that higher drug concentrations were required in DRG neurons.Growing evidence suggests that functional Kv7/M channels are expressed in peripheral sensory neurons and fibers and that their activity strongly contributes to fiber excitability + currents contribute to the analgesic actions of CXB. Thus, if all three CXB analogues with different potencies of COX-2 inhibition relieve pain similarly, it would strongly suggest that K+ channel activation is a target for the analgesic action of CXB. In this regard, DMC is particularly valuable, given that DMC, like CXB, activates Kv7/M channels but lacks COX-2 inhibitory activity. We found that CXB, UMC and DMC could attenuate inflammatory pain induced by BK. CXB and UMC, both inhibitors of COX-2, seemed more effective than DMC , DMC (100 mM) and RTG (100 mM) were dissolved in DMSO and stored at −20°C. The other chemicals were all purchased from Sigma .The use of animals in this studied was approved by the Animal Care and Ethical Committee of Hebei Medical University  under the International Association for the Study of Pain (IASP) guidelines for animal use. All surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering.2). The cells were seeded on glass coverslips in a 24-multiwell plate and transfected when 60–70% confluence was reached. For transfection of six wells of cells, a mixture of 3 µg Kv7.2 and Kv7.3 in pcDNA3 (1.5 µg for each), pEGFP-N1 cDNAs and 3 µl Lipofectamine 2000 reagent  were prepared in 1.2 ml DMEM and incubated for 20 min according to the manufacturer’s instructions. The mixture was then applied to the cell culture wells and incubated for 4–6 h. Recordings were made 24 h after transfection and the cells were used within 48 h.The HEK293 cell line was purchased from American Type Culture Collection . HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics in a humidified incubator at 37°C : 160 NaCl, 2.5 KCl, 5 CaCl2, 1 MgCl2, 10 HEPES, 8 glucose, pH 7.4. A low-profile perfusion chamber fed by gravity perfusion system was used for solution exchange .Patch electrodes were pulled from borosilicate glass and fire-polished to a final resistance of 1–2 MΩ when filled with internal solution. An axon 700B  patch clamp amplifier was used for voltage clamp experiments. All recordings were performed using the amphotericin B  perforated patch technique. The internal pipette solution contained (in mM): 150 KCl, 5 MgClMale Sprague Dawley rats (180–220 g) were randomly grouped and allowed to acclimatize for at least 20 min to the environment prior to the experiment. All experimenters were blinded to the treatment allocation and were only unblinded once the study had finished.The right hind paw of the animal received an intraplantar injection (50 µl) of BK  and the nocifensive response  were recorded using a video camera for 30 min. To study the effects of drugs on BK-induced nociceptive behavior, animals were pre-injected with CXB analogues or RTG. After 5 min, BK and the drug were co-injected into the same site of the hind paw. Control animals were injected with solvent  instead of the tested drugs. All drugs were diluted in saline from stock solution and applied at a volume of 50 µl at a concentration of 100 µM.Mechanical withdrawal thresholds were measured using calibrated von Frey filaments (a set of monofilaments made from nylon filaments of varying diameter)  applied to the plantar surface of the paw. Testing was initiated with an Evaluator Size 5.07 (10 g). If the animal withdrew the paw, the next weaker hair was applied. In the case of no withdrawal, the next stronger hair was applied. The cut-off was Evaluator Size 6.10 (100 g).To test for thermal hyperalgesia, radiant heat was applied to the plantar surface of a hind paw from underneath a glass floor using a ray of light from a high-intensity lamp bulb. The paw withdrawal latency was recorded automatically when the paw was withdrawn from the light .CCI was used as a model of neuropathic pain. Animals were randomly divided into 4 groups that received either CXB, DMC, RTG or solvent treatment. After one day of environment acclimatization, basic mechanical and thermal withdrawals were assessed. The surgeries were performed one day later. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (10–20 mg/kg). The left hind leg was shaved and cleaned using 70% ethanol. The sciatic nerve was exposed by blunt preparation of connective tissue at the mid-thigh level, proximal to the sciatic trifurcation. Four non-absorbable sterile surgical sutures (0.1 mm) were loosely tied around the sciatic nerve, 1–1.5 mm apart. The skin was sutured and the animal was transferred to a recovery cage. CCI rats received the vehicle (0.5% sodium carboxymethyl cellulose) or CXB, DMC or RTG treatment (30 mg/kg/day) by intragastric administration twice a day in a volume of 1 ml from 1 day to 14 days after the surgery. Mechanical and thermal withdrawals were tested at 1, 5, 10 and 14 days after surgery using the methods described above.2+(A1−A2)/(1+(x/x0)p), where x is the drug concentration, and p is the Hill coefficient. The current activation curves were generated by plotting the normalized tail current amplitudes against the step potentials and were fitted with a Boltzmann equation: y = A/{1+exp[(Vh−Vm)/k]}, where A is the amplitude of relationship, hV is the voltage for half-maximal activation, Vm is the test potential, and k is the slope factor of the curve. All data are reported as the mean ± standard error of the mean (SEM). Differences between groups were assessed by a Student’s t-test or one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test. The differences were considered significant if P ≤0.05.The concentration-response curve was fitted by logistic equation: y = A


These nanoscale features have the ability to impact cell adhesion, migration, proliferation, and lineage commitment. Significant advances have been made in deciphering how these nanoscale cues interact with stem cells to determine phenotype, but much is still unknown as to how the interplay between physical and chemical signals regulate in vitro and in vivo cellular fate. This review dives deeper to investigate nanoscale platforms for engineering tissue, as well use the use of these nanotechnologies to drive pluripotent stem cell lineage determination.Tissue engineering utilizes cells, signaling molecules, and scaffolds towards creating functional tissue to repair damaged organs. Pluripotent stem cells (PSCs) are a promising cell source due to their ability to self-renewal indefinitely and their potential to differentiate into almost any cell type. Great strides have been taken to parse the physiological mechanisms by which PSCs respond to their microenvironment and commit to a specific lineage. The combination of physical cues and chemical factors is thought to have the most profound influence on stem cell behavior, therefore a major focus of tissue engineering strategies is scaffold design to incorporate these signals. One overlooked component of the  In recent years, tissue engineering has emerged as a potential method for treating numerous diseases and regenerating damaged cells. By applying engineering approaches to knowledge of biological systems, a tissue engineered substitute can be generated to restore, replace, or maintain partial if not entire organ function . The keyTowards this effort of fully directing cell behavior, various biomaterials as well as different cell types and signaling molecules have been investigated. Recent studies have examined the ability of mechanical signals to influence stem cell lineage commitment since cells in the body reside in different tissue niches with variable mechanical properties that effect cellular function. From this information, biomaterials have been designed to harness tissue-specific mechanical properties to guide stem cells to the targeted cell type. Specifically, nanoscale platforms are utilized since they offer a unique ability to mimic the physical cues that cells receive from their microenvironment. Researchers have developed materials with tunable matrix elasticities, nanotopographies, and nanoscale patterns that have the ability to manipulate cell phenotype. This review explores current developments in the use of nanotechnology to drive cell function determination. The following section briefly describes common cell types used in tissue engineering applications, then discusses the interactions between cells and different biomaterials with nanoscale features.Major sources of cells for tissue engineering include adult stem cells, progenitor cells, embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs). Adult stem cells, or mesenchymal stem cells (hMSCs), are multipotent and generally derived from adipose tissue (AD-hMSCs) or the bone marrow (BM-hMSCs). When exposed to the correct chemical signals, AD-hMSCs can differentiate towards osteogenic, chondrogenic, adipogenic, myogenic, and hepatic lineages, as well as become endothelial cells -5. Bone Progenitor cells are lineage-committed and maintain the tissue in which they