Design and preparation of NMN nanoparticles based on protein-marine polysaccharide with increased NAD+ level in D-galactose induced aging mice model.

Nicotinamide mononucleotide (NMN) is being investigated for its ability to address the decline in NAD+ level during aging. This study aimed to construct a delivery system based on ovalbumin and fucoidan nanoparticles to ameliorate the bioaccessibility of NMN by increasing NAD+ level in aging mouse model. The NMN-loaded ovalbumin and fucoidan nanoparticles (OFNPs) were about 177 nm formed by the interplay of hydrogen bonds between ovalbumin and fucoidan. Compared with free NMN, NMN-loaded OFNPs intervention could obviously improve the antioxidant enzyme activity of senescent cell induced by D-galactose. The NMN-loaded OFNPs treatment could ameliorate the loss of weight and organ index induced by senescence, and maintain the water content for the aging mice. The Morris maze test indicated that hitting blind side frequency and escape time of NMN-loaded OFNPs group decreased by 13% and 35% compared with that of free NMN group. Furthermore, the NMN-loaded OFNPs significantly alleviated the age-related oxidative stress and increased the generation of NAD+ 1.34 times by improving the bioaccessibility of NMN. Our data in this study supplied a strategy to enhance the bioavailability of NMN in senescence treatment.

A Magnesium Binding Site And The Anomeric Effect Regulate The Abiotic Redox Chemistry Of Nicotinamide Nucleotides.

Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.

Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor.

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.

Reduced nicotinamide mononucleotide is a new and potent NAD+ precursor in mammalian cