Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy) and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR) spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200–900 cm−1), mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.High hydrostatic pressure (HHP) is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50–900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of  High hydrostatic pressure (HHP) is a food processing technology used to maintain the quality attributes of fresh foods, while extending their shelf life through the inactivation of pathogenic and spoilage microorganisms, as well as of endogenous enzymes. In fact, HHP has been proposed as an alternative to thermal processing due to the less detrimental effects it shows on food quality and organoleptic properties ,2,3.Staphylococcus aureus, Listeria monocytogenes, Salmonella spp. and verocytotoxigenic Escherichia coli (VTEC), are among the bacterial species most extensively studied with this aim [E. coli strains in HHP resistance have been described, with some strains being inactivated by pressures as low as 200 MPa, whereas others can survive exposures to 600 MPa [Great research efforts have been made in recent years to evaluate the safety of HHP-treated foods by studying the inactivation kinetics by HHP of pathogenic microorganisms in food and food models . The foothis aim . VTEC, cthis aim . Some stthis aim ,7. Howev 600 MPa ,9.Despite much effort in recent years, the main cellular targets and the mechanisms of bacterial killing by HHP have not yet been fully identified . KnowledE. coli strains after HHP treatments of different intensities. For this purpose, the uptake of the fluorescent probe, propidium iodide (PI), and the loss of intracellular contents were used as indicators of membrane damage, while morphological alterations were analyzed by TEM and global changes in cellular biochemical features were assessed by FTIR spectroscopy.This study aimed to determine the morphological and physico-chemical changes occurring in cells of Escherichia coli E218/02 and Escherichia coli C-600 were exposed to different pressure-time combinations . Whereas HHP treatments at 50 MPa for 24 h did not give rise to significant reductions in the bacterial population, exposure to 300 MPa for 5 min resulted in the inactivation of 4.5 and 4.7 log cycles for E. coli E218/02 and E. coli C-600, respectively, and treatment at 600 MPa for 5 min gave rise to a 6.8 and 7.3 log reduction, respectively (E. coli strains at the pressure-time combinations assayed.Stationary-phase cultures of ectively A. No sur260) and 280 nm (OD280) of cell-free filtrates from untreated and treated samples (260 and OD280 of a similar magnitude for both E. coli strains. A further increase in pressure intensity (600 MPa) did not result in a further increase in OD260 and OD280 values, which were similar to those observed for cells treated at 300 MPa. No significant differences in OD260 and OD280 values were observed between E. coli E218/02 and E. coli C-600 at any of the treatment conditions.The leakage of intracellular nucleic acids and proteins after HHP treatment at 300 MPa for 5 min and 600 MPa for 5 min was assessed by monitoring the optical density at 260 nm  by untreated and HHP-treated populations . PI enteE. coli E218/02 and E. coli C-600 were assessed by using transmission electron microscopy (E. coli strains at the two HHP treatment intensities (300 and 600 MPa for 5 min).The effects of HHP treatment on the ultrastructure of croscopy . Untreat1  and w4  showed the lowest differentiation indexes (DI) and, consequently, the highest reproducibility, which demonstrates a good standardization of growth conditions and preparation of samples.The changes in the molecular composition after HHP treatment were determined by using FTIR spectroscopy . Althoug4 region was the most discriminant one. HCA of this spectral region divided the samples into two main groups separated with a linkage distance of 0.25, one including untreated samples (cluster 5) and samples treated at 50 MPa (cluster 4) and other comprising samples treated at higher pressures with lethal effects on bacterial cells (300 (cluster 3), 600 and 900 MPa (clusters 4 and 5))  of the second derivative spectra demonstrated that the w and 5)) . No signE. coli cells.HHP represents a novel food processing technology that can be used as an alternative to conventional heat treatments in order to guarantee microbial inactivation and food safety, while causing minor effects on the quality and sensorial attributes of foodstuffs. The current study demonstrates the efficacy of HHP treatments to inactivate VTEC. Although recent investigations have focused on the elucidation of the mechanisms of bacterial inactivation and death by HHP ,12,13,14260 and OD280 of cell-free filtrates. This leakage of intracellular components was of a similar magnitude at 300 and 600 MPa and suggests that HHP may cause disruption in the cellular envelopes. The vast majority of HHP-treated (300 and 600 MPa) cells were also stained after incubation with the dye PI, as shown by fluorescence microscopy observations. This fact evidences that their membrane was damaged, allowing for the uptake of this dye. Thus, membrane integrity tests showed that HHP treatments disrupt the cell envelopes, rendering them more permeable. These findings are in agreement with previous studies that demonstrated the occurrence of losses of physical integrity of outer and inner membranes after exposure to high pressures [4 spectral region . Changes observed in these spectral regions suggest the presence of compositional or conformational alterations in some components of the external cell envelopes occurring in response to high pressures. Nonetheless, changes in FTIR spectra were minor in comparison to those previously described for E. coli cells exposed to a range of lethal stress conditions [The ability of high pressures to damage the bacterial membrane was assessed by measuring the release of intracellular contents and the uptake of PI (dye that enters the cell when membrane integrity is compromised and binds to intracellular nucleic acids) following exposure to 300 and 600 MPa for 5 min. Treatment at both pressure intensities gave rise to a leakage of intracellular nucleic acids and proteins, as shown by the increase in ODressures ,12,13,14ressures ,21,22. Fressures ,26,27. Fnditions .E. coli treatment at 300 MPa gave rise to bacterial inactivation and significant structural and physico-chemical modifications. Treatment at 600 MPa caused a higher level of bacterial inactivation, but did not give rise to any additional structural/compositional alterations. This suggests that bacterial damage occurs once a threshold of pressure is reached and that different levels of damage recovery could happen as a function of the treatment intensity.Ultrastructural observations, membrane integrity tests and FTIR spectroscopy analysis showed that structural and physico-chemical alterations caused by HHP treatments were not dependent on the pressure intensity of the lethal HHP treatment. E. coli strains were used throughout this study, Escherichia coli E218/02, a VTEC O157:H7 strain originally isolated from a dry-fermented sausage involved in an outbreak in Sweden [Escherichia coli C-600, a non-pathogenic laboratory strain. Cultures were maintained in cryovials at −80 °C. Bacteria were resuscitated in tubes containing 10 mL of brain heart infusion  by incubation at 37 °C for 24 h followed by streaking on BHI agar plates, which were then incubated under the same conditions. Precultures were prepared by transferring an isolated colony from a plate into a test tube containing 10 mL of sterile BHI followed by incubation at 37 °C for 24 h. Precultures were subsequently used to inoculate 50 mL of sterile BHI with approximately 103 cells/mL, followed by incubation at 37 °C for 24 h, which resulted in a stationary phase culture with approximately 109 cells/mL. This culture was subsequently used for HHP treatments.Two n Sweden , and Escg for 5 min, and the cellular pellets were resuspended in 10 mL of PBS. Afterwards, suspensions (4 mL) were placed in heat-sealed sterile plastic pouches before pressurization. The pressure-time combinations tested were 50 MPa for 24 h, 300 MPa for 5 min, 600 MPa for 5 min and 900 MPa for 5 min. HHP treatments were performed in a Model FPG 7100:9/2C Series Foodlab . Pressurization water and the vessel were at 12 °C ± 1 °C before pressure treatment. The transmission fluid was a mixture of water and 30% propylene glycol. The adiabatic temperature increased 2–3 °C for every 100 MPa; thus, the maximum temperature range under 600 MPa and 900 MPa was 24–30 °C and 38–39 °C, respectively. The time taken to achieve the maximum holding pressure (900 MPa) was 50 s, and the decompression time was about two seconds. Pouches were removed immediately after treatment and aseptically opened. Ten-fold serial dilutions were produced in sterile 0.1% (w/v) peptone solution; suitable dilutions were plated on BHI agar, and viable cells were enumerated following the incubation of plates at 37 °C for 48 h (longer incubation times did not show any influence on the count).Aliquots of 10 mL of the bacterial cultures were centrifuged at 8000× 8 cells/mL. Afterwards, 1 µL of PI  was added, and the mix was incubated in the dark at room temperature for 10 min. Finally, the cell suspension was centrifuged; the cellular pellet was suspended in PBS, and samples were analyzed by Epi-fluorescence microscopy  with a high-pressure mercury lamp  and filter cube  blue excitation filter combination  with spectral profiles as follows: Excitation (EX) filter 450–490 nm, Dichroic Mirror (DM) filter 505 nm, Barrier (BA) filter 520 nm. The cells that stained red were considered as non-viable.Control untreated cultures and bacterial cultures exposed to the different pressure-time regimes were subsequently diluted in PBS to achieve a final cellular concentration of ~10Aliquots of 3 mL of cell cultures exposed to different pressure-time regimes were filtered through a 25-mm-diameter, 0.22-µm pore size Millex-GS syringe filter , and the presence of nucleic acids and proteins in the cell-free filtrate was checked by measuring the absorbance at 260 nm and 280 nm, respectively , as described elsewhere [E. coli cells exposed to 300 and 600 MPa for 5 min were harvested by centrifugation and fixed in 2.5% glutaraldehyde -PBS for 3 h at 4 °C. Bacteria were then washed three times with PBS, and cells were treated with osmium tetroxide (TAAB Laboratories)-1% PBS for 45 min at room temperature in darkness. Afterwards, three new washings with PBS were performed. Subsequently, cells were pelleted in bacteriological agar , and pellets were dehydrated in ethanol solutions of increasing concentrations and embedded in an epoxy resin , which was polarized by its incubation for 48 h at 60 °C. Finally, ultrathin sections were collected onto copper grids and stained with uranyl and lead. Microscopic observations were carried out on at least ten different microscope fields per treatment condition, using several microscope magnifications of a JEOL 1010 microscope  at 80 kV [at 80 kV .