reside . These pin vivo germ layer establishment [in vivo and in vitro tissue development from hESCs can be overcome by engineering a three-dimensional (3D) microenvironment from which the undifferentiated cells can receive cues and thus differentiate towards specific lineages.hESCs and iPSCs overcome limitations of hMSCs and progenitor cells by offering the ability to self-renew indefinitely as well as differentiate into any cell type of the body . Embryonlishment . While tiPSCs were first created by introducing four pluripotency transcription factors  to a mouse fibroblast cell, after which the fibroblast exhibited properties of undifferentiated hESCs . These sOne barrier for using hESCs and iPSCs in regenerative medicine is that teratoma formation in implanted tissue can occur when cells have not fully and uniformly differentiated into the target tissue ,28. TherAlthough hESCs and iPSCs are promising cell sources for tissue engineering applications and invaluable tools for studying developmental biology, there are still many fundamental aspects of PSC biology that are unknown. Specifically, researchers are striving to understand and deconstruct the mechanisms by which the microenvironment effects lineage determination, as well as cell phenotype and function.The native microenvironment is composed of the extracellular matrix (ECM), which is a network of proteins that provides physical and chemical cues determining cell behavior -32. Cellin vivo tissue structure provides cues to cells at a nanoscale. Furthermore, cells tend to respond to microscale fiber scaffolds the same way that they do when cultured on a 2D polystyrene cell culture plate. Cell morphology becomes fat, which causes a lopsided attachment of focal adhesions [Past biomaterial design has focused on microscale technologies to drive stem cell lineage commitment, but the dhesions . Therefodhesions , and thedhesions . The folin vivo microenvironment is composed of channels, pores, and ridges that provide physical cues to cells at a nano level [The no level . Knowledno level -43. ThesThis technique can form a network of polymer fibers down to the size of 10 nm . To genein vivo.The general method of soft lithography uses elastomeric stamps to print nanoscale polymers on a surface -54. Pattin vivo microenvironment. Similar diffusive transport also occurs in hydrogel cell culture platforms, and hydrogels can be designed to incorporate cell adhesion ligands and other biologically relevant components [Hydrogels are a popular tissue engineering scaffold with proven success in medicine and biological research due to their tunable tissue-like properties -65. The mponents . Althougmponents ,67. Furtin vitro 3D cell microenvironment to add roughness [m-aminobenzene sulfonic acid) and polyethylene glycol in which neurons exposed to more positively charged groups exhibited greater neurite length and had additional growth cones [Carbon nanotubes (CNTs) possess ideal qualities for tissue regeneration strategies such as tunable chemical and mechanical properties, electrical conductivity, cytocompatibility, and nanoscale dimensions that serve as topographical cues . Furtheroughness -71. Whenoughness . Severaloughness -76, myoboughness  as well oughness , but litoughness . A studyoughness . CNTs cath cones . Since C-18 liters [in vitro microenvironment is optimized. Microfluidic platforms have been used extensively to study cell biology, specifically cellular adhesion forces [This technique allows for precise regulation of fluid flow and microenvironmental geometry, usually in the form of channels with similar dimensions to that of the cell type under investigation. Volumes can easily be controlled to levels of 108 liters , and then forces , the cytn forces , and then forces -85. ThisAs described in the previous section, several methods for tuning biophysical signals for influencing stem cell behavior have been explored. Nanoscale signals such as shear stress, strain, material elasticity, topographical variation, and cell shape all affect cellular function and lineage specification. This section discusses how each of these factors govern PSC behavior; specifically cell adhesion, proliferation, alignment, and differentiation.in vivo microenvironment. Muscle contraction and relaxation, bone compression and decompression, cell migration, fluid flow, and tissue regeneration all cause variations of mechanical forces in the body. The ECM also has a range of elastic moduli that generate physical stimuli for attached cells through focal adhesions. These mechanical cues are transduced through focal adhesion kinase (FAK) and Src family signaling [in vitro with hopes of determining what physical forces, separately or in combination, control lineage commitment.Cells experience mechanical cues such as stress and strain in the ignaling . Furtherignaling ,86,87. AThe effect of mechanical cues on cell function has been studied for many cell types, and has established general knowledge regarding cell behavioral responses ,88-91. Rin vivo, most commonly exerted on cells in the circulatory system [Shear flow has also been investigated as a mechanical cue for PSCs since it is a dynamic stress found y system . Mouse Ey system . This roy system ,98. Furty system .A recent study demonstrated that matrix elasticities of 1 kPA, 8 kPa, and 25 kPa lead hMSCs respectively towards neurogenic, myogenic, and osteogenic lineage . This diESCs are generally cultured on stiff 2D cell culture plates. Studies have shown that cell traction and colony stiffness increase when ESCs are grown on traditional rigid substrates, which also correlates with the downregulation of Oct3/4 in mouse ESCs ,103. CelStem cell shape regulates physiology, controls proliferation, and ultimately governs lineage specification , 105. Ce2 and 75 μm2 fibronectin islands to show that size directly controls apoptosis and proliferation, respectively [One of the first experiments demonstrating the impact of cell size on behavior used 20 μmectively . Furtherectively . Patternectively . These fin vivo; for example proteins in the ECM are generally arranged in a fibrous manner with these topographical properties. These fibrillar networks are approximately 10-100 nanometers but can be several microns [Topography plays a key role in cell maintenance and function. Nanoscale architecture has grooves, ridges, pits, and pores  microns ,113, and microns . Nanotop microns . Surface microns . When ce microns . Trough  microns ,115-122. microns . This, aTopography is a powerful tool since, not only is cytoskeleton tension altered like in cell shape experiments, but also entire molecular arrangement and dynamic organization of cellular adhesion mechanisms are affected . PolymetAnother study used UV-assisted capillary force lithography to create 350 nm ridge/groove pattern arrays, then demonstrated the ability of the surface topography to direct hESCs towards neuronal lineage in the absence of differentiation-inducing soluble factors . FurtherMWNT films were employed to investigate the response of hESCs to surface roughness. hESC colonies favored rougher surfaces for attachment, exhibited fattened morphology with standard colony size, and retained pluripotency when cultured on MWNT films . A similIn another study, surface nanoroughness of silica-based glass wafters was altered and hESCs were placed on the various substrates in single cells. hESCs on the control glass surface demonstrated highly branched morphology with many cytoplasmic extensions, while cells on the nanorough glass were compact with few, short flapodia. Cells on a rough surface patterned with square-shaped smooth islands favored attachment to the smooth glass instead of the nanorough areas and expressed pluripotent marker Oct3/4, and hESCs placed on an exclusively rough surface spontaneously differentiated. Proliferation of hESC colonies was determined by placing cells on smooth glass and nanorough substrates, and it was determined that doubling time of hESCs on the control surface was 41 hours compared to a slower 71 hour doubling time of colonies on the rough surface . In oppoin vivo microenvironmental cues in differentiation studies to parse which factors control lineage commitment. Trough carefully designed scaffolds and substrates, scientists have advanced the understanding of how nano-microenvironmental cues define cell behavior. By gaining this knowledge, stem cell differentiation can be further specified by combining nano-architecture and insoluble factors with other important biochemical cues.Tissue engineering has demonstrated the ability to generate desired cell types by combining the knowledge of biomaterials, stem cells, and signaling factors. The use of hPSCs in regenerative medicine has immense potential for treating numerous ailments, but experimental methods for solely and completely creating the desired tissues is necessary to avoid teratoma formation. Towards this goal, researchers have focused on mimicking


