CPX-351 exploits the gut microbiota to promote mucosal barrier function, colonization resistance, and immune homeostasis.

ABSTRACT: CPX-351, a liposomal combination of cytarabine plus daunorubicin, has been approved for the treatment of adults with newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes, because it improves survival and outcome of patients who received hematopoietic stem cell transplant compared with the continuous infusion of cytarabine plus daunorubicin (referred to as "7 + 3" combination). Because gut dysbiosis occurring in patients with AML during induction chemotherapy heavily affects the subsequent phases of therapy, we have assessed whether the superior activity of CPX-351 vs "7 + 3" combination in the real-life setting implicates an action on and by the intestinal microbiota. To this purpose, we have evaluated the impact of CPX-351 and "7 + 3" combination on mucosal barrier function, gut microbial composition and function, and antifungal colonization resistance in preclinical models of intestinal damage in vitro and in vivo and fecal microbiota transplantation. We found that CPX-351, at variance with "7 + 3" combination, protected from gut dysbiosis, mucosal damage, and gut morbidity while increasing antifungal resistance. Mechanistically, the protective effect of CPX-351 occurred through pathways involving both the host and the intestinal microbiota, namely via the activation of the aryl hydrocarbon receptor-interleukin-22 (IL-22)-IL-10 host pathway and the production of immunomodulatory metabolites by anaerobes. This study reveals how the gut microbiota may contribute to the good safety profile, with a low infection-related mortality, of CPX-351 and highlights how a better understanding of the host-microbiota dialogue may contribute to pave the way for precision medicine in AML.

Inflammation, mitochondrial dysfunction and physical performance: a possible association in older patients with persistent atrial fibrillation-the results of a preliminary study.

BACKGROUND: Atrial fibrillation (AF) is associated with chronic inflammation, a hallmark of ageing process. The aim of this study was to determine interleukin-6 (IL-6)-associated variables, also exploring acylcarnitines, expression of mitochondrial abnormalities. METHODS: We evaluated 22 controls and 50 patients with persistent AF. IL-6 and acylcarnitines were measured with ELISA kits and mass spectrometry techniques. RESULTS: IL-6 concentration (mean: 3.9 ± 3.1 pg/mL) was lower in controls and increased in AF patients, especially with heart failure. The CHA2DS2-VASc, the MMSE and the SPPB scores were 3.8 ± 1.6, 28 ± 2 and 9.4 ± 2.1. Thirteen acylcanitines correlated with IL-6. At multivariable analysis, IL-6 was directly associated with C4-OH-a short-chain acylcarnitine, fibrinogen and alanine aminotransferase values, and with hyperuricemia. An inverse association existed with calcium concentration and SPPB score. CONCLUSIONS: In older AF patients, IL-6 correlated with acylcarnitines and lower physical performance. Alterations in energy production, reduced physical function and inflammation could contribute to frailty development.

Steryl ferulates composition in twenty-two millet samples: Do "microwave popping" and fermentation affect their content?

Climate change has led to rediscovery of minor drought tolerant grains such as millet. Among its bioactive molecules, steryl ferulates have been poorly explored. Steryl ferulates composition of was investigated by high performance liquid chromatography-diode array detector-tandem mass spectrometry and high resolution tandem mass spectrometry in twenty-two millet samples and also in some fermented and microwave heated samples. Six compounds were found in Panicum, Pennisetum, Eleusine and Setaria genera, with a prevalence of campestanyl and sitostanyl ferulate. The lowest steryl ferulates content was found in Panicum, with values ranging from 2.98 ± 0.04 µg/g to 8.72 ± 0.41 µg/g. Foxtail millet and finger millet showed the highest amount with 46.07 ± 5.20 µg/g and 85.29 ± 4.30 µg/g, respectively. As for pearl millet, microwave heating and fermentation increased steryl ferulates by two (33.77 ± 0.88 µg/g) and five (75.83 ± 1.25 µg/g) times, with respect to the untreated sample. Microwave heating and fermentation could be used to increase steryl ferulates in millet.