−1 with an interval of 1 cm−1. The spectral resolution was 4 cm−1. The final spectra were achieved averaging 20 scans. FTIR experiments were performed in triplicate. A software application developed for the Perkin-Elmer environment was used for transformation, including normalization , smoothing and second derivative. After transformation, spectra were recorded in ASCII format and processed [−1, influenced by functional groups of membrane fatty acids (w1); the window between 1800 and 1500 cm−1, affected by amide I and amide II groups belonging to proteins and peptides (w2); the window between 1500 and 1200 cm−1, mixed region influenced by proteins, fatty acids and phosphate-carrying compounds (w3); the window between 1200 and 900 cm−1, which is informative mostly for the carbohydrates and polysaccharides in the cell wall (w4); and the window between 900 and 700 cm−1, named the true fingerprint, because of very specific spectral patterns (w5).Control cells and cells exposed to 50, 300, 600 and 900 MPa were harvested by centrifugation and suspended in 50 µL of PBS, placed (15 µL) in a ZnSe window and stove dried . Infrared spectra were obtained with a FTIR spectroscope  equipped with a Deuterated Triglycerine Sulfate (DTGS) detector. Measurements were recorded over the wavelength range of 4000 to 700 cmrocessed . The whoDI) [−1) and independently for the ranges described (w1 − w5), according to the equations.ry1y2 is the Pearson’s correlation coefficient, y1i and y2i are the individual absorbance values of the two spectra to be compared, n is the number of data points in the given range and y1 and y2.To study variability between replicates and within windows, samples were processed, in independent experiments, yielding three replicates for each HHP treatment condition. The differentiation index (DI)  was calcDI may adopt values between zero and 2,000, with zero for identical spectra, 1000 for completely non-correlated and 2000 for completely negatively non-correlated spectra [The three replicates for each combination  are considered and the mean is obtained.  spectra .The spectral data were subjected to multivariate statistical methods  and Factor Analysis (FA)). Pearson’s product moment correlation coefficient was used to measure the similarity between spectra, and strain clustering was achieved using Ward’s algorithm. All of the analyses  were carried out with the Statistica for Windows, v. 7.0, program .E. coli cells to HHP causes alterations in their physical integrity, with cellular enlargement, membrane damage or detachment, DNA and protein denaturation and leakage of intracellular contents, minor changes in molecular composition are evident, and these are focused in the carbohydrates and polysaccharides of the cell wall. These observations serve to enhance the currently-available knowledge on the mechanisms of bacterial inactivation by HHP and may be valuable to design more effective food preservation regimes based on the integration of mild HHP into a multi-target hurdle technology concept. For instance, our results show that HHP compromises membrane integrity, which suggests that mild HHP might increase the efficacy of food-grade antimicrobials, such as nisin, or other natural antimicrobials that otherwise might be unable to enter the cell.Our findings evidence that while exposure of


Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. There is no disease-specific difference in viral strains, and it is unclear how HTLV-1 causes such different diseases manifesting as lymphoproliferation or inflammation. Although some progress has been made in therapies for these diseases, the prognosis for ATL is still dismal and HAM/TSP remains an intractable disease. So far, two regulatory proteins of HTLV-1, Tax and HBZ, have been well studied and shown to have pleiotropic functions implicated in viral pathogenesis. Tax in particular can strongly activate NFκB, which is constitutively activated in HTLV-1-infected cells and considered to contribute to both oncogenesis and inflammation. However, the expression level of Tax is very low in vivo, leading to confusion in understanding its role in viral pathogenesis. A series of studies using IL-2-dependent HTLV-1-infected cells indicated that IL-10, an anti-inflammatory/immune suppressive cytokine, could induce a proliferative phenotype in HTLV-1-infected cells. In addition, type I interferon (IFN) suppresses HTLV-1 expression in a reversible manner. These findings suggest involvement of host innate immunity in the switch between lymphoproliferative and inflammatory diseases as well as the regulation of HTLV-1 expression. Innate immune responses also affect another important host determinant, Tax-specific cytotoxic T lymphocytes (CTLs), which are impaired in ATL patients, while activated in HAM/TSP patients. Activation of Tax-specific CTLs in ATL patients after hematopoietic stem cell transplantation indicates Tax expression and its fluctuation in vivo. A recently developed anti-ATL therapeutic vaccine, consisting of Tax peptide-pulsed dendritic cells, induced Tax-specific CTL responses in ATL patients and exhibited favorable clinical outcomes, unless Tax-defective ATL clones emerged. These findings support the significance of Tax in HTLV-1 pathogenesis, at least in part, and encourage Tax-targeted immunotherapy in ATL. Host innate and acquired immune responses induce host microenvironments that modify HTLV-1-encoded pathogenesis and establish a complicated network for development of diseases in HTLV-1 infection. Both host and viral factors should be taken into consideration in development of therapeutic and prophylactic strategies in HTLV-1 infection. H. H20]. HSuch spontaneous induction of HTLV-1 expression in culture of primary PBMCs is observed in asymptomatic HTLV-1 carriers, HAM/TSP patients and about half of ATL patients. In the remaining ATL cases, the ATL cells do not express Tax even after culture because of genetic alterations and epigenetic silencing of HTLV-1 gene expression .The mechanism of the on/off switch of HTLV-1 Tax expression in vitro and in vivo is not well understood. Requirement of TORC family proteins in long terminal repeat (LTR)-driven Tax expression  and decrNegative regulation of HTLV-1 expression may involve type-I IFN responses. Although HTLV-1-transformed cells such as MT-2 cells are known to be resistant to type I IFN , IFNα suFurthermore, when IL-2-dependent HTLV-1-infected cells are co-cultured with stromal cells, viral expression becomes undetectable, yet recovers again when the infected cells are re-isolated and cultured alone  , 88. An Constitutive NFκB activation in Tax-negative ATL cells is associated with increased NF-κB-inducing kinase (NIK) . DownregAnother candidate that may mediate activation of NFκB and ISG could be pattern recognition receptors (PRRs) that recognize various pathogen-associated molecular patterns (PAMPs) and potentially mediate NFκB and IRF3 downstream . In de nCombination therapy with azidothymidine and IFNα (AZT/IFNα) has been used for ATL patients , 98 and HTLV-1-specific T-cell responses are observed in most asymptomatic HTLV-1 carriers and HAM/TSP patients, but impaired in ATL patients. Tax is the major target antigen for T-cells , 25. EnvDespite Tax expression being undetectable by serological methods in vivo, evidence of Tax antigen presentation in ATL patients was obtained in ATL patients following hematopoietic stem cell transplantation (HSCT). Because frequent relapse is one of the reasons for poor prognosis in ATL patients, HSCT following chemotherapy is recommended in Japan and has been shown to achieve long-term survival in one-third of recipients , 115. InAnti-tumor effects of CD8+ Tax-specific CTLs have been shown in an animal model, in which HTLV-1-infected lymphomas in nude rats were eradicated by adoptive transfer of syngeneic Tax-specific CTLs that had been induced by vaccines using Tax-coding DNA  or Tax pThe first anti-ATL therapeutic vaccine was designed to activate CD8+ Tax-specific CTLs by using Tax peptides as antigen and autologous DCs as adjuvant Fig. b 28, 11. The TaxIn the initial pilot study, three ATL patients who were in stable conditions after other therapies except for HSCT were subcutaneously injected with Tax peptide-pulsed autologous DCs three times at fortnightly intervals. All three patients showed clear proliferative responses of Tax-specific CTLs after vaccination without severe adverse effects . ClinicaThese favorable clinical outcomes of the Tax-DC vaccine indicate the significance of Tax-specific CTLs in maintenance of remission, although they may become ineffective when Tax-negative ATL clones emerge.In ATL patients, half of the cases retain the ability to express Tax in ATL cells, while the other half lose this ability . TherefoIn ATL patients whose ATL cells lack Tax expression, Tax-specific CTLs cannot directly attack ATL cells. However, these CTLs may still control subdominant HTLV-1-infected cell clones that retain the ability to express Tax, because there are multiple HTLV-1-infected cell clones and the dominant ATL clones sometimes differ among tissues and change during the disease course , 126.Since it takes several weeks to induce immune responses, the Tax-targeted vaccine cannot be the first line therapy for aggressive types of ATL. In the clinical studies, Tax-DC vaccines were administered in ATL patients as a maintenance therapy after chemotherapy. Indolent types of ATL such as smoldering and chronic ATL may be more responsive to the vaccine, as ATL cells likely retain the ability to express Tax more frequently compared with aggressive ATL. Although HAM/TSP patients may also retain the ability to express Tax, HAM/TSP patients usually have active Tax-specific CTLs and less likely benefit from additional vaccines.Once its safety and efficacy are confirmed, Tax-targeted vaccines might potentially be applied for prophylaxis of ATL development in the future. A small proportion of ACs exhibit insufficient Tax-specific CTL response and elevated proviral load , both ofThe complexity of disease mechanisms in HTLV-1 infection results from the host immune responses in concert with HTLV-1-encoded genes (Fig. The extremely low but not silent Tax expression in vivo can be partly explained by type I IFN and ISG, which suppress viral expression mainly at a post-transcriptional level, presumably maintaining HTLV-1 expression at low equilibrium levels in various tissues in vivo. The presence of an IFN signature in HTLV-1-infected cells implies continuous innate immune stimulations going on in these cells, which might also contribute to pathogenesis in HTLV-1 infection.While Tax protein is undetectable by serological means in vivo, Tax-specific CTLs still seem to recognize HTLV-1-infected cells to some extent. The results of a clinical study of a Tax-targeted therapeutic vaccine in ATL patients indicated a greater impact of Tax-specific CTLs on immune surveillance of HTLV-1 infected cells than expected, again suggesting the presence of Tax expression in vivo. Although further investigation is required, this opens up a new door to early therapy and prophylaxis against ATL.