In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation, these bacteria develop mutual metabolic dependence with the host legume. In most legumes, the rhizobia infect post-mitotic cells that have lost their ability to divide, although in some nodules cells do maintain their mitotic capacity after infection. Here, we review what is currently known about legume symbiosomes from an evolutionary and developmental perspective, and in the context of the different interactions between diazotroph bacteria and eukaryotes. As a result, it can be concluded that the symbiosome possesses organelle-like characteristics due to its metabolic behavior, the composite origin and differentiation of its membrane, the retargeting of host cell proteins, the control of microsymbiont proliferation and differentiation by the host legume, and the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells. Different degrees of symbiosome evolution can be defined, specifically in relation to rhizobial infection and to the different types of nodule. Thus, our current understanding of the symbiosome suggests that it might be considered a nitrogen-fixing link in organelle evolution and that the distinct types of legume symbiosomes could represent different evolutionary stages toward the generation of a nitrogen-fixing organelle. Symbiosis between different organisms has played a key role in evolution and in fact, the term “symbiogenesis” is an evolutionary concept that refers to “the appearance of new physiologies, tissues, organs, and even new species as a direct consequence of symbiosis”  form. IT grows inwardly until it reaches the nodule primordium cells. The intracellular mode of infection occurs in most of the rhizobia-legume symbioses studied and it is tightly controlled by the host. Intercellular infection may take place via natural wounds, where lateral roots emerge through epidermal breaks (crack infection), or it may occur directly between epidermal cells or between an epidermal cell and an adjacent root hair  and humans (13). Moreover, ancestral bacterial protein transport routes coexist with the evolving mitochondrial protein import machinery in R. americana. Accordingly, Reclinomonas mitochondria may represent a “connecting link” between the metazoan mitochondria and their ancestral bacterial progenitors ; and the third, possibly induced by the action of pluricellular plant-based ecosystems   in their cytoplasm. As in the case of rhizobia-legume symbioses, the host and microsymbiont are strictly separated by a host-derived membrane in these species , the extra- or intracellular location of the microsymbiont, the presence or absence of segregation to daughter cells and of vertical transmission  and the fern Azolla, the diazotroph microsymbiont resides extracellularly in a mucilaginous sheath in the dorsal cavities of Azolla leaves. The cyanobacteria's filaments enter into the fern's sexual megaspore, allowing the microsymbiont to be transferred vertically to the next plant generation. While it retains its photosynthetic capacity, it seems that these diazotroph cyanobacteria have lost their capacity to survive as free-living organisms  or intercellularly, depending on the host plant species. Frankia induces the formation of multi-lobed, indeterminate nodules, which are modified adventitious secondary roots formed from the root pericycle. Nodule infected cells become full of branching Frankia hyphae surrounded by a perimicrobial membrane of host origin, forming vesicles in which nitrogen fixation takes place  but rather, it arises from unique zones of cell division in the root cortex  is the only non-legume plant that can establish effective nodule symbiosis with rhizobia. This symbiosis is a case of convergent evolution and it occurred more recently than that of legumes. From a phylogenetic and taxonomic point of view, Parasponia is closer to some actinorhizal plants that belong to the Rhamnaceae, Elaeagnaceae, and Rosaceae families, than to legumes  does not involve root hairs but rather, crack entry or root erosion and an intercellular IT. This IT protrudes into the host plant cell by plant membrane invagination, forming the so-called fixation thread. Fixation-thread, that remains in contact with the plasma membrane, are the equivalent to a symbiosome in legumes and to arbuscules in arbuscular mycorrhizal (AM) roots . Recently, Neorhizobium and Pararhizobium have been proposed as new genera . Similarly, the most recent genus Ensifer diverged about 200 Mya  genes I and II allowed the time of divergence among the α-proteobacteria genera of rhizobia to be estimated . Rhizobia are among the α-proteobacteria with the largest genomes , and in fact, lateral gene transfer is the primary source of genetic diversity in rhizobia , and the largest of all rhizobia  can perform photosynthesis and fix nitrogen in symbiosis or in free-living conditions  are included in the Rosid I clade  began their spread about 39 Mya and thus, it is the genistoid and dalbergioid that have the oldest origin within the papilionoids  membrane and the peribacteroid space  and an intrinsic tonoplast protein of the Nod26 group  and a nucleotide translocator , as well as mitochondrial processing peptidases, probably located in the symbiosome lumen  that is expressed in a nodule-specific manner in Lotus japonicus, is essential for nodule symbiosis  and alfalfa (E. meliloti), and in the determinate nodules of soybean (Bradyrhizobium japonicum), display metabolic dependence on the host for branched-chain amino acids , a legume family of nodule-specific cysteine-rich (NCR) peptides are targeted to the endosymbiotic bacteria. These peptides are responsible for the bacteroid differentiation that involves the induction of endopolyploidy, cell cycle arrest, terminal differentiation, and a loss of bacterial viability. It was recently demonstrated that a nodule specific thyoredoxin (Trx s1) is targeted to the bacteroid, controlling NCR activity and bacteroid terminal differentiation , all of which form indeterminate nodules  carry polyploid and enlarged bacteroids, and that these plants also express NCR peptides. However, these peptides are not homologous to NCR peptides from IRLC legumes, suggesting an independent evolutionary origin  or non-swollen  bacteroids, suggesting that the effects on bacteroid differentiation might have changed during the evolution of the Lupinus genus .Interestingly, species within the genus Lupinus spp. and Genista tinctoria (genistoid legumes), and also in certain dalbergioid legumes . All these legumes are infected by Bradyrhizobium spp. through epidermal infection or crack infection, and the infected zone of their nodules has no uninfected cells  also contain dividing infected cells (Vega-Hernández et al., Infected nodule cells are usually post-mitotic and do not divide further. However, one of the most interesting and quite unusual traits for eukaryotic cells is found in certain legume nodules whose host cells can divide after being infected by rhizobia Figures , 3. The L. albus (Fedorova et al., L. albus nodules, symbiosomes are segregated equally between the two daughter cells when the host plant cell divides, just like other cell organelles, e.g., mitochondria (González-Sama et al., Mitochondria and plastids divide in the plant cytoplasm, and cytoskeletal elements not only secure their distribution and movement but also, their correct partitioning between the daughter cells at cytokinesis King, . SymbiosIt has been established that a key event in the evolution from a free-living bacteria to an organelle is the loss of bacterial genes and their transfer to the nucleus of the plant host, a fate that occurred during mitochondrial and chloroplast evolution (Douglas and Raven, In the case of nitrogen-fixing rhizobia, the absence of gene transfer to the nucleus may be due to the low oxygen concentrations required by the nitrogenase enzyme, which would generate poor ROS production and mutation rates (Allen and Raven, No gene loss or genome reduction has been observed in viable symbiotic rhizobia. Symbiotic rhizobia that do not undergo terminal differentiation are still capable of existing as free-living bacteria. Accordingly, they must be equipped with a number of genes to survive in different environments and to compete with other microorganisms. Moreover, in nodules containing swollen terminally differentiated bacteroids, some non-differentiated bacteria inhabit the apoplastic space and consequently, all the genes necessary for independent life are still retained (Stêpkowski and Legocki, An evolutionary pathway has been proposed in symbiotic systems to shift from free-living organisms to facultative symbiosis and to ecologically obligatory symbiosis, usually involving genome expansion (Provorov et al., While it has been postulated that organelle development cannot occur in differentiated multicellular organisms (McKay and Navarro-González, TC, EF, JP, and ML wrote the manuscript. All the authors read and approved the final version of 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. The reviewer OS and handling Editor declared their shared affiliation.