Optimizing therapeutic outcomes of immune checkpoint blockade by a microbial tryptophan metabolite.

BACKGROUND: Despite the great success, the therapeutic benefits of immune checkpoint inhibitors (ICIs) in cancer immunotherapy are limited by either various resistance mechanisms or ICI-associated toxic effects including gastrointestinal toxicity. Thus, novel therapeutic strategies that provide manageable side effects to existing ICIs would enhance and expand their therapeutic efficacy and application. Due to its proven role in cancer development and immune regulation, gut microbiome has gained increasing expectation as a potential armamentarium to optimize immunotherapy with ICI. However, much has to be learned to fully harness gut microbiome for clinical applicability. Here we have assessed whether microbial metabolites working at the interface between microbes and the host immune system may optimize ICI therapy. METHODS: To this purpose, we have tested indole-3-carboxaldehyde (3-IAld), a microbial tryptophan catabolite known to contribute to epithelial barrier function and immune homeostasis in the gut via the aryl hydrocarbon receptor (AhR), in different murine models of ICI-induced colitis. Epithelial barrier integrity, inflammation and changes in gut microbiome composition and function were analyzed. AhR, indoleamine 2,3-dioxygenase 1, interleukin (IL)-10 and IL-22 knockout mice were used to investigate the mechanism of 3-IAld activity. The function of the microbiome changes induced by 3-IAld was evaluated on fecal microbiome transplantation (FMT). Finally, murine tumor models were used to assess the effect of 3-IAld treatment on the antitumor activity of ICI. RESULTS: On administration to mice with ICI-induced colitis, 3-IAld protected mice from intestinal damage via a dual action on both the host and the microbes. Indeed, paralleling the activation of the host AhR/IL-22-dependent pathway, 3-IAld also affected the composition and function of the microbiota such that FMT from 3-IAld-treated mice protected against ICI-induced colitis with the contribution of butyrate-producing bacteria. Importantly, while preventing intestinal damage, 3-IAld did not impair the antitumor activity of ICI. CONCLUSIONS: This study provides a proof-of-concept demonstration that moving past bacterial phylogeny and focusing on bacterial metabolome may lead to a new class of discrete molecules, and that working at the interface between microbes and the host immune system may optimize ICI therapy.

A Shifted Composition of the Lung Microbiota Conditions the Antifungal Response of Immunodeficient Mice.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1-/- mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

Surfactant-Assisted Distal Pulmonary Distribution of Budesonide Revealed by Mass Spectrometry Imaging.

Direct lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle (n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa (n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs (n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lung.

Proteomics of arthropod soluble olfactory proteins.

Shotgun proteomics is a very sensitive bottom-up approach used to study complex mixtures of proteins through a combination of high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS). This technique has been successfully applied to the study of olfactory appendixes of insects and other arthropods. Since extracting soluble proteins from tissues and processing them for shotgun proteomics is quite straightforward, the technique has proved to be very suitable for studying soluble olfactory proteins, such as odorant binding proteins (OBPs), chemosensory proteins (CSPs) and Niemann-Pick type 2 proteins. Here, we describe the main principles and methods at the basis of shotgun proteomics, including its use to quantify proteins through label-free quantification. We also provide operational protocols for the experimental workflow steps, i.e., insect dissection, protein extraction, protein enzymatic digestion, sample purification, HPLC-MS analysis, search of mass spectral data against polypeptide databases and first steps in the analysis of the search results.

Identification of the Nicotinamide Salvage Pathway as a New Toxification Route for Antimetabolites.