The pleiotropic influence ß-arrestins exert on these receptors regulates a breadth of physiological functions, and additionally, ß-arrestins are involved in the pathophysiology of numerous and wide-ranging diseases, making them prime therapeutic targets. In this review, we briefly describe the mechanisms by which ß-arrestins regulate GPCR signaling, including the functional cellular mechanisms modulated by ß-arrestins and relate this to observed pathophysiological responses associated with ß-arrestins. We focus on the role for ß-arrestins in transducing cell signaling; a pathway that is complementary to the classical G protein-coupling pathway. The existence of these GPCR dual signaling pathways offers an immense therapeutic opportunity through selective targeting of one signaling pathway over the other. Finally, we will consider several mechanisms by which the potential of dual signaling pathway regulation can be harnessed and the implications for improved disease treatments. The 7TMR superfamily is responsible for transducing a wide variety of extracellular signals into intracellular functions. More than three decades ago, it was shown that ligand activation of 7TMRs resulted in intracellular signaling through receptor coupling to heterotrimeric guanine nucleotide or G proteins . Thus, 7in vitro studies show that ß-arrestins terminate G protein-mediated cell signaling by translocating and binding to GRK-phosphorylated serine and threonine residues in the GPCR third intracellular loop or C terminal tail. Once bound, ß-arrestins sterically prevent further receptor-G-protein coupling  . This adin vitro experimental conclusions that ß-arrestins play an important role in regulating normal physiological and pathophysiological responses. Despite the lethality resulting from elimination of both ß-arrestins, single ß-arrestin-knockout (ß-arrestin-KO) mice are quite normal and perturbations of homeostasis are often required to observe an effect of the absence of ß-arrestin expression.With the development of mice deficient in either ß-arrestin1 or ß-arrestin2 , evidencThe first published study examining normal physiologic responses in ß-arrestin2-KO mice involved exogenous opioid administration as the homeostatic perturbation . Bohn etThe first study demonstrating a role for ß-arrestin in disease pathogenesis used a murine model of asthma . This stß-arrestins trigger physiological responses through scaffolding of cell signaling proteins such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), proto-oncogene tyrosine protein kinase Src (c-Src family tyrosine kinases), phosphoinositide 3-kinases, protein kinase B (AKT), c-Jun N-terminal kinases (JNK3) and elements of nuclear factor κb (NF κb) .Although convenient to characterize GPCR signaling as two distinct signaling pathways, one that is G-protein dependent/ß-arrestin-independent and the other that is ß-arrestin-dependent/G protein-independent, the reality is more complex. We recently showed that P-p38 signaling, and associated chemotaxis, of T helper type 2 cells is partially dependent on ß-arrestin2 and that this ß-arrestin2-dependent signaling is downstream of Gαi . In thatAs predicted by experimental findings, the ß-arrestin-dependent signaling pathway, like its G protein (ß-arrestin-independent) counterpart, regulates a wide variety of important cellular responses including cell development, growth and survival, immune cell function, protein translation, and neuronal signaling . The abiß-arrestin versus G-protein signaling in disease.The discovery that a single GPCR subtype can couple to different transduction proteins and produce multiple cellular responses has led to development of ligands that can preferentially “bias” the receptor toward one pathway. Some of these “biased ligands” preferentially target the ß-arrestin pathways (by either activation or inhibition) and have shown promise in drug development. These advances will be discussed in the section titled Consistent with the broad range of physiological processes modulated by ß-arrestins, the upregulation of ß-arrestin expression is associated with many diseases. Whether or not changes in expression are adaptive or maladaptive remain to be determined. For example, in a mouse model of cardiac dysfunction, enhanced cardiac ß-arrestin2 expression mitigated adverse cardiac remodeling ; whereasInappropriate modulation of cell survival can lead to cancer and fibrotic diseases, while abnormalities in immune cell function have implications for autoimmunity, infection, and the inflammatory component of many diseases. For an in depth review of the role for ß-arrestins in disease, please refer to the work by 2AR-ß-arrestin2 signaling to cSrc-mediated cytoskeletal reorganization  components which are mainly derived from fibroblasts and myofibroblasts . If highfibrosis . Mice wie effect . Expresse effect . Taken te effect . These rThe range of diseases in which ß-arrestins play a role is very broad. For example, ßarrestin plays a role in brain function. A positive correlation between brain ß-arrestin (ß-arrestin1 and 2) levels (both protein and mRNA) and Alzheimer’s disease diagnosis, severity, and amyloid burden has been demonstrated by several investigators . Also, G1 receptor overactivation , a neurodegenerative illness where loss of dopaminergic neurons from the nigrostriatal system affects locomotion, chronic treatment with L-DOPA, a dopamine precursor, induces dyskinesias by Dtivation . These ativation . Using Pactivity , supportAs discussed above, data from the last 2 decades have shown that a single GPCR subtype can couple to different transduction proteins and produce multiple cellular responses. These observations have resulted in a new era of pharmacology where, relative to the endogenous hormone or neurotransmitter, ligands can selectively or at least preferentially activate one of the diverse responses produced by a single GPCR subtype. This phenomenon has been termed “ligand-directed trafficking of receptor stimulus” or “biased signaling.” In many cases, the therapeutic effect of a ligand is mediated by one pathway and the adverse effects by another pathway. Thus, rational development of biased ligands has become an active part of modern drug discovery.1 receptor ligands in congestive heart failure (CHF)  can be termed as universally beneficial or detrimental. The (patho)-physiological effects of any pathway will always be disease-specific, time-dependent, and indeed often cell-specific.To date, the diseases with the most clinical data in support of the development of biased drugs are: the use of ß-adrenergic receptors (ßAR) and angiotensin ATre (CHF) ; μ-opioin asthma . There iectively , sphingoectively , and ade disease , among o1 receptor  and the ß-arrestin-dependent pathway , and either stimulate, or at least not antagonize the receptor conformation that promotes ß-arrestin-dependent signaling. Perhaps the most compelling evidence for the advantage of this ligand profile as ideal is the observed therapeutic advantage of carvedilol in CHF (2AR (ß2ARTYY) where the mutation renders the ß2AR unable to bind G proteins; carvedilol was the only beta-blocker that retained the ability to activate ERK1/2  mediate pain relief through Gi/o activation, while ß-arrestin-dependent signaling induces respiratory depression and constipation . For exa2 receptor antagonist used to reduce gastric acid secretion in acid-peptide disorders, may have greater therapeutic efficacy compared to other H2 receptor antagonists  of the nanobody Nb80, and inhibit ß2AR-G protein coupling (Alternative or complementary ways to bias GPCR signaling include allosteric modulation of GPCR as well as the use of pepducins and/or nanobodies. Nanobodies, camelid antibody fragments, have been developed that preferentially bind to and stabilize the human ßrmations . Rasmussruitment . They idruitment . As nanocoupling .2AR, has been reported to be completely ß-arrestin–biased in primary adult murine cardiomyocytes, possibly enhancing cardioprotective effects for CHF therapy (s-biased ß2AR signaling, has been shown to be a potential asthma therapy candidate as ß-arrestins are believed to be responsible for the symptoms associated with asthma (i-biased pepducin, ATI-2341, has been developed for the chemokine receptor CXCR4 (in vivo is currently constrained because pepducins lack a targeting mechanism in multilayered tissues and thus are limited to cells in close proximity to the circulating pepducins (Another approach to biasing receptor signaling involves pepducins, first developed by  therapy . Intraceh asthma . Anotheror CXCR4 . It is nepducins . Howeverepducins .Given the functional versatility of ß-arrestins, they are aptly suited to effectively and broadly regulate cell signaling and resultant physiological and pathophysiological processes. The therapeutic potential of targeting ß-arrestins is enormous, since disease-specific treatments could increase the safety and efficacy of GPCR-targeted therapeutics. On the other hand, given that a single ß-arrestin subtype can modulate dozens of GPCRs, this may pose problems in drug discovery. The search for ß-arrestin modulators will not be easy given the complexity of GPCR signaling pathways and the pleiotropy of ß-arrestin functions making precise targeting of paramount importance. Despite these challenges, there are several ligands preferentially targeting ß-arrestin signaling now in clinical trials and more in development. Thus, we will hopefully soon have answers as to the impact of these ligands in future therapies.JW and RB contributed to the conceptualization of the subject matter. JW, RB, ELG-R, and AH reviewed the literature and wrote sections 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.