The relatively simple molecular structure of hydrogen-bonded (HB) systems is often belied by their exceptionally complex thermodynamic and microscopic behaviour. For this reason, after a thorough experimental, computational and theoretical scrutiny, the dynamics of molecules in HB systems still eludes a comprehensive understanding. Aiming at shedding some insight into this topic, we jointly used neutron Brillouin scattering and molecular dynamics simulations to probe the dynamics of a prototypical hydrogen-bonded alcohol, liquid methanol. The comparison with the most thoroughly investigated HB system, liquid water, pinpoints common behaviours of their THz microscopic dynamics, thereby providing additional information on the role of HB dynamics in these two systems. This study demonstrates that the dynamic behaviour of methanol is much richer than what so far known, and prompts us to establish striking analogies with the features of liquid and supercooled water. In particular, based on the strong differences between the structural properties of the two systems, our results suggest that the assignment of some dynamical properties to the tetrahedral character of water structure should be questioned. We finally highlight the similarities between the characteristic decay times of the time correlation function, as obtained from our data and the mean lifetime of hydrogen bond known in literature. It is not common to find in Nature phenomena more intriguing than those related to the hydrogen bond interaction. This interaction dictates indeed not only the overall behaviour in organic molecules as DNA or proteins, but also that of systems as different as aqueous solutions and alcohols, which are undoubtedly the two classes of liquids having the larger impact on Life and daily Human activity and development, with innumerable applications in today’s life. The accurate knowledge of their properties is crucial to establish a firmer ground for the development of more refined liquid state theories, but it also has much broader societal implications. The full understanding of their microscopic behaviour is, however, far from being achieved even for the two most studied members of these families, namely water and methanol. This is mainly due to the presence of the hydrogen bond that induces strongly directional intermolecular interactions, absent in other non associated liquids. Comparing water to methanol at mesoscopic scales, as done in this paper, is rather natural and common. Methanol is indeed the alcohol having most analogies with water, their difference simply consisting in the replacement of a proton by a methyl group, that bestow on alcohols their amphiphilic character and different solvent properties. The continuous hydrogen bond (HB) breaking and formation is supposed on one hand to determine the overall behaviour of both liquids, and on the other to be at the root of their different structural, dynamical, and solvating properties. Liquid water, a system intensively studied in the search for a rationale for its unique properties and many unsolved anomalies235τHB, ranging from 1–2 psτHB ~ 1 ps, have been estimated for RT water by incoherent neutron scattering experimentsHowever, the accepted and sharp structural distinction between these two liquids appears to fade according to the findings of recent experiments on water791081213The investigation of the wavevector dependence of the THz excitations in liquid water, and their possible dispersive nature, can be readily performed by inelastic neutron (INS)222324252628Q primarily with longitudinal movements only, therefore not allowing us to directly examine the transverse character of a given mode. Consequently, any experimental signature of a shear wave propagation reflects a longitudinal-transverse (L-T) mixing2727293536Unfortunately, THz spectroscopic methods couple at low S may then be computed and analysed in a much broader exchanged wavevector Q and energy E = ħω range, where ħ and ω represent the reduced Planck constant and the angular frequency, respectively. Within this approach, the neutron data are thus essential to benchmark, a posteriori, the ability of simulation, which in principle deals with a model system, to provide a realistic description of the dynamics in the actual system under study through the use of a reliable interaction potential.In order to perform a reliable characterization of the dynamical response of the sample, the synergy between numerical and experimental techniques is essential. Once the simulation results for the spectrum of density fluctuations are validated by the experimental ones, the dynamic structure factor ω(Q) has often been performed using the neutron Brillouin scattering technique. This technique has to satisfy two main requirements. (i) Low scattering angles: because of long range disorder, collective phenomena show up indeed in these systems only at very low Q, in the so called pseudo first Brillouin zone which extends from the position of the main peak of the static structure factor down to the lowest possible Q, ideally to Q = 0. (ii) High energy incident neutron beam: since the collective excitations typically travel at velocities of the order of few thousands of m/s in liquids, the use of high energy (~10–100 meV) thermal neutrons is mandatory. Satisfying both conditions is extremely demanding at experimental level, because it implies the detection of high energy neutrons scattered from the sample at low angles: the contamination of the direct beam is unavoidable, and tight collimations must be used at the price of a strong flux decrease.The study of the collective dynamics in amorphous systems and the determination of the dispersion law BRIllouin SPectrometer BRISPT = 298 K. Measured spectra were reduced, with standard procedures, to the intensity, Iexp scattered by the sample, which is a quantity that contains, in addition to the sought-for single-scattering contribution, also a non negligible component due to multiple scattering (MS).The neutron Q, E) domain than in the experimental case and quantities that cannot be measured directly, such as: (i) the center-of-mass (CM) dynamic structure factor and (ii) the longitudinal and transverse currentsQħ.The MD simulations allow us to access a broader  once the MS and instrumental resolution are taken into account, and emphasizes the overall accuracy of both measurements and calculations. More details on the experimental and calculated quantities can be found in the Sections Methods and Supplementary Information.Q, E) range, the more so if one realises that measurements with different neutron wavelengths and resolutions amount to independent determinations. This provides a solid ground to model the spectral response of the sample by the CM dynamic structure factor SCM determined from the simulations. Complying with standard notation for the spectral variable, we now switch from the energy E to the angular frequency ω.The examples shown in SCM is taken as the correct quantity representing the collective translational dynamics, which, however, is not unaffected by the molecular asymmetry and the anisotropy of the interaction, also due to the HB. The computed SCM are plotted in Q values. It is worth noticing that the highest-frequency mode, visible in the simulated spectra, was out of the experimental energy window and could not be covered by measured INS spectra.As in previous works on molecular fluids4445SCM reads:Consistently with the long-debated case of water which, in recent experimental studies25Methods section and, for more detail, to refs ω = 0 describing the quasi-elastic response arising from the combined effects of thermal and viscous relaxations, while the frequency of the two other lines is related to the frequency ωVE of the propagating sound wave. As to the DHO components, each of them contributes to SCM through a further pair of Brillouin lines located at ωD1,2.For the explicit definitions and detailed properties of the two models used in SCM are shown in −1Q range, while a model with only a DHO function was preferred at the lower or larger Qs. Best fit values of ω(Q) reported in ωs(Q), spans the intermediate energy values and exhibits the typical trend expected for a sound mode. The excitation propagates with an apparent propagation velocity ~2750 m/s, much larger than the adiabatic sound velocity (cs = 1100 m/s), followed by the overdamping and bending down to a vanishing frequency around the position of the first maximum of the static structure factor, Qp ≈ 17 nm−1. Conversely, both DHO components of SCM display a softer mode not clearly dispersive, which remains underdamped in the whole Q range.Examples of best fits of JL and JT, respectively. The study of JL = (ω/Q)2SCM represents an alternative to the inspection of SCM, since it is a positive variable vanishing both at infinite and zero frequencies and, unless null everywhere, it must reach at least a maximum in betweenQ limit, the position of the maximum tends towards the frequency of the dominant acoustic mode and it is customarily assumed to provide a reasonable estimate of this even at finite Q values. Conversely, although the JT cannot be rigorously approximated by any known analytic function, the general features of its shape provide a meaningful characterisation of the transverse nature of the dynamics, and the frequency of the corresponding modes.Prior