Interest in the modulation of nicotinamide adenine dinucleotide (NAD) metabolome is gaining great momentum because of its therapeutic potential in different human disorders. Suppression of nicotinamide salvage by nicotinamide phosphoribosyl transferase (NAMPT) inhibitors, however, gave inconclusive results in neoplastic patients because several metabolic routes circumvent the enzymatic block converging directly on nicotinamide mononucleotide adenylyl transferases (NMNATs) for NAD synthesis. Unfortunately, NMNAT inhibitors have not been identified. Here, we report the identification of Vacor as a substrate metabolized by the consecutive action of NAMPT and NMNAT2 into the NAD analog Vacor adenine dinucleotide (VAD). This leads to inhibition of both enzymes, as well as NAD-dependent dehydrogenases, thereby causing unprecedented rapid NAD depletion, glycolytic block, energy failure, and necrotic death of NMNAT2-proficient cancer cells. Conversely, lack of NMNAT2 expression confers complete resistance to Vacor. Remarkably, Vacor prompts VAD formation and growth suppression in NMNAT2-positive neuroblastoma and melanoma xenografts. Our data show the first evidence of harnessing the entire nicotinamide salvage pathway for antimetabolic strategies.

Steroidal and non-steroidal third-generation aromatase inhibitors induce pain-like symptoms via TRPA1.

Use of aromatase inhibitors (AIs), exemestane, letrozole and anastrozole, for breast cancer therapy is associated with severe pain symptoms, the underlying mechanism of which is unknown. The electrophilic nature of AIs suggests that they may target the transient receptor potential ankyrin 1 (TRPA1) channel, a major pathway in pain transmission and neurogenic inflammation. AIs evoke TRPA1-mediated calcium response and current in rodent nociceptors and human cells expressing the recombinant channel. In mice, AIs produce acute nociception, which is exaggerated by pre-exposure to proalgesic stimuli, and, by releasing sensory neuropeptides, neurogenic inflammation in peripheral tissues. AIs also evoke mechanical allodynia and decreased grip strength, which do not undergo desensitization on prolonged AI administration. These effects are markedly attenuated by TRPA1 pharmacological blockade or in TRPA1-deficient mice. TRPA1 is a major mediator of the proinflammatory/proalgesic actions of AIs, thus suggesting TRPA1 antagonists for the treatment of pain symptoms associated with AI use.

Telomerase activated thymidine analogue pro-drug is a new molecule targeting hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although hepatectomy and transplantation have significantly improved survival, there is no effective chemotherapeutic treatment for HCC and its prognosis remains poor. Sustained activation of telomerase is essential for the growth and progression of HCC, suggesting that telomerase is a rational target for HCC therapy. Therefore, we developed a thymidine analogue pro-drug, acycloguanosyl-5'-thymidyltriphosphate (ACV-TP-T), which is specifically activated by telomerase in HCC cells and investigated its anti-tumour efficacy. METHODS: First, we verified in vitro whether ACV-TP-T was a telomerase substrate. Second, we evaluated proliferation and apoptosis in murine (Hepa1-6) and human (Hep3B, HuH7, HepG2) hepatic cancer cells treated with ACV-TP-T. Next, we tested the in vivo treatment efficacy in HBV transgenic mice that spontaneously develop hepatic tumours, and in a syngeneic orthotopic murine model where HCC cells were implanted directly in the liver. RESULTS: In vitro characterization provided direct evidence that the pro-drug was actively metabolized in liver cancer cells by telomerase to release the active form of acyclovir. Alterations in cell cycle and apoptosis were observed following in vitro treatment with ACV-TP-T. In the transgenic and orthotopic mouse models, treatment with ACV-TP-T reduced tumour growth, increased apoptosis, and reduced the proliferation of tumour cells. CONCLUSIONS: ACV-TP-T is activated by telomerase in HCC cells and releases active acyclovir that reduces proliferation and induces apoptosis in human and murine liver cancer cells. This pro-drug holds a great promise for the treatment of HCC.

The effects of CA IX catalysis products within tumor microenvironment.

Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment.

Reactivity and biological properties of a series of cytotoxic PtI2(amine)2 complexes, either cis or trans configured.