Letizia Mencarini e Daniele Vignoli, Genitori cercasi. L’Italia nella trappola demografica [Parents Wanted. Italy in the Demographic Trap], Milano: Egea; 2018. 149 pages, ISBN 978-88-8350-279-8 This well-written, clearly articulated, and documented book of Mencarini and Vignoli deals with one of the central questions of many contemporary populations: the “birth dearth,” fruit of a downward spiral of low fertility, declining number of potential parents, and declining number of births. The mechanisms of this negative spiral, or demographic trap, in the authors’ definition—1.035 million babies were born in Italy in 1964, more than double of the 0.458 million of 2017—are well known to the specialists, but remain obscure to the general public. This widespread ignorance is also the consequence of the failure of specialists to disseminate and communicate to the general public the results of their research, leaving this vital function in the hands of the media.In Chapter 1, the Italian demography is compared with that of the major European countries. Italy, with Spain, has the lowest fertility, the highest mean age of mothers at the birth of their children, the highest life expectancy, and the larger excess of deaths over births. Germany, that shared a demographic situation similar to that of Italy and Spain, has undergone an impressive recovery of fertility in the last few years. The demography of France and of the UK is, on the other hand, in a situation of equilibrium. Chapter 2 retraces the course of fertility after the postwar recovery until the 1960s, and the continuous decline from the 1970s to the turn of the century, the illusionary mini-recovery in the first decade of the new millennium, and the further decline during the Great Crisis. Much of the decline has been the consequence of a continuous postponement of the first birth and of the substantive increase of the mean age at childbearing. Fertility decline is smoother when cohorts are observed, with the number of children per woman falling to 1.6 for cohorts born around 1970. Childlessness, for the same cohorts, has increased to over 20%, often because of a conscious decision of mothers or of a prolonged postponement of the decision to have a first baby. In the last decade—in Italy, the crisis of the economy has been more severe than elsewhere—the age structure of women in reproductive age has changed as the numerous women born during the 1960s and 1970s have been replaced by the increasingly less numerous ones born in the 1980s and 1990s, when total fertility had fallen consistently below 1.5 (Chapter 3). The authors conclude that the postponement of births in the life cycle and the decline of the numbers of potential mothers have had a depressive effect on births larger than that determined by the crisis and by the worsening of the standard of living. So the “trap” has been closing up, low fertility has produced a declining number of potential mothers, and this downward trend is set to continue in the future, with a negative effect on the number of births, even if a recovery takes place as hypothesized by current population projections (Chapter 4). A recovery of fertility is in the realm of possibilities, also because surveys consistently show that Italian women, and men, desire or consider ideal a number of children around two, the largest gap between aspirations and realization in European countries (Chapter 5). But why Italian fertility is so low? . According to the authors, the traditional explanations based on the cost-benefit analysis of Beckerian inspiration, or on the “second demographic transition” paradigm, seem to lose strength in the “postmodern” societies of the new millennium (Chapter 6). Although delayed, the many transformations that have changed the traditional identity of the family in Europe since the 1970s are taking hold in Italy: more women at work, more unions and less marriages, an increasing number of children born outside the marriage, and more divorces and separations. But this transition has failed to be sustained by an adequate transformation and adaptation of the welfare system, and, above all, by an egalitarian revolution in gender roles, and more so inside the family than in society at large.Mencarini and Vignoli are not sanguine about the future, although they agree that a mix including a reform of the welfare system, efficient policies sustaining women in the labor market , immigration, and  more egalitarian gender relations might help the country to open the “demographic trap” in which is the prisoner. One factor equally important in determining the structural low fertility, and that the authors do not mention, is the long permanence of children in the family and the delay in which the full economic autonomy is reached by young people, and, as a consequence, the delay in taking crucial decisions—such as forming a stable union or having a child. After all, in a cost-benefit approach, the cost of a child that becomes autonomous at 20 is much lower than the cost of a child that leaves the parents’ house at 30!Policymakers’ attention rapidly decays the farther the horizon of a decision is. But, as Mencarini and Vignoli rightly say, before forming a decision and taking action, it is necessary to know the facts. Now, thanks to this book, the facts are clear and the cards are on the table. But will policymakers play the game?Massimo Livi Bacci26 XI 2018


Drosophila Makorin RING finger protein 1 gene (Mkrn1) functions in the metabolic regulation of oogenesis. Mkrn1 was endogenously expressed at high levels in ovaries and Mkrn1 knockout resulted in female sterility. Mkrn1-null egg chambers were previtellogenic without egg production. FLP-FRT mosaic analysis revealed that Mkrn1 is essential in germline cells, but not follicle cells, for ovarian function. As well, AKT phosphorylation via insulin signaling was greatly reduced in the germline cells, but not the follicle cells, of the mutant clones in the ovaries. Furthermore, protein-rich diet elevated Mkrn1 protein levels, without increased mRNA levels. The p-AKT and p-S6K levels, downstream targets of insulin/Tor signaling, were significantly increased by a nutrient-rich diet in wild-type ovaries whereas those were low in Mkrn1exS compared to wild-type ovaries. Taken together, our results suggest that nutrient availability upregulates the Mkrn1 protein, which acts as a positive regulator of insulin signaling to confer sensitivity and tissue specificity in the ovaries for proper oogenesis based on nutritional status.Reproduction is a process that is extremely sensitive to changes in nutritional status. The nutritional control of oogenesis via insulin signaling has been reported; however, the mechanism underlying its sensitivity and tissue specificity has not been elucidated. Here, we determined that  Reproduction is an energetically expensive process because it involves massive cell proliferation and growth that requires synthesis of various proteins. Because of this high-energy demand, reproduction is tightly regulated by nutrient availability to ensure that reproduction only happens when there is a surplus of nutrients. When nutrients are scarce, reproduction is compromised to preserve energy for survival. Reproductive pauses in response to starvation are common among species of the animal kingdom; this reproductive dormancy can be quickly reversed by nutritional status –3.Drosophila ovaries are composed of ovarioles that consist of sequentially developing egg chambers [Foxo gene [chambers . At the chambers , 6. The oxo gene . After toxo gene .InR) and insulin substrate protein 1 (chico) genetic mutation cause female sterility [in vivo, despite evidence that insulin is involved in androgen production, gonadotropin signaling, PCOS (Polycystic Ovary Syndrome), and obesity-induced infertility [The hormonal system of organisms relays nutritional information to organs. Insulin and insulin-like growth factor signaling is one of the most important hormonal systems for nutritional regulation. Insulin is released upon nutrient uptake and subsequently regulates processes that require energy, such as cellular growth, metabolism, and reproduction , 10. Femterility , 13. Alsterility . Insulinterility , 15. Howertility .InRE19, lnk4Q3/6S2, and chico1 cause reduction, but not complete abolition, of insulin signaling without exhibiting defects in vital organs; however, oogenesis is greatly compromised [Irs-2 in mice also causes female sterility with other major defects [Nutrient availability has been shown to delay or accelerate reproductive capabilities , 18. Thepromised , 13, 19. defects . Further defects . HoweverDrosophila, the ovaries require TOR activity for the proliferation and maintenance of germline cells [Drosophila, PRAS40, the inhibitor of TOR signaling pathway, affects the interaction of the insulin and TOR signaling only in ovaries [The target of rapamycin (TOR) signaling pathway is another highly conserved nutrient sensing signal that is required for cell growth and proliferation , 23. Whine cells . Hence, Drosophila, ecdysone, a steroid hormone that controls molting in insects, is required for reproductive processes. Developing egg chambers degenerate without ecdysone signaling [Drosophila [Steroid hormone signaling is also important for reproduction. In ignaling , 25. In osophila , 27. In osophila –30.Mkrn1 is a Drosophila homologue of mammalian makorin family and encodes RING zinc finger proteins with ubiquitin ligase activity. In mammals, Mkrn1 reportedly destabilizes many substrates including p53, p21, PPARγ, hTERT, PTEN, APC, and AMPK [Drosophila Mkrn1 is involved in the proper timing of the larval to pupal transition and affects final body size by regulating ecdysone synthesis in the prothoracic gland suggesting Mkrn1 plays a role in the transition from growth to maturation [Mkrn1 null mutant (Mkrn1exS) flies are sterile, and further demonstrated that Mkrn1 is required for Drosophila oogenesis. Furthermore, Mkrn1 was strongly expressed in ovaries and was upregulated by a protein-rich diet. Mkrn1 null female flies exhibited vitellogenesis failure, which also occurs when flies were starved or nutrient signaling was perturbed. Insulin/Tor signaling was greatly reduced in Mkrn1exS ovaries, implying a role as a positive regulator in the insulin/Tor signaling pathway. Collectively, our data support the notion that Mkrn1 functions as a tissue-specific regulator of the insulin/Tor signaling pathway to activate oogenesis in ovaries in a nutrient-sensitive manner.and AMPK –36, and turation . Here, wMkrn1 and Mkrn1exS mutant Drosophila strains were generated as previously described [Mkrn1 gene deletions—Df(3L)BSC418/TM6C (BDSC 24922) and Df(3L)BSC419/TM6C (BDSC 24923)—were used in this study. NRE-EGFP (BDSC 30727) was used to examine Notch signaling and PRAS40KO (BDSC 76339) was used to examine epistatic interactions with Mkrn1exS.The escribed . Two linDrosophila stocks were maintained on a standard cornmeal food at room temperature except for the experimental groups that required nutrient-poor and -rich diet conditions. The nutrient-poor diet contained 10% sucrose and 2% agar, whereas the nutrient-rich diet contained 10% sucrose, 2% agar, and yeast paste. Females were cultured for 1 day under different nutritional conditions less than 8 hours after eclosion.All FRT80B Mkrn1exS strain was generated by FRT80B and Mkrn1exS recombination followed by crossing with hsflp; FRT80B, ubi-GFP females. F1 generation, one-day-old, female flies were heat-shocked for 1 hr at 37°C twice a day for 3 consecutive days and kept in a 25°C incubator. Ovaries were dissected and subjected to immunostaining 10 days after the first shock.For FLP-FRT mosaic analysis, the Mkrn1-forward, 5’-GACGTGCGGCATCTGCTTTG-3’; Mkrn1-reverse, 5’-TGTTTGGCCTGACGCCATGT-3’; Phm-forward, 5’-GCTTGCATTTCCGAGACGAT-3’; Phm-reverse, 5’-ACGATCATCGAACCACCCTT-3’; E74-forward, 5’-CAAACCGAAGCTGGAGATGG-3’; and E74-reverse, 5’-TCGTCCACTTGATGAAACGC-3’. Cbp20 or actin mRNA levels were used to normalize gene expression levels using the following primer sequence: cbp20-forward, 5’-GTATAAGAAGACGCCCTGC-3’; cbp20-reverse, 5’-TTCACAAATCTCATGGCCG-3’; actin-forward, 5’-CATGTTTGAGACCTTCAACACCCC-3’; actin-reverse, 5’-GCCATCTCCTGCTCGAAGTCTAG-3’. Data