to a deeper discussion of these results, it is extremely useful to inspect the MD outputs, displayed in Q; by increasing Q, we observe a gradual merging of the two spectra that become coincident at Q ≥ 15 nm−1, except for the intrinsic difference in the ω → 0 limit. The evidence of such progressive merging of the two polarisation states has been, to the best of our knowledge, never shown before, and it can be presumably seen as the effect on the currents of the transition from the continuum to the single particle regime: at low Q, when the system is probed over mesoscopic (few tens of Å) distances including many neighbours, the two polarisation states fully probe the anisotropic nature of the dynamics. Upon increasing Q and approaching, although still far by reaching, the single particle limit, this asymmetry gradually disappears together with the signs of any distinction between longitudinal and transverse dynamics.As evident from SCM, both currents in JT clearly shows at all the Q investigated the presence of bumps related to excitations of transverse nature. The frequencies corresponding to these local JT maxima, ωTl(Q), ωTi(Q), ωTh(Q), for the low, intermediate and high energy mode respectively, are compared to the frequencies obtained from the SCM in SCM.Similarly to what has been observed for the ωTl(Q) has a slight, though evident, Q dependence, with a slope close to the methanol adiabatic sound velocity, but with the important difference that ωTl(Q) does not show a positive dispersion as ωs(Q) does. Therefore, the analysis of JT allows to attribute to the lower energy mode, ωD1(Q), a transverse and acoustic nature. The two transverse excitations at higher energy, in particular ωTi(Q), are substantially Q-independent. Both are detectable at almost all Qs, and follow the typical behaviour of optic modes, as revealed by the non vanishing Q = 0 extrapolation of their characteristic frequency. The optic-like mode at ωTi(Q) ~ 20 rad ps−1 is present in both JL and JT in SCM probably because the corresponding frequency was too close to that of the longitudinal acoustic mode, and therefore hard to separate from it. The values obtained for ωTh(Q) confirm the presence of a third mode at high energy, equivalent to ωD2(Q). Its persistence in a broad Q domain is clearly inferred when the currents are analysed.Even more clearly than in Q ≥ 15 nm−1. This seems consistent with the idea that being movements essentially localised and non-propagating, their dominant frequency becomes independent from both amplitude and direction of the exchanged momentum. This makes the mere concept of a mode polarisation ill-defined.Analogously to what has been observed for the current spectra of SCM of methanol are even harder to detect in any spectroscopic method probing the total S as often hidden by intramolecular dynamics. In x-ray spectroscopy, the difficulty is further complicated due to the typical long-tailed energy resolution function. In the IXS study of ref. S spectra were analysed with a model fit function that intrinsically excludes the presence of two excitations and reduces to a simplified model composed of a single DHO supplemented by a single central Lorentzian line. While the neutron study of ref. SCM through MD computations was correctly recognized in ref. The ensemble of these findings reverses the interpretation of the collective dynamics of methanol. Due to their intrinsic weakness, the inelastic components of Qp values: this suggests a universal nature of dispersion in HB liquid dynamicsQ’s in water, probably owing to its larger Qp. In addition to this, we find the important result that methanol, as water, exhibits a second excitation at lower frequencies. This mode is only slightly dispersive but with a possible transverse acoustic origin that also in water has been suggested on the ground of a low Q linear dispersion with a slope smaller than, yet somewhat close to, the adiabatic sound velocity of water−1 range at Q ≥ 4 nm−1. This mode has been frequently related to a coupling between longitudinal and transverse dynamics arising in systems, like water, having a tetrahedral coordination25Our findings suggest some similarities of the methanol dynamics with the long debated case of liquid water242527Q > 5 nm−1 as in methanol, but at higher energies, was also observed in the recent simulation of supercooled TIP4P/2005 water32Even closer resemblances can be found in supercooled water, which shows a similar triple mode structure as reported in both an experimentalAnalogies have been found also in the normal mode analysis of a few hydrogen bonded liquidsQ-constant frequency modes are to be connected to the symmetric and asymmetric stretching of the HB connecting them. Therefore, in analogy with the picture proposed in ref. Based upon the results of ref. JL at low Qs rapidly vanishes being gradually taken over by a low frequency transverse mode at high Qs. A similar trend was previously observed by IXS measurements in waterQ range a viscoelastic behaviour characterised by propagating sound modes which are increasingly damped with growing Q, until they become overdamped in a rather narrow range around the position of the main peak in the static structure factor Qp, where the sound propagation is arrested. This behaviour, already known to be a common property of a large variety of liquids454649SVE, is also shared by methanol. In fact, if the acoustic mode dispersion ωs(Q) of methanol is compared to that of its non-hydrogen-bonded analog, i.e. methane, one sees that the two curves have substantially the same shape, differing only by a constant factorMoreover, we notice in Q and the longitudinal and transverse waves propagating in water  and methanol .The main conclusion of our work is thus represented by the discovery of a methanol collective dynamics much richer and complex than known so far, and displaying features bearing evident similarities to those found in liquid water. Our data indirectly suggest that relevant dynamical features typically attributed to the tetrahedral coordination in water survive also in a non-tetrahedral liquid and it seems tempting to interpret the overall dynamics found for methanol as a sort of distinctive behaviour in HB systems. τ reported in Methods). The lifetimes corresponding to the three inelastic excitations shown in τ1 = 1/z1 ~ 1 ps, and τ2 = 1/z2 ~ 2 – 3 ps, respectively, at Q ≤ 15 nm−1.At a more speculative level, we may try to provide additional insight into the dynamics in methanol by recalling the strong asymmetry introduced by the HB, and assuming that its lifetime affects the decay time of all vibrational excitations implying HB bonds (such as e.g. the bending of O-O-O triplets). We recall indeed that our model is the frequency counterpart of time correlation functions having decay constants that reflect damping factors of both the VE and DHOs contributions in Eq. 147τ’s reach an almost constant level at Q ~ 15 nm−1, which implies that localisation of all the vibrations occurs at the same Q where the longitudinal and transverse modes become almost undistinguishable. At Q ≤ 15 nm−1, we find that the two additional VE decay times, τ1 and τ2, have values similar to the HB mean lifetime obtained from simulations on methanol17τ1 and τ2 that we have found in parallel simulations ran with the methanol HB interaction turned off.All the We finally remark that the dynamic scenario emerging from the present results seems compatible with the one observed in the Fast Infrared Spectroscopy study of the water OH stretching dynamics, where the coexistence of fast HB local oscillation (~200 fs) with a slower (~1 ps) correlation due to collective motions was provedT = 298 K. A deuterated sample (CD3OD) was chosen in order to maximise coherent scattering and the visibility of collective dynamics. The measurements were carried out with two values of the incident-neutron wavelength (λ0 = 1 and 2 Å) for a more efficient coverage of the  region while keeping the neutron speed sufficiently larger than the adiabatic sound speed in methanol (cs = 1100 m/sGexp(E) with a measured full width at half maximum of 3.0 and 0.7 meV for the two neutron wavelengths. The scattering angle range (1° ≤ θ ≤ 14°) allowed by the BRISP setup made possible the exploration of dynamical excitations at wave vectors between Q = 2 and 16 nm−1, the upper bound being close to the position of the main peak in the static structure factor Qp = 17 nm−1.The small-angle spectrometer BRISP58CH = 1.09 Å, rCO = 1.41 Å, rOH = 0.945 Å) by means of the SHAKE algorithm, while the remaining intramolecular motions such as bond-angle bending and rotation of the methyl group around the axis of the C-O bond were treated using a harmonic and a cosine potential, respectively, with the corresponding OPLS-AA parameters which are reported in the Supplementary Information. This model has proven to be able to reproduce in a satisfactory way the density and heat of vaporization of liquid methanolQ < 24 nm−1, and ω up to ~350 rad ps−1.MD simulations have been performed using the DL POLY 2.20 packageIt is also worth noting that MD simulations have also been carried out with the H1 potential in ref. The VE contribution in bVE makes the inelastic Lorentzian lines asymmetric with respect to frequency of sound excitations ωVE with a damping zVE, and (ii) two Lorentzian quasi-elastic terms proportional towhere the parameter