Six diiodido-diamine platinum(II) complexes, either cis or trans configured, were prepared, differing only in the nature of the amine ligand (isopropylamine, dimethylamine, or methylamine), and their antiproliferative properties were evaluated against a panel of human tumor cell lines. Both series of complexes manifested pronounced cytotoxic effects, with the trans isomers being, generally, more effective than their cis counterparts. Cell cycle analysis revealed different modes of action for these new Pt(II) complexes with respect to cisplatin. The reactivity of these platinum compounds with a number of biomolecules, including cytochrome c, two sulfur containing modified amino acids, 9-ethylguanine, and a single strand oligonucleotide, was analyzed in depth by mass spectrometry and NMR spectroscopy. Interestingly, significant differences in the reactivity of the investigated compounds toward the various model biomolecules were observed: in particular we observed that trans complexes preferentially release their iodide ligands upon biomolecule binding, while the cis isomers may release the amine ligands with retention of iodides. Such differences in reactivity may have important mechanistic implications and a relevant impact on the respective pharmacological profiles.

Cuticular hydrocarbons rather than peptides are responsible for nestmate recognition in Polistes dominulus.

A colony of social insects is like a fortress where access is allowed only to colony members. The epicuticular mixture of hydrocarbons has been widely reported to be involved in nestmate recognition in insects. However, recent studies have shown that polar compounds (mainly peptides) are also present, mixed with hydrocarbons, on the cuticle of various insects, including the paper wasps of the genus Polistes. As these polar compounds are variable among Polistes species and are perceived by the wasps, this cuticular fraction could also be involved in nestmate recognition. Through MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time of Flight) mass spectrometry analysis, we assessed, for the first time, the intercolonial variability of the cuticular polar fraction of Polistes dominulus in order to evaluate its reliability as source of nestmate recognition cues. We then tested through behavioral assays the importance of the 2 isolated fractions (apolar and polar) in nestmate recognition by presenting them separately to colonies of P. dominulus. Our results showed that the cuticular polar compounds are not colony specific and they are not used by paper wasps to discriminate nestmates from non-colony members. On the contrary, we confirmed that the isolated cuticular hydrocarbons are the chemical mediators prompting nestmate recognition in paper wasps.

Vitamin D3 quantification in a cod liver oil-based supplement.

A reliable, accurate and reproducible method to quantify vitamin D3 (Vit. D3) in oily dietary supplements was developed after three Vit. D3 intoxications were diagnosed as reasonably resulting from a dietary administration of a cod liver oil based supplement. Liquid chromatography coupled to mass spectrometry operating in atmospheric pressure chemical ionization conditions (LC-APCI) and by using a deuterium labelled internal standard resulted to be an effective technique to reach the analytical aim. Due to the complexity of the oily matrix, the new analytical approach required a solid phase extraction step prior to analysis. The amount of Vit. D3 declared on the label of the cod liver oil based supplement for each soft capsule is 1.5µg. Consequently, the method was developed to quantify Vit. D3 amounts in the range 1-5µg/mL. To improve reliability of obtained data, both MS and MS/MS acquisition methods were employed. The method was evaluated by measuring the characteristic parameters such as linearity, precision, accuracy and robustness and cross checked against a certified pharmaceutical preparation. The LC-APCI-MS and MS/MS methods were applied in order to assess the Vit. D3 content in the dietary supplements taken by the intoxicated patients, found about three order of magnitude higher than that declared. The Vit. D3 content of other batches of the same commercial product was found as declared.

Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor gamma-independent regulation of nucleophosmin.