were analyzed and quantified using Rotor Gene 6000 software.Adult female heads, thoraxes, abdomens, and ovaries were dissected. From these tissues, total RNA was isolated using QIAzol lysis reagent (QIAGEN) and reverse-transcribed with oligo-dT primers using Prime Script reverse transcriptase (TAKARA). Quantitative real-time PCR was performed using a Rotor Gene 6000 (QIAGEN) with SYBR Premix Ex Taq . The following primers were used: Drosophila Mkrn1 [Drosophila were prepared using lysis buffer  with freshly added 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, protease inhibitor (Sigma), 1 mM Na3VO4, and 0.25 mM NaF . For p-AKT and p-S6K analysis, phosphatase inhibitor cocktail 2 and 3 (Sigma) were used instead of Na3VO4 and NaF. The protein extracts were resolved by SDS-PAGE and the resulting blots were probed using the following primary antibodies: rabbit anti-Mkrn1, 1:3000 [We used our previously generated antibody against la Mkrn1 . Protein, 1:3000 ; rabbit , 1:3000 ; mouse aThe ovaries of female adult flies were dissected in PBS; and approximately twenty ovaries for each sample were analyzed by immunostaining. The ovaries were fixed in 4% paraformaldehyde and rinsed with 0.5% PBST solution (1× PBS containing 0.5% Triton X-100). The fixed ovaries were incubated for 1 hr in blocking solution comprised of 0.5% PBST and 10% horse serum. Primary antibodies were diluted in blocking solution and incubated with samples overnight at 4°C followed by washing with 0.5% PBST. The samples were subsequently incubated with secondary antibodies diluted in blocking solution overnight at 4°C. The ovaries were washed and stained with hoechst33342 , mounted on slides, and incubated with Phalloidin-TRITC  in PBS (2.5 ug/ml) for 30 min to stain the actin filaments. Confocal images were obtained with a LSM710 or LSM800 Microscope (Zeiss) and processed with Zen software (Zeiss). The following antibodies and final dilutions were used: rabbit anti-Mkrn1, 1:3000 ; goat anRapamycin (Sigma) was dissolved in ethanol and diluted to a final concentration of 1 μM in PBS. Ovaries were dissected from female files 6 to 8 hours after eclosion and incubated in PBS with or without rapamycin for 1 hour, or 2 hours. After incubation, the ovaries were immediately processed for western blot analysis.Mkrn1-null flies  are come glands . To dete abdomen , which iertility .Mkrn1exS flies were much smaller than control ovaries and did not contain mature eggs BSC418/Mkrn1exS and Df(3L)BSC419/Mkrn1exS, were sterile. Moreover, ovarian phenotypes and size were indistinguishable among the Df(3L)BSC418/Mkrn1exS, Df(3L)BSC419/Mkrn1exS, and Mkrn1exS homozygotes, confirming that this allele is the true null allele of Mkrn1 , and broad (br) during the ME switch. We introduced Notch reporter NRE-EGFP, in which Notch response element drives expression of EGFP, into control and Mkrn1exS flies. NRE-EGFP expression was stronger around stage 6. The onset pattern of NRE-EGFP expression was indistinguishable in control and Mkrn1exS flies  and ecdysoneless cell cycle regulator (ecd) mutants, which are similar to the Mkrn1exS phenotype [Mkrn1exS larvae [Mkrn1exS ovaries. In adults, ovaries can also produce ecdysone [phantom (phm), an enzyme required for ecdysone synthesis, and found its transcript levels to be slightly reduced in Mkrn1exS ovaries compared to controls  as a readout of insulin signaling pathway activation. p-AKT levels were measured by western blot analysis of ovarian protein extracts from one-day-old female flies to ensure that both control and mutant flies had similar sized ovaries at the same stages of oogenesis for proper molecular comparison. We found that AKT phosphorylation was greatly reduced in Mkrn1exS ovaries compared to control, indicating that insulin signaling is reduced in Mkrn1exS ovaries  in egg chambers harboring insulin and TOR signaling pathway mutant germline clones, such as InR hypomorph, chico1, and S6K mutants [InR, chico, S6K, and Mkrn1 mutant germline clones suggests that Mkrn1 has a regulatory role in insulin signaling in germline cells. Collectively, we think that germline cells might be protected from degeneration induced by a reduction in insulin signaling caused either by the Mkrn1 mutation or by other components of the insulin signaling pathway, but not the follicle cells.Clonal analysis of the ne cells , indicat mutants . The simMkrn1 expression is highly enriched in ovaries. This explains why Mkrn1exS flies do not exhibit alterations in body size while chico, lnk, and InR hypormorphs exhibit a reduction in body size in addition to the female-sterile phenotype. This implies that Mkrn1 is a tissue-specific modulator of the insulin signaling pathway. However, the significance of Mkrn1 enrichment in ovaries remains unclear. According to our current study, as well as others, we can infer that tissue-specific differences in the dynamics and strength of insulin signaling is required based on the organ for proper homeostasis. For example, in developing flies, brain growth is spared during nutrient restriction. This tissue-specific effect is achieved by strong Alk expression in the brain, which regulates the TOR and insulin signaling pathways [Unlike key components of the insulin signaling pathway, pathways . In adulpathways . Converspathways .Drosophila PRAS40 supports the tissue specific regulation of the nutrient signaling pathway [Mkrn1 is a nutrient-dependent, tissue-specific regulator of insulin/Tor signaling in the ovaries. Tissue-specific modulators of insulin signaling account for the different responses of organs in mammals as well. Prior studies have shown reduced insulin sensitivity in the ovaries of lean mice, and increased insulin sensitivity in the ovaries of obese mice compared to peripheral metabolic tissues. This could be due to enhanced regulation of insulin signaling by Irs-1 and Irs-2 in the ovaries compared to only Irs-1 regulation in the periphery [Irs-2 caused female infertility in mice [Ovary-specific factors or mechanisms are required to trigger events that are restricted to the ovaries by ubiquitous nutritional changes or by the genetic manipulation of insulin signaling. A prior study of  pathway . The auteriphery . To this in mice .Mkrn1exS flies. However, an energy-dependent role of AMPK would only be critical in follicle cells and dispensable in the germline cells of Drosophila ovaries [Mkrn1exS flies. Pten is a key regulator of the insulin signaling pathway and s destabilized by Mkrn1. Interestingly, Mkrn1 functions as a positive-feedback regulator of the PI3K/AKT signals in cervical cancer progression because it is stabilized by AKT-mediated phosphorylation and it destabilizes Pten, which leads to further activation of the insulin signaling pathway [Mkrn1exS flies could account for the decreased level of AKT phosphorylation. However, the absence of a reliable Drosophila Pten antibody and the lethal Pten mutation phenotype made it difficult for us to test this idea. Thus, whether or not Mkrn1 regulates the insulin signaling pathway through Pten or other target(s) during oogenesis remains to be determined.The question of how Mkrn1 regulates insulin signaling in response to nutritional status thus remains. The potential molecular function of Mkrn1 as a ubiquitin E3 ligase has been studied. The Mkrn1 substrates identified include p53, p21, PPARγ, hTERT, Pten, APC, and AMPK –36. Amon ovaries . This wo pathway . Hence, mkrn1 null mutant. For example, APC was recently identified as an Mkrn1 substrate and functions in WNT signaling [Drosophila, WNT signaling is required for stem cell maintenence [Drosophila ovaries, the increase in APC in Mkrn1exS would theoretically lead to the degradation of Armadillo, a Drosophila homologue of mammalian β-catenin. However, this was not the case in Mkrn1exS. Armadillo levels were increased in Mkrn1exS compared to controls Click here for additional data file.


Weissella and aerobic bacteria, while lower Pantoea and Enterobacter compared with the control material. The FT event promoted the development of Lactobacillus in silage microflora, inducing more intense lactic fermentation after an initial short lag. The aerobic bacteria were suppressed immediately after the onset of ensiling. Microbiomes of the two silages tended to be almost similar after 2 days of ensiling. However, a small number of aerobic bacteria tended to revitalize in the FT silage with prolonged ensiling time, indicated by apparent abundances of Acinetobacter and Pseudomonas at the end of ensiling. The results obtained here suggest that the FT event could promote the development of Lactobacillus during ensiling and the control of aerobe revitalization need to be concerned with silages made from the freeze-damaged forages.Freezing damages in forages represents a major economic loss to agriculture. This study was conducted to investigate the effects of freeze–thaw (FT) event on microbial community dynamics of red clover silage. Results showed that the FT-treated material displayed higher proportions of  In boreal and temperate regions, forage crops are frequently exposed to freezing temperature during autumn, spring, and mild winter. This leads to reduction in field productivity and, particularly, problem in forage utilization due to altered physical properties after thawing . Direct Trifolium pratense L.) is a leading legume forage widely cultivated in boreal and temperate regions. Compared to alfalfa (Medicago sativa L.), red clover is characterized by rapid spring establishment and superior performance on acid and wet soils , and harvested on November 12, 2017. The growth stage was at late bud to early bloom. The stubble was about 10 cm above ground level. After harvest, red clover was immediately transferred to laboratory and chopped to a theoretical length of 1∼2 cm with a forage chopper. The chopped red clover was equally divided into two groups after thoroughly mixing. The first group was untreated control. The second group was frozen in a commercial refrigerator (−20°C) for 2 h and then thawed for 1 h at room temperature (10∼16°C). This FT process was repeated three times. After that, these forages (approximately 500 g for each bag) were picked into thick polyethylene bags (200 μm thick), vacuumed, and stored in a light-blocking box at room temperature. CK is abbreviation of untreated control silage and FT is FT treated silage. These silos were opened after 1, 2, 4, 8, and 30 days of ensiling, respectively. There were three replicates for each treatment per day. After opening, fermentation quality, microbial population, and microbial community were determined.g, and the supernatant was reserved for organic acid  analysis. The quantification of organic acid was conducted using an Agilent 1260 HPLC system equipped with a refractive index detector . Nitrogen compounds including free amino acid (FAA-N), ammonia (NH3-N) and non-protein nitrogen (NPN) were measured in the extracts using the method presented by Approximately 35 g silage sample was extracted in 70 ml of deionized water at 4°C for 24 h to obtain the extract. The pH of the extract was measured with an electrode pH meter . The extract was centrifuged for 10 min at 10,000 × The plant tissue damage degree (TTD) was measured in the ensilage materials according to the method of –1 to 10–9 in sterilized water. The LAB was counted on de Man, Rogosa and Sharpe agar medium, incubated in an anaerobic incubator at 37°C for 3 days. Yeasts were enumerated on potato dextrose agar after aerobic incubation at 28°C for 2 days. The yeasts were distinguished from moulds and other bacteria by colony appearance and cell morphology. The aerobic bacteria were estimated by using nutrient agar after aerobic incubation at 30°C for 24 h.Ten grams of sample was blended with 90 mL of sterilized water and serially diluted from 10For the molecular analysis of the microbial communities, genomic DNA was extracted through the following steps: 10 g of silage sample was mixed with 90 mL of sterile 0.85% NaCl solution, and treated with a table concentrator at 120 r/m for 2 h. After filtering with carbasus, and the liquor was centrifuged at 10,000 r/m for 10 min at 4°C. The supernatant was discarded, and the deposit was suspended in 1 mL of