z1,2with damping The DHO model is expressed by a stripped-down version of the same spectral profile, where only the two inelastic termsD1,2 and ΓD1,2 being the frequency and damping of the excitations, respectively. The characteristic lifetime τ1,2 of a given excitation is simply obtained as the reciprocal of the respective damping.survive, ΩHow to cite this article: Bellissima, S. et al. The hydrogen-bond collective dynamics in liquid methanol. Sci. Rep.6, 39533; doi: 10.1038/srep39533 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies. Parasponia are tropical tree species belonging to the Cannabis family (Cannabaceae) and are known as the only non-legume plants that can establish a nitrogen-fixing endosymbiosis with rhizobium  diverged about a 100 million years ago  signals  and CALCIUM AND CALMODULIN-DEPENDENT PROTEIN KINASE (PanCCaMK) – commit conserved functions in the Parasponia and legume LCO signaling pathways  expression (HISTIDINE KINASE 4 (HK4) that in legumes is essential for nodule organogenesis (ETHYLENE INSENSITIVE 2 (EIN2) that is a negative regulator of nodulation in legumes . Pots were half-filled with agraperlite  and watered with modified EKM medium  (Mesorhizobium plurifarium BOR2 (OD600 = 0.025) (All experiments were conducted using  thereof . P. andeNa2MoO4]  and plac= 0.025) .NPTII) and an Arabidopsis thaliana codon-optimized variant of Cas9  (M. truncatula (Mt4.0v1) (Populus trichocarpa (v3.0) (1. Protein sequences of P. andersonii (PanWU01x14_asm01_ann01) and Trema orientalis (TorRG33x02_asm01_ann01) were obtained from www.parasponia.org   , M. trunMt4.0v1)  and Popua (v3.0)  were obtonia.org . These sTAIR102)  and M. tT v7.017  implemenT v7.017  implemenP. andersonii was performed using A. tumefaciens strain AGL1  of A. tumefaciens were used. Bacteria were scraped from plate and resuspended in 25 ml of infiltration medium , chosen based on previous study (RNA was isolated from snap-frozen root tips ∼2–3 cm) as described by us study . All pri–3 cm as post hoc tests. Statistical analyses were performed using IBM SPSS Statistics 23.0 .Statistical differences were determined based on one-way ANOVA and Tukey P. andersonii, we first determined the most optimal conditions for regeneration of non-transgenic tissue. We compared regeneration efficiencies of nine tissue explant types in combination with 11 different media, including the propagation and root-inducing media previously used for P. andersonii . When 2 mm in size, transgenic calli were separated from tissue explants, which stimulated shoot formation . Two to three months after the start of transformation, a single shoot was selected from each explant to ensure that the transgenic lines represent independent transformation events. These shoots can be genotyped and vegetatively propagated  targeting PanHK4 and PanNSP2 and single sgRNAs targeting PanEIN2 and PanNSP1 were placed under an A. thaliana AtU6 small RNA promoter . Genotyping of regenerated shoots showed that >85% contained the Cas9 gene, indicating successful transformation. Potential mutations at any of the target sites were identified through PCR amplification and subsequent sequencing of the PCR product. This revealed mutations at the target site in about half of the transgenic shoots examined, of which the majority were bi-allelic (Table 1). Most mutations represent small insertions and deletions but also larger deletions and inversions were identified, some of which occur in between two target sites , cotton (Gossypium hirsutum), and citrus (Citrus clementina)  P. andersonii as well as control transgenic lines . In contrast, leaf abscission was not observed on Panein2 mutant trees . This demonstrates that Panein2 mutants are indeed ethylene insensitive.To characterize the resulting mentina) . We explPanein2 mutant trees revealed an additional non-symbiotic phenotype. These trees form bisexual flowers containing both male and female reproductive organs . In contrast, WT P. andersonii trees form unisexual flowers that contain either stamens or carpels . This suggests that ethylene is involved in the regulation of Parasponia sex type.Inspection of  carpels   . Therefore, we conclude that PanHK4-mediated cytokinin signaling is required for regulation of P. andersonii secondary growth.Cytokinins are important regulators of cambial activity, as shown in uloides) . To deteM. truncatula previously identified a set of genes downregulated in roots of Mtnsp1 and Mtnsp2 mutants  and MORE AXILLARY BRANCHING 1  that are putatively involved in strigolactone biosynthesis . We noted that Pannsp1 mutant lines differ in the level of PanD27 and PanMAX1 expression. Both genes have an intermediate expression level in Pannsp1–6 and Pannsp1–13, compared to Pannsp1–39 and Pannsp2 mutants . The three Pannsp1 mutant lines differ from each other in the type of mutations that were created. Pannsp1–6 and Pannsp1–13 contain a 1 bp insertion and 5 bp deletion close to the 5′-end of the coding region, respectively. These mutations are immediately followed by a second in-frame ATG that in WT PanNSP1 encodes a methionine at position 16. In contrast, Pannsp1–39 contains a large 232 bp deletion that removes this in-frame ATG . This suggests impaired nodule development in P. andersonii ein2 mutants.The phenotype of the root . Panein2Panein2, Panhk4, and Pannsp1–6/Pannsp1–13 mutant nodules, we sectioned ∼10 nodules for each mutant line and studied these by light microscopy. Wild-type P. andersonii nodules harbor an apical meristem, followed by several cell layers that contain infection threads  . These cells are followed by cells that are filled with fixation threads . The general cytoarchitecture of Panhk4 and Pannsp1–6/Pannsp1–13 mutant nodules does not differ from that of WT or transgenic control nodules , suggesting that these are functional. In contrast, in Panein2 mutant nodules intracellular infection is hampered . Most (>75%) Panein2 mutant nodules harbor only infection threads as well as large apoplastic colonies . Some mutant nodules, harbor cells that contain fixation threads. However, even in the best nodules, fixation thread formation is severely delayed and many cells in the fixation zone still show vacuolar fragmentation . This shows that ethylene signaling is required for efficient fixation thread formation in P. andersonii nodules.To determine the cytoarchitecture of gure 7A) . Below tnsp1, nsp2 and ein2, whereas no effect on nodule formation was found by knocking out hk4 in P. andersonii. Interestingly, we uncovered a novel role for the ethylene signaling component EIN2 in intracellular infection of P. andersonii nodules.Taken together, these data reveal symbiotic mutant phenotypes for Parasponia can provide insights into ‘core’ genetic networks underlying rhizobium symbiosis , making generative propagation of multiple mutant lines logistically somewhat challenging. An alternative to generative propagation is in vitro maintenance of transgenic lines. Additionally, the fast and efficient transformation procedure presented here will allow recreation of a particular mutant in less than 6 months.An advantage of the pagation . This alpagation . Most mupagation . Since cmpatible . Howeverllinated . This coPanhk4 and Panein2 showed symbiotic phenotypes that differ from corresponding legume mutants. P. andersonii Panhk4 mutants form nodules with a WT cytoarchitecture, indicating that these nodules are most likely functional. Analysis of stem cross-sections showed that Panhk4 mutants possess a reduced procambial activity. Similar phenotypes are observed in homologous mutants in A. thaliana (arabidopsis histidine kinase 4 (ahk4), whereas it is completely abolished in the ahk2 ahk3 ahk4 triple mutant  and melon (Cucumis melo) (Parasponia species.is melo) . Moleculis melo) , 2015. IEIN2 knockout mutations result in different phenotypes between Parasponia and legumes. In legumes, ethylene negatively regulates rhizobial infection and root nodule formation (M. truncatula ein2 mutant (named sickle) that forms extensive epidermal infection threads and clusters of small nodules . However, all three mutants are affected in transcriptional regulation of strigolactone biosynthesis genes PanD27 and PanMAX1 . The three Pannsp1 mutant lines differ from each other in the type of mutations that were created. Pannsp1–6 and Pannsp1–13 contain small deletions that are immediately followed by a second in-frame ATG that in WT PanNSP1 encodes a methionine at position 16. In contrast, Pannsp1–39 contains a larger deletion that removes this in-frame ATG  that interferes with hormone homeostasis (P. andersonii symbiosis genes to determine to what extent legumes and Parasponia use a similar mechanism to establish a nitrogen-fixing symbiosis with rhizobium.Taken together, we showed that on e.g.,  but has eostasis . The proP. andersonii genes used in this study can be found in Supplementary Table www.parasponia.org.All datasets analyzed for this study are included in the manuscript and the supplementary files. Gene identifiers for all Conceptualization, AvZ and RG; Methodology, AvZ, MH, SL, and WK; Investigation, AvZ, TW, MSK, LR, FB, MH, SL, EF, and WK; Formal analysis, AvZ, TW, and EF; Visualization, AvZ; Writing – original draft, AvZ; Writing – review and editing, AvZ and RG; Funding acquisition, TB and RG; Supervision, RG.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.