UNLABELLED: Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelial cancers, including hepatocellular carcinoma (HCC). The effective anticancer properties and the underlying molecular mechanisms of these drugs in vivo remain unclear. In addition, the primary biological target of TZD, the ligand-dependent transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), is up-regulated in HCC and seems to provide tumor-promoting responses. The aim of our study was to evaluate whether chronic administration of TZD may affect hepatic carcinogenesis in vivo in relation to PPARgamma expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARgamma-expressing and PPARgamma-deficient mouse models of hepatic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes by differential in gel electrophoresis and mass spectrometry analysis. Identified TZD targets were confirmed in cultured PPARgamma-deficient hepatocytes. TZD administration in hepatitis B virus (HBV)-transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in the liver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARgamma deletion in hepatocytes of HBV-transgenic mice (Tg[HBV]CreKOgamma) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARgamma-deficient hepatocytes. TZD inhibited NPM expression at protein and messenger RNA levels and decreased NPM promoter activity. TZD inhibition of NPM was associated with the induction of p53 phosphorylation and p21 expression. CONCLUSION: These findings suggest that chronic administration of TZD has anticancer activity in the liver via inhibition of NPM expression and indicate that these drugs might be useful for HCC chemoprevention and treatment.

[Au2(phen(2Me))2(µ-O)2](PF6)2, a Novel Dinuclear Gold(III) Complex Showing Excellent Antiproliferative Properties.

A novel dioxo-bridged dinuclear gold(III) complex with two 2,9-dimethylphenanthroline ligands was synthesized and thoroughly characterized. Its crystal structure was solved, and its solution behavior assessed. Remarkably, this compound revealed excellent antiproliferative properties in vitro against a wide panel of 36 cancer cell lines, combining a high cytotoxic potency to pronounced tumor selectivity. Very likely, these properties arise from an innovative mode of action (possibly involving histone deacetylase inhibition), as suggested by COMPARE analysis. In turn, electrospray ionization-mass spectrometry studies provided valuable insight into its molecular mechanisms of activation and of interaction with protein targets. Gold(III) reduction, dioxo bridge disruption, coordinative gold(I) binding to the protein, and concomitant release of the phenanthroline ligand were proposed to occur upon interaction with superoxide dismutase, used here as a model protein. Because of the reported results, this new gold(III) compound qualifies itself as an optimal candidate for further pharmacological testing.

CSF proteomic analysis in patients with normal pressure hydrocephalus selected for the shunt: CSF biomarkers of response to surgical treatment.

The aim of our pilot study was to investigate, by a proteomic approach, the expressed differences in cerebrospinal fluid (CSF) protein patterns in order to aid in the diagnosis and treatment of normal pressure hydrocephalus (NPH). Seventeen patients with NPH, selected by Intracranial-Pressure monitoring (ICPmo), underwent implantation of a shunt and after 6 months were clinically re-evaluated. Thirteen patients improved, whereas four did not. During ICPmo CSF was collected and its proteoma was analyzed by 2D gel electrophoresis and mass spectrometry. The over-expression of alpha2HS glycoprotein, alpha1 antichimotrypsin and alpha1beta glycoprotein and the under-expression of glial fibrillary acidic protein, apolipoproteins (AIV, J and E), complement C3c, anti-thrombin, alpha2 antiplasmin and albumin seem to be associated with a positive response to surgery. Most of these proteins have been reported to be altered in Alzheimer disease, supporting the hypothesis of a possible link between these two nosological entities.

Exploring proteins in Anopheles gambiae male and female antennae through MALDI mass spectrometry profiling.

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.

Peculiar mechanistic and structural features of the carboplatin-cytochrome c system revealed by ESI-MS analysis.

Carboplatin (CPT), today the most important platinum(II) anticancer drug, manifests an extreme kinetic inertness, in vitro, at physiological pH; the actual mechanisms for its activation inside cells are still poorly understood. We show here that horse heart cytochrome c reacts with CPT, leading to the formation of stable platinum/protein adducts. The two major CPT-cytochrome c species resulting from the aforementioned reaction were characterised by electrospray ionisation mass spectrometry (ESI-MS). Notably, both these adducts have the ability to react with guanosine 5'-monophosphate (5'-GMP), giving rise to the respective cytochrome c-CPT-5'-GMP ternary complexes. Additional ESI-MS measurements on enzymatically cleaved cytochrome c adducts suggest that protein platination probably occurs at Met65. The mechanistic implications of these findings are discussed.