sterile 0.85% NaCl solution. The liquor was centrifuged at 12,000 r/m for 10 min at 4°C, and the supernatant was discarded. The pellet was used for DNA extraction. Total DNA were extracted with TIANamp Bacteria DNA isolation kit. The agarose gel electrophoresis and Nano Drop 2000 118 UV-vis spectrophotometer  were used to check the quality of DNA after extraction.Approximately 10 ng of DNA isolated from each sample was used for amplification. The primers were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT- 3′) targeting the V3-V4 regions of 16S rRNA genes.The PCR reaction system consisted of 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mmol/L dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. These reactions were performed by thermocycler PCR system  under the following condition: a prior denaturation at 95°C for 30 min, followed by 27 cycles of denaturation 30s at 95°C, annealing at 55°C for 30 s, elongation at 75°C for 45 s, and a final extension at 72°C for 10 min. The PCR products were extracted by 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit . To reduce PCR deviation, PCR reaction for one treatment was performed in triplicate.1). The RDP Classifier (version 2.22) in the Silva (Release1283) database was applied to perform sequence-level taxonomic assignment using confidence threshold of 70%. The non-metric multi-dimensional scaling (NMDS) was conducted using the Vegan software based on the unweighted uniFrac distance of genus.The DNA samples were sequenced with an Illumina MiSeq 133 PE300 platform . To control sequencing quality, sequences with scores lower than 20 were discarded based on the QIIME quality control process (version 1.7.0). The operational taxonomic units (OTUs) at 97% similarity level were clustered using Usearch  factorial arrangement of treatments by using the SPSS 19.0. Duncan’s multiple comparison was used for the means separation. Significant differences were declared when P < 0.05) in FAA-N and NPN concentrations and TDD in the fresh material. The numbers of all enumerated microbes, particularly aerobic bacteria, increased (P < 0.05) in FT material compared with those in CK material.The characteristics of red clover forages upon ensiling are shown in P < 0.05) LAB number was observed in FT material compared with CK material, the FT event seemed to result in a transitory lag in the fermentation process during the initial 1 day of ensiling, as indicated by higher (P < 0.05) pH and lower (P < 0.05) lactic acid concentrations in FT silage compared with those in CK silage at day 1. After 1 day of ensiling, more intense lactic fermentation was observed in FT silage, as indicated by lower (P < 0.05) pH and higher (P < 0.05) lactic acid concentration. At the end of ensiling, the lower (P < 0.05) pH and higher (P < 0.05) lactic acid concentrations indicated better fermentation quality of FT silage compared with CK silage.As presented in Approximately 1,837,106 valid sequences were obtained after removing unqualified sequences, with an average length of 449 bp per sequence for bacteria. These reads were clustered into 361 OTUs at 97% sequence identity. The rarefaction curve  and GoodThe dynamic variance of bacterial community of the two silages during ensiling was observed by NMDS analysis. As shown in Firmicutes, Proteobacteria, and Cyanobacteria) were detected at high abundance in fresh red clover  were detected abundant in the FT material.Three phyla  until the end of ensiling; the relative abundance of Proteobacteria phylum decreased sharply to <12.7%. Relative to CK silage, the FT event increased the relative abundance of Firmicutes and decreased the abundance of Proteobacteria at day 1 of ensiling, while the bacterial community structures of the two silages became similar after 2 days of ensiling. Phyla Actinobacteria and Bacteroidetes in the FT silage decreased to undetectable level after the onset of ensiling. However, their relative abundances became apparent again at the end of ensiling.After the beginning of ensiling, Pantoea (63.8%), followed by Enterobacter (11.3%), Weissella (9.72%) and Pseudomonas (7.04%), and Lactococcus (3.08%) and Pediococcus (2.25%). Compared with CK material, the abundant presence of various aerobic bacteria, such as Pseudomonas (14.1%), Cyanobacteria (6.40%), Rhizobium (2.86%), Acinetobacter (2.85%), Comamonas (1.93%), Sphingomonas (1.67%), Methylobacterium (1.24%) as well as some unclassified genera (11.9%), was found in FT material. Furthermore, it was observed that the relative proportions of genera Pantoea (26.6% vs. 63.8%) and Enterobacter (4.71% vs. 11.3%) deceased in the material microflora, whereas genera Lactobacillus (1.14% vs. undetectable level) and Weissella (20.3% vs. 9.72%) increased.To further understand the effects of FT event on microbial community dynamics during ensiling, bacterial community structures of the two silages were examined at genus level . The mosWeissella proliferated quickly, reaching to 65.7% of the bacterial community at day 1. However, its abundance decreased thereafter with prolonged ensiling time. The relative abundance of Pediococcus increased after the onset of ensiling and peaked (26.4%) at day 4 of ensiling. Along with the changes in Weissella and Pediococcus abundances, the relative abundance of Lactobacillus increased and became dominant after 4 days of ensiling. After the onset of ensiling, the all aerobic bacteria quickly decreased to undetectable levels. With respect to the Pantoea, they also dropped to marginal levels in the bacterial community. However, members of Enterobacter were still readily apparent throughout ensiling. Compared with CK silage, the FT event reduced Enterobacter (3.29% vs. 9.41%) at the day 1 of ensiling. In addition, it was observed that the relative proportions of Lactobacillus were always higher in FT silage compared with CK silage before 4 days of ensiling.After the onset of ensiling, Acinetobacter (2.74%) and Pseudomonas (1.65%) became apparent again in FT silage at the end of ensiling.With prolonged ensiling time, microbiomes of the two silages were mostly constituted by LAB species and tended to be almost similar after 2 days of ensiling. However, genera In practice, the FT event frequently occur before harvest or during silage making, leading to physical damages and vigorous growth of various bacteria in forage crops. It is known that microbial communities originally present on ensilage material play an important role in fermentation quality . This meAs indicators of protein hydrolysis , the incDespite the FT procedures were conducted under conditions where as possible as to avoid soil and water contamination, the numbers of all enumerated microbes, particularly aerobic bacteria, and increased significantly in the FT material compared with the CK material upon ensiling. The increased microbial counts were assumed to link the increased nutrients release that favored proliferation of indigenous microbes and likely some airborne bacteria .Legume forages are generally regarded as difficult to ensile because of high buffering capacity and low sugar content . ClostriUnder anaerobic conditions, LAB ferment soluble carbohydrates to organic acids, mainly lactic acid, results in decline in silage pH . The fasBacteria present in fresh forage crop and silage can be simply classified into three groups based on their contributions to silage quality and abilities to thrive in anerobic environment: LAB, which are desirable bacteria contributing to the production of lactic acid and pH decline; undesirable facultative aerobes, which compete with LAB for nutrients during ensiling and contribute little to the pH decline; strict aerobes, which are also undesirable bacteria whereas remain active only before oxygen is depleted in silo .Proteobacteria and Firmicute were two most abundant phyla in fresh red clover, comprising 99.2% of the microflora. After the FT event, the relative abundance of Firmicute increased from 16.2 to 28.2%. The increased Firmicute was attributed to increased abundance of Weissella, which belong to Firmicute. Weissella, an obligatory heterofermentative LAB species, are major components of the microflora in various types of forage crops  and Pseudomonas (1.65%) became apparent again in FT silage at the end of ensiling. Acinetobacter and Pseudomona are aerobic, non-fermenting bacteria, which can be found in different environments .TS designed the experiments. JL, LC, SW, and ZD conducted the experiments. ZD analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thus, we found that the non-phenolic lignin can react with chlorine dioxide to form AOX more easily than phenolic lignin.Adsorbable organic halogen (AOX) is generally formed by the reaction of residual lignin in pulps with chlorine dioxide during bleaching. Lignin has a complex structure. Different functional groups and bonds are present in lignin structures. Phenolic hydroxyl is one of the important functional groups in lignin, and it significantly influences the chemical properties and reactivity. To study the effect of phenolic hydroxyl on AOX formation, vanillyl alcohol (VA) was selected as the phenolic lignin model compound, and veratryl alcohol (VE) was selected as the non-phenolic lignin model compound in this study. The kinetics of AOX formation by the reaction of VA or VE with chlorine dioxide was studied. The effects of pH, chlorine dioxide, lignin model compound concentration and reaction temperature on AOX formation are discussed. The activation energies of the reaction of VA and VE with chlorine dioxide are 16 242.47 J mol AOX cannot be easily degraded; thus, it can remain in the environment for a long time. AOX has adverse effects on plant growth and animal endocrine systems  is the concentration of H+ (mol l−1), [ClO2] is the concentration of ClO2 (mol l−1), [C] is the concentration of VA or VE (mol l−1), a, b and c are the reaction orders of H+, ClO2 and the lignin model compound, respectively.The chlorine dioxide dose, lignin model compound concentration, pH and reaction temperature have considerable influence on AOX formation during pulp bleaching. To assess the effect of different factors on the AOX formation rate, a kinetic model of AOX formation by using a lignin model substance and reacting it with chlorine dioxide was established by referring to the previous studies ,28,35. TEquation (3.2) was derived from the logarithm of equation (3.1):3.2.−1 and a concentration of ClO2 of 124.00 mmol l−1. The amount was enough for sufficient reaction progress for VA or VE, which was similar to that during an actual chlorine dioxide bleaching stage. The pH was 2.00, 2.50, 3.00, 3.50 or 4.00.The effects of pH on AOX formation upon the reaction of VA/VE with chlorine dioxide are shown in a). AOX primarily formed within 10 min, which indicated that VA can easily react with chlorine dioxide under acidic conditions. The curve of AOX formation gradually flattened as the reaction proceeded owing to the consumption and decomposition of chlorine dioxide and the oxidation and degradation of VA.For the reaction of VA with chlorine dioxide, the AOX formation increased as the pH increased a. AOX prb). The AOX formation rate obviously varied within 10 min, which suggested that AOX can be easily formed by reacting VE with chlorine dioxide at low pH. Owing to the reactions that occurred under acidic conditions, ClO2 converted to HClO in the chlorine dioxide solution at lower pH. In addition, HClO further converted to Cl2 as the pH decreased below 2.00 [2 with residual lignin during chlorine dioxide bleaching. Therefore, there was a considerable amount of HClO and Cl2 that were converted from ClO2 in the chlorine dioxide solution at the beginning of the reactions under low pH conditions. A considerable amount of AOX was formed by the reaction of VE with HClO and Cl2. Meanwhile, HClO and Cl2 were rapidly consumed and ClO2 was gradually decomposed. The curve for AOX formation was thus stable.For the reaction of VE with chlorine dioxide, AOX formation was higher at lower pH values b. The AOlow 2.00 . AOX wasW/dt) and log(H+) was determined by the linear regression analysis. The regression equations for log(dW/dt) − log(H+) are shown in a was thus determined. For the reaction of VA with chlorine dioxide, the slopes of the three fitting equations were 0.07, 0.07 and 0.06. Parameter a1 in the kinetic model of the reaction of VA with chlorine dioxide to form AOX was determined by averaging these three values; thus, a1 was determined to be 0.07. For the reaction of VE with chlorine dioxide, the slopes of the three fitting equations were 0.06, 0.06, and 0.06. Parameter a2 in the kinetic model of the reaction of VE with chlorine dioxide to form AOX was determined by averaging these three values; thus, a2 was determined to be 0.06. The reaction orders of the proton are 0.07 and 0.06. The values show that the acidities of the reaction solutions did not affect AOX formation. However, the activity of chlorine dioxide was affected by acidity .ClO in chlorine dioxide solution. It was converted to HClO and Cl2 when the value of pH was low. As a consequence, the AOX formation increased as pH value decreased. This corresponds to the results shown in Other conditions were maintained, when only the pH was changed. Equation (3.3) can be obtained by rearranging equation (3.2):3.3.