Dairy branch had a combined 35% share of the above consumption. As shown by the data obtained from the Polish Central Statistical Office, the majority of dairy plants use its own source of water, so this branch is also important water producer in Poland. Water used for dairy industry should meet the requirements of at least drinking water quality, so the factories need to treat the water. This paper analyses the correlations between selected technical process, equipment profiles and water quality, and consumption in two types of dairy factories (DF). The first one DF-1 processes approx. 50,000 L of milk, and the second, DF-2 processes approx. 330,000 L of milk per day. The water taken from the wells needs to be pre-treated because of iron and manganese concentration and due to specific requirements in various industrial processes. As a result of this work, we have managed to propose technological solutions in the context of water consumption rationalization. The proposed solutions aim at improving water and wastewater management by reducing the amount of consumed water by industry.Food industry is one of the most important and fastest growing sectors of economy in Poland. This sector is also characterized by high demand for the resources, particularly for water. Polish food industrial plants consumed 793 hm At the same time, in 2014, Polish industrial sector generated more than 7876 hm3 of sewage, out of which 734.5 hm3 falls in the dairy industry, as analyzed further in this article . However, given the fact that a number of chemicals in concentrations normally found in water can cause negative health effects with long-term exposure, toxic substances having the ability to accumulate in the body, as well as substances with carcinogenic properties, are also subject to strict rigor and are eliminated from water intended for human and animal consumption. This is due to sanitary standards on water quality, among which the most important legislation in the European Union being the Directive of the Parliament and the EU Council on the quality of water intended for human consumption, specifying the standard chemical, physical, and biological properties which must be met by water supplied and used in food plant production , and parameters such as color, turbidity, total number of bacteria, total organic carbon, taste, and smell. Although the parameters have no direct impact on consumer health, the aim is to determine the effectiveness and quality of water treatment process, which is referred to as auxiliary indicator parameter. It should be noted that the standards for a number of indicators applicable under the Directive are more restrictive than the WHO recommendations ; the flow and load of the sewage; existing practices and procedures for water management; and analysis of the obtained results. The procedure for water quality sampling and the appliances used for water quality parameter measurements were determined according to recommendations of Polish State Sanitary Inspection. The spectrophotometer manufactured by HACH Co. model DR 2000  was applied for water quality analysis of ammonium, nitrate, nitrite, iron, and manganese turbidity and color. The hydrogen ion concentration (pH) was measured with a pH meter CP—411 , and the electrical conductivity was measured using a conductivity meter—CC—411 . Hardness level was determined by titration following accredited testing procedure. The concentration of total count of microorganisms was conducted as per PN-EN ISO 6222:2004. The measurement of smell has been conducted via a simplified method based on the norm PN-EN 1622. Some data was taken from direct interview with the employees of the company. Furthermore, the correlations between selected technical process and production factors, equipment profiles, water quality, and consumption are also presented. The collected data was compared with the data available in literature. It should be noted that the analyzed companies had diverse production profiles and management procedures.Qd = 127 m3/day, the average hourly demand is Qh = 5 m3/h, whereas the maximum is Qmaxd = 140 m3/day. Because of increased color and iron concentration, the water is subject to pre-treatment process. Following the treatment, it is transported into a clean water tank with the capacity of V = 48 m3, and through the use of pressure tank, the water is directed to the factory water supply system. The wastewater produced in the process of filter backwashing is discharged into industrial treatment plant.The first factory (DF-1) processes approx. 50,000 L of milk per day. The DF-1 is characterized by own drill well and additional water supply system connection with the municipal network, so that in case of accident or increased production, the water could be charged from municipal water supply. The presented dairy cooperative uses water for technological purposes, washing plant and equipment, steam generation and heating, as well as partly for social purposes. Average daily water demand is 5 and suspended solids are the main pollutants that arise from leaks, spills, and removal of adhering materials during cleaning and sanitizing of equipment, cleaning and sanitizing solution with water washing and cooling water.DF-1 is in the possession of a permit for the uptake of groundwater. The individual amounts of pollutants in the wastewater discharged into the sanitary sewage system determine other water permit disposed by the facility. At the production facility, there are three types of sewer network, namely the sanitary sewage system, storm water network, and the technological wastewater network. The storm water system is used to drain rainwater from the site. Dairy effluent is pre-treated in industrial wastewater treatment plant. Dairy processing discharge wastewater characterized by high organic load due to the presence of milk components. Wastewater are characterized by high changes in temperature or pH. Other parameters of importance for dairy effluent are high COD, TSS, N, and P. As indicated also by Borbon et al. , high BOQavd = 2.16 m3/day, the hourly average Qavh = 90 m3/h, and the hourly maximum Qmaxh = 180 m3/h. Raw water from wells is transported under pressure by pumps for drinking water treatment plant, where it is subject to processes of removing iron and manganese and chlorination. Then, it is pumped into two underground reservoirs with a capacity of 500 m3 each and subsequently distributed throughout the whole plant as treated drinking water. Water used for the supply and charging of boilers requires additional preparation. This includes processes such as filtration, softening, and reverse osmosis. The DF-2 factory makes use of CIP systems, which limits the consumption of raw water by reusing the water from the last rinse of equipment to the first rinse cycle. DF-2 also owns water permit for underground water uptake and wastewater permit to discharge wastewater. If whey from the cheese-making process is not used as a by-product and discharged along with other wastewaters, the organic load of the resulting effluent is further increased  processes approx. 330,000 L of milk per day. At DF-2, the water supply system is based on its own underground water intake. Water intakes are located approximately 0.75–1.00 km from the plant. Noteworthy is the fact that a slight amount of water produced by the industry can be sold to the third parties. Water intake, as in the case of a DF-1, includes a defined zone of primary and intermediate protection. DF-2 owns a water permit for groundwater intake originating from their own sources serving for production and social purposes, as issued by the head of district. The consumption amounts are as follows: the daily average The dairy processing plants manufacture various dairy products where the primary ingredient is raw milk. In this paper, the presented case studies covered DF-1—milk production and DF-2—cheese production line. The processes taking place at the milk plant include milk receipt and filtration of raw milk; separation of complete or part of the milk fat due to standardization of market milk; pasteurization and homogenization, if required; followed by packaging and storage, including refrigerated storage. In the cheese production, line milk is separated into skim milk and cream, then pasteurized, followed by specific processes depending on the desired product. The product is packaged and stored before being distributed.Selected processes employed for manufacturing of various products are indicated in Fig. The basic element of proper water management in the food industry is to ensure adequate water quality. This is a prerequisite for prevention of incidents arising from security threats. Risk prevention constitutes of detection and identification (recognition). Risk control in both factories is carried out by specially developed programs to monitor the quality of raw water and treated water. Such programs provide appropriate steps throughout the whole chain: supply, production, and distribution of water. The most important, however, insignificant from the point of view of the analysis of sanitary