−1 and a concentration of ClO2 of 24.80, 49.60, 73.40, 99.20 or 124.00 mmol l−1.The effects of chlorine dioxide dose on AOX formation upon the reactions of VA/VE with chlorine dioxide are shown in 2, as shown in figure 2a. As the concentration of ClO2 increased from 24.80 to 49.60 mmol l−1, the AOX formation increased by approximately three times. This indicated that the dosage of chlorine dioxide is an important factor that affects AOX formation. The curve of AOX formation is relatively smooth under low ClO2 concentration conditions owing to the quantities of HClO and Cl2 being much smaller in the chlorine dioxide solution. Within 5 min of the reaction of VA with chlorine dioxide, HClO and Cl2 reacted with VA rapidly to form AOX, and they were consumed completely. The rate of AOX formation changed considerably with the increase in chlorine dioxide dosage after 5 min of reaction time.The AOX formed by reacting VA with chlorine dioxide significantly increased owing to the increase in the concentration of ClOb). The AOX formation rate of the reaction of VE with chlorine dioxide was faster than that of VA with chlorine dioxide. With an increase in the concentration of ClO2, the AOX formation rate was promoted. AOX was primarily formed within 5 min.For the reaction of VE with chlorine dioxide, the AOX formation significantly increased as the concentration of chlorine dioxide increased b. The AO2 was changed. Equation (3.4) can be obtained by rearranging equation (3.2):W/dt) − log(ClO2) were determined by linear fitting of the data (b was determined by the slope of the regression equations of log(dW/dt) − log (ClO2). For the reaction of VA with chlorine dioxide, the slopes of the three fitting equations were 0.94, 0.94 and 0.93. Parameter b1 in the kinetic model of the reaction of VA with chlorine dioxide to form AOX was determined by averaging these three values, and it was b1 = 0.94. For the reaction of VE with chlorine dioxide, the slopes of the three fitting equations were 0.87, 0.85 and 0.82. Parameter b2 in the kinetic model for the reaction of VE with chlorine dioxide to form AOX was determined by averaging these three values, and it was b2 = 0.85.Other conditions were maintained, and only the concentration of ClOthe data . Paramet3.4.2 of 124.00 mmol l−1 and a concentration of VA or VE of 31.00, 46.50, 62.00, 77.50 or 93.00 mmol l−1.The effects of the concentration of VA or VE on AOX formation by the reaction of VA/VE with chlorine dioxide are shown in a, for the reaction of VA with chlorine dioxide, the AOX formation rate was slower when the concentration of VA was below 62.00 mmol l−1 for the first 10 min, and it increased with increasing VA concentration. This suggested that the concentration of VA could significantly affect the AOX formation. For the reaction of VE with chlorine dioxide, the concentration of VE also had a significant effect on the AOX formation rate. The AOX formation rate was remarkably higher during the first 2 min. Thus, the rate and content of AOX formed by reacting VE with chlorine dioxide were higher than those of VA at the same concentration for the lignin model compound.As shown in figure 3W/dt) − log(C) were determined by linear regression analysis of the experimental results (c was determined by the slopes of the log(dW/dt) − log(C) regression equations. For the reaction of VA with chlorine dioxide, the slopes of the three fitting equations were 0.33, 0.35 and 0.39. Parameter c1 in the kinetic model of the reaction of VA with chlorine dioxide to form AOX was determined by averaging these three values, and c1 = 0.36. For the reaction of VE with chlorine dioxide, the slopes of the three fitting equations were 0.30, 0.30 and 0.29. Parameter c2 in the kinetic model of the reaction of VE with chlorine dioxide to form AOX was determined by averaging these three values, and c2 = 0.30.Other conditions were maintained, and only the concentration of VA or VE was changed. Equation (3.5) could be obtained by rearranging equation (3.2): results . Paramet3.5.−1, a concentration of VA or VE of 62.00 mmol l−1 and a reaction temperature of 323, 328, 333, 338 or 343 K.The effects of reaction temperature on AOX formation by the reaction of VA/VE with chlorine dioxide are shown in k, can be obtained:According to the Arrhenius equation, the reaction rate constant, a1, b1 and c1 and the concentrations of H+, ClO2 and VA were included in equation (3.8) to calculate log (k). Linear fitting equations for log(k) − 1/T were obtained by linear regression analysis of the experimental results (A1) = 4.87. The pre-exponential factor was obtained; A1 = 73 824.40. The slopes of the linear regression equations were −899.55, −849.89 and −795.92. The average of the slopes was −848.45. The reaction activation energy of VA with chlorine dioxide was thus determined to be E1 = 16 242.47 J mol−1, which indicated that the activation energy of the reaction of VA with chlorine dioxide was small, and AOX was quickly formed.Equation (3.8) was derived from equations (3.2) and (3.7): results . For the+, ClO2 and VA are not integers, which indicates that the AOX formation from the reaction of VA with chlorine dioxide is a complex multivariate reaction. The reaction order of H+ is significantly small; thus, H+ may play a catalytic role in the reaction.The kinetic model of AOX formation from the reaction of VA with chlorine dioxide is shown asa2, b2 and c2 and the concentrations of H+, ClO2 and VE were included in equation (3.8) to calculate the log (k). For the reaction of VE with chlorine dioxide, the average intercept of the linear regression equations was 3.01, log (A2) = 3.01. The pre-exponential factor was determined to be A2 = 1012.05. The slopes of the linear regression equations were −14.70, −14.54 and −14.37, respectively. The average of the slopes was −14.54. The reaction activation energy of VE with chlorine dioxide was determined to be E2 = 281.34 J mol−1, which is significantly lower than the reaction activation energy of VA with chlorine dioxide, which indicated that VE can easily react with chlorine dioxide to form AOX. This was because the hydrogen atoms on the benzene ring of VE were prone to be replaced by chlorine atoms to form AOX. However, oxidation and degradation of benzene ring were the dominant reactions when chlorine dioxide reacted with VA.Parameters + also had little effect on AOX formation, whereas the concentration of ClO2 significantly affected the AOX formation. In addition, the effect of the concentration of VE on AOX formation was more remarkable than that of VA, which suggested that in an actual bleaching process, increasing the proportion of non-phenolic structures in the lignin may promote AOX formation during chlorine dioxide bleaching.The kinetic model of AOX formation by the reaction of VE with chlorine dioxide is shown in3.6.R2 of the fitting equation was 0.94, which indicated that the kinetic model was in a good agreement with the experimental results. For the reaction of VE with chlorine dioxide, the relationship between the predicted content of AOX formation and the experimental data is shown in R2 of the fitting equation was 0.93, and the kinetic model was able to effectively predict AOX formation.The predicted AOX formation content from the reaction of lignin model compounds with chlorine dioxide could be calculated by the established kinetic model. For the reaction of VA with chlorine dioxide, the relationship between the predicted content of AOX formation and the experimental data is shown in 4.2, pH, temperature and lignin model compound concentration on the AOX formation rate were studied. A kinetic model for AOX formation upon the reaction of VA/VE with chlorine dioxide was established. The activation energies for the reaction of VA or VE with chlorine dioxide were obtained. The results showed that the reaction activation energy for VE with chlorine dioxide was significantly lower than that of VA with chlorine dioxide, which indicated that the non-phenolic lignin model compound can react with chlorine dioxide to form AOX more easily than the phenolic lignin model compound.The effects of ClO


We present a rare case of crusted scabies in an human T‐cell lymphotropic virus type 1 (HTLV‐1) infected woman prior to onset of adult T‐cell leukemia/lymphoma (ATL). We highlight the importance of this rare form of scabies as a prediagnostic sign of ATL, requiring high suspicion and monitoring of possible symptoms for early detection of ATL. Further hematological investigation was carried out with bone marrow biopsy and computed tomography scan, showing intraabdominal lymphadenopathy that led to the diagnosis of ATL associated with HTLV‐1 infection. Hepatitis C also recurred.A recurrence of crusted skin lesions was observed, this time on the scalp and ears. She received antiviral therapy with zidovudine, acyclovir, and alpha‐interferon against HTLV‐1. Subsequently, cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy considerably improved the symptoms. Skin lesions cleared again with topical permethrin 5% plus oral ivermectin. One year after treatment, the patient is in good health.3Brazil has one of the highest prevalence rates of scabies worldwide. Regarding HTLV‐1, 2.5 million Brazilians are estimated to be infected.Crusted scabies is strongly associated with HTLV‐1 virus, and recent studies show that this infestation is also a predictive marker for ATL. In a study in French Guiana, an HTLV‐1 endemic area, four of six HTLV‐1 seropositive patients had concomitant ATL when CS was diagnosed, or developed ATL a few months later, suggesting that the occurrence of CS in these patients is a sign of ATL‐related immunosuppressionStrongyloides stercoralis intestinal hyperinfection, or its skin form of larva currens (migrans), is common and always localized; therefore, when disseminated, it may be associated with HTLV‐1.Human T‐cell lymphotropic virus was discovered only in 1980, when it was isolated from a patient with lymphomaFor dermatologists, a chronic, unresponsive, recurrent, severe, exudative, foul‐smelling atopic eczema with crusting should raise suspicion. HTLV‐1 tests are required in resistant forms of childhood eczema. In cases of unresponsive seborrheic dermatitis with chronic nasal secretion and presence of opportunistic infections,This case supports previous but scarce reports in the literature demonstrating this rare form of scabies as a prediagnostic sign of ATL, requiring a high degree of suspicion of HTLV coinfection and careful monitoring of possible symptoms in order to detect the early development of ATL.None declared.CB is the principal investigator, provided medical care, made the diagnosis and treated the patient. This author conceived the study and takes overall responsibility for the integrity of the content of the manuscript. PB conducted a critical review of the literature, critically revised the manuscript, and supervised the study. MSW assisted in the design of the study, conducted a literature search and wrote the first draft of the report, revised the manuscript for important intellectual content and wrote the discussion. All authors read and approved the final manuscript.


However, most neurotrophic factors also have important functions outside of the nervous system. Especially essential renal growth and differentiation regulators are glial cell line-derived neurotrophic factor (GDNF), bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs). Here we discuss how trophic factor-induced signaling contributes to the control of ureteric bud (UB) branching morphogenesis and to maintenance and differentiation of nephrogenic mesenchyme in embryonic kidney. The review includes recent advances in trophic factor functions during the guidance of branching morphogenesis and self-renewal versus differentiation decisions, both of which dictate the control of kidney size and nephron number. Creative utilization of current information may help better recapitulate renal differentiation  Trophic factors, also known as neurotrophic factors, have important functions outside of the nervous system , especiaNephron endowment is established during the embryonic period of an individual and critically dictates renal health and function in adults; hence, a detailed understanding of the signals and events regulating renal differentiation is a necessity. Our current understanding of mammalian kidney development derives from experiments in mice, and to a lesser extent in rats and zebrafish . The greKidneys, like lungs, mammary and salivary glands, prostate, and seminal vesicles develop through branching morphogenesis. The characteristic growth and shape of these organs is achieved via novel branch generation that is highly specific for each organ. Thus, the pattern of epithelial branching during the differentiation of lungs is very different from that in kidneys and is largely dictated by the signals derived from the surrounding cells in the nascent mesenchymal compartment .Renal development in mammals is a multistage process during which three spatially and temporally distinct kidneys, the pronephros, mesonephros, and metanephros are formed Figure . These aThe definitive kidney, the metanephros, begins forming when metanephric mesenchyme (MM) cells induce outgrowth of the UB from the nephric duct at E10.5 in mice (weeks 4–5 of gestation in humans). Subsequent renal morphogenesis is guided by reciprocal inductive interactions between the MM and the UB. After the initial UB outgrowth, it secretes signals that induce the MM to condense around the growing UB tip, forming the cap mesenchyme (CM). This is the beginning of CM-guided UB branching morphogenesis that happens rapidly until mid-gestation and slows down after E15.5 .UB branching is reiterated for multiple rounds to form the entirety of the collecting duct system . ThroughNephrons are derived from a multipotent, self-renewing progenitor population of CM cells wrapping around the UB tip . The sizin utero (by week 36), while in mice it lasts until post-natal day three (P3) (Tsc1) as a possible regulator of progenitor aging  . The mecor aging .in vivo and studies of organ explants suggest that the main arterial network in the kidney develops independently of the glomerular vasculature. Initially, OSR1-positive precursors give rise to glomerular vascular progenitors, while at later stages in development, FLK1-positive vascular progenitors are present in the UB tip niche  (n humans  and laten humans  as an orn humans . GDNF ren humans . Additio and 51) .Ret or Gfrα1 demonstrated that signaling through GDNF-RET-GFRα1 is critical for kidney development, as these mice also present renal agenesis or severe renal hypoplasia  receptors Figure  to regulTight regulation of FGF signaling, or RTK in general, takes place at many levels, and occurs both extra- and intracellularly. It involves heparin/heparin sulfate proteoglygans (HSPG), non-TK FGFR (FGFL1), FRS2α, and Sprouty proteins . HeparinFgfr1 and -2 IIIb splice variants while their ligands are usually expressed by the mesenchyme  RTKs and nine Eph-receptor-interacting proteins (ephrins) comprises the largest RTK family in mammals . Based oRegulation of vasculature development section.Activation of Eph/ephrin signaling cascades occurs exclusively upon direct contact of juxtaposed cells. Forward signaling in Eph-expressing cells triggers a large number of signal-mediating, kinase-dependent or -independent pathways, while reverse signaling in the ephrin-expressing cell causes focal adhesion kinase activation, cytoskeletal changes, and/or transcriptional responses . The celThe TGFβ superfamily of secreted proteins contains at least 30 members and includes Activins, Nodals, BMPs, and Growth and Differentiation Factors (GDFs). Due to its large presentation in living organisms, it also has major functions in many diverse contexts, including developmental, homeostatic, and disease processes where members of this signaling family control basically all cell biology aspects . For theTGFβ2, similarly, to its family members, is synthesized as a precursor and forms homodimers to activate a heteromeric receptor complex composed of type I or II transmembrane serine/threonine kinase receptors . RegardlThe intracellular mediators, the SMAD proteins, are shared in TGFβ and BMP pathways and include both receptor-regulated SMADs  and common mediator SMADs . The function of SMAD2/3 is negatively regulated by inhibitory SMADs (SMAD6 and -7), which block their interaction with the receptors. Also SMAD-independent signaling, mainly through small guanosine triphosphatases , MAPK, and PI3K pathways, may mediate TGFβ response in context-dependent situations .GDNF signaling via Ret and GFRα1 is critical for kidney development and underscores the relevance of reciprocal signaling between the UB and the MM in coordinating the balance of branching and nephrogenesis  of the UB, and Ret-expressing cells give rise to the entire collecting duct system  and another actin depolymerizing factor Destrin (Dstn) demonstrated that organization of epithelial cell cytoskeleton is important for new branch formation  mouse models with Ret and Etv4/Etv5 mutant cells demonstrated that GDNF signaling at the UB tip is required for tip cells to remain at the tip. Therefore, loss of  the tip . These d the tip . These sal cells . It remaFGF signaling The earliest events in ureteric bud formation and outgrowth are well-known to depend on GDNF signaling , hallmarking the major event in nephron differentiation in rats . The speated NPs , 2015.FGF signaling has become evidently one of the major regulatory pathway in NP maintenance and differentiation. Pax3Cre-mediated simultaneous loss of Fgfr1 and -2 results in the failure to establish proper MM and thus, supports FGFs essential function in the formation of the initial NP pool  and thus highlighting the importance of FGF signaling in establishing nephrogenic potential. On the other hand, +/-; Fgf20-/-Fgf9  compound mutants show premature NP differentiation and greatly diminished total nephron number suggesting that FGF signaling activated by these ligands is needed to maintain undifferentiated status in progenitors. Also, studies with some of the FGF signaling regulators support its role in maintaining the NP population while additionally reveal control of cellular processes such survival and proliferation  may have an important role in the arrangement of the renal vasculature. It has been shown that peritubular capillary development is mediated by angiopoietin-2, which antagonizes Ang1-dependent Tie2 signaling by the endothelia to promote VMC differentiation . Vasculaigration . It is pigration , and theigration .in vivo and traditional in vitro experiments have demonstrated that RAS/MAPK, PI3K/AKT, and PLCγ cascades are essential for UB morphogenesis s Figure . MAPK sis Figure . Very brg kidney .RAS-GDP is activated by various mitogens or growth factors to become RAS-GTP which has many downstream pathways, including RAF-MEK-ERK . RAS-GTPin vitro kidney cultures, which showed that inhibition of MEK proteins results in decreased tip cell proliferation and abnormal branching morphogenesis  as secondary messengers. The class I PI3Ks mediate trophic factor-induced RTK and G protein-coupled receptor signaling, and through PIP3 recruit and activate, e.g., PI3K-dependent kinase-1 (PDK1), AKT, and small GTPases ( GTPases . AKT is  GTPases . Activat GTPases . Elevate GTPases .in vivo mutagenesis of Ret Y1062 docking site, PI3K is also required for normal UB branching (Etv4 and -5, requires normal PI3K activation but does not, at least at the mRNA level, depend on MAPK/ERK activity (Pten, an antagonist of PI3K activity, suggests that the UB branching pattern is shaped by PI3K activation (Based on chemical inhibition and ranching . The essactivity . FGF-stiactivity . Though tivation .Of the other intracellular cascades activated downstream of RTK signaling, PLCγ and SRC pathways appear important for UB morphogenesis. Isoform specific mutations in tyrosine 1015 activating PLCγ causes renal abnormalities related to UB branching, but only in the context of the RET51 isoform . ChemicaSMAD pathway Without activated TGFβ superfamily signaling, the SMAD proteins, the intracellular mediators of this pathway, are shuttling constantly between the cytoplasm and nucleus ( nucleus . Upon th nucleus . Additio nucleus .in vitro culture of NPs supports the requirement for BMP and Rho kinase pathways as the addition of BMP7 and ROCK inhibitor together with WNT activation and FGF supply enables their maintenance (Several SMAD proteins localize to NPs while being downregulated in the induced nephron precursors . This suntenance .RTK activated intracellular pathways WNT/β-catenin signaling is the major driver that pushes NPs to differentiate (in vitro cultured kidneys (rentiate . Additiorentiate . The com kidneys ,b. It sh kidneys . We showSince the identification of the first trophic factors in the 1970s, remarkable progress has been made toward identifying additional members of this large group of signaling molecules and understanding the varied mechanisms by which they exert their trophic roles. These mechanisms often present redundancy and/or synergism in a tissue- and time-dependent manner. Two somewhat overlapping lines of research are now providing a stepping stone to better understand the role of these trophic factors in kidney development. On the one hand, there has been a significant effort from the McMahon lab and others to characterize human kidney development not only at an anatomical level but also at the molecular level using single-cell sequencing . ComplemAll authors contributed equally to the writing of this review 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.