water security threats is a parameter that requires constant monitoring, namely—iron. Iron as a component of water-bearing rocks occurs in waters of almost 80% underground sources. Harmless from the point of view of health, it causes significant trouble in production processes, particularly affecting negatively the quality of dairy products. The most important parameters of the quality of raw water in individual plants with reference to the current standards are presented in Table Since at both plants the problem of excessive concentration of iron in raw water has been recognized, both analyzed plants are equipped with iron removal system. Iron removal systems operate on the basis of the same unit processes, namely, aeration, which aims to oxidize ferrous iron, as well as rapid filtration, in an attempt to stop the precipitated iron compounds. In order to ensure microbiological safety after the iron removal process, at both plants, the water is being disinfected with the use of chlorine. Analysis of archival data and interviews with employees at the plant lead to the conclusion that the operating treatment system ensures the required security level, both in terms of quantity and quality of water used in the manufacturing process. As previously mentioned, the process water, which may have contact with food, must meet strict quality requirements, which prevents it from being reused.Nonetheless, the water used for washing machines and halls, especially when used for the first washing, is not the subject of such restrictive preconditions. This case was adopted at DF-2 where the CIP system has been implemented. After the completion of the technological process, clearing machines, and equipment from the product and its detachment from supply tanks, the rinsing is conducted in a closed circuit. The process of cleaning the line begins with pre-wash, which removes the remaining product from washed surfaces. Cleaning agent containing many contaminants is removed from the system. Thereafter, the preliminary heavily contaminated water is transported directly to the treatment plant. The initial washing is followed by proper wash with the use of detergents. The solution remaining from last washing is passed to the pre-wash tank at the washing station. The process ends with rinsing with clean water. Considering that such water may have contact with food production, it must meet the requirements for drinking water.Other requirements are set for the water used for steam production. In the dairy industry, the technological steam is used not only as a heat carrier, but also, and perhaps primarily as a disinfecting agent in the process of sterilization and pasteurization. Some processes require direct contact of steam with the product . If in the production process the steam being used has direct contact with food products, the parameters of water used for steam production should correspond to the parameters of drinking water, including the boiler water used for steam boilers. Such water must meet the requirements defined by the manufacturers of boilers, which usually relate to the hardness of water. The requirements depend on the design of a boiler and increase accordingly with its operating pressure.At both study sites, there are water softener stations which effectively remove hardness to the levels below 0.01 mval/l, supporting the operation of boilers. In addition, water in the boiler system is adjusted through the use of chemicals that do not have toxic, carcinogenic, mutagenic, or harmful properties and are thermostable under normal operating conditions of the boiler. These substances are approved by the National Institute of Health for having contact with food. Both basic ingredients of these formulas and complementarity-stabilizing additives are a category of chemicals listed in food additives. This approach of both companies creates the comfort of safety, both from the point of view of the construction of the boiler and steam quality, and thus the quality of dairy products.Another problem regarding water management at the company is to ensure the supply of water to maintain greenness in the summer, and the provision of sufficient quantities of water in case of fire. In these cases, the requirements for water quality are not as restrictive, since this part of water management depends on the quantity of water available. At both plants during the summer months, the maintenance of green areas was carried with raw water and additionally with treated wastewater which quality allowed for such use Zahuta . RegardiDairy processors are aggressively challenged to conserve water necessitating the need for not only reducing water consumption but also to employ measures for recovery and recycling of process water without compromising on the hygienic quality and safety of the products. In Poland, there are norms of water consumption specified by the Minister of Infrastructure Regulation of 14 January 2002 on determining the average water consumption standards. Specific standards of the average water consumption for dairy products are presented in Table In the literature, Flemmer , Perry  and SteiRespective processes have been analyzed at the plants in order to evaluate water consumptions. Water consumption indicators broken down by technological processes in both analyzed companies are presented in Tables The diversity of indicators at individual factories was discovered to be resulting from the diversity in the type of production. When comparing the data with the available literature data as presented in the introduction, at Table The data presented indicate a high potential of this industry to use systems reuse water. According to literature, data recovery of water used can save up to 20–40% of the total costs associated with the production of water (Milani et al. This is an essential fact because Poland is in the process of major legislative changes and the introduction of significant increase in fees for the use of water from primary sources. Therefore, any change will translate into tangible economic savings and improve enterprise competitiveness.Undoubtedly, water consumption is also influenced by the nature of dairy production. Most water is required at plants that produce milk powder and cheese, a little less is consumed at plants producing drinking milk. In order to compare the levels of water consumption for both of analyzed plants, the average water consumption indicators have been calculated: the average for years 2011–2014 and for 2015 only, after the introduction of water management industry changes. The data are presented in Table When taking into account data of the year by year period, we may observe reduction in water consumption per liter of product. These improvements are attributed to developments in process control and cleaning practices. During the research period on presented case studies has been implemented the system of simple changes and lower water consumption in the washing process. Additionally, some percent of wastewater has been used for green area irrigation, and all leaks have been monitored. At DF-1, 10% decrease in water consumption indicator has been achieved. At DF-2, almost 15% presumably because DF-2 uses CIP and only a slight modification to CIP has resulted in high reduction of fresh water use. Significant impact of controlling and optimizing cleaning parameters on water consumption was described also by Wojdalski et al. . PracticThe above data allows us to draw very optimistic forecasts and conclusions, namely that the existing measures aimed to reduce water consumption from primary sources do bring the expected results.The problems of environment protection in the industrial sectors are becoming more and more relevant, with strict legal requirements that imply considerable investments. This encourages researchers to look for new systemic solutions and methodologies to improve efficiency of water management. Industrial plants are specific in terms of quantity and quality of treated water, applied technologies and technical solutions, and specific operational regime. Thus, the decision about selecting the most appropriate type of technology is very individual and should be accurate for selected industrial plant.Management strategies for improving water productivity of dairy production have to start at the source of the water. Since water treatment technology significantly influences the total consumption of water at the industrial plants, we should properly select the source of water and unit processes, so that water consumption for technological purposes is as low as possible.3 water/m3 of milk intake (Wojdalski et al. Further technologies used in production require large quantities of water, used either for washing machine, cooling system, or product processing. The possible ways these operations can be modified or employee practices changed to reduce water use are identified and discussed. The role of management in processing water and waste control is an important factor for rational water management in the industry. In the literature, we could find a water consumption rate even of 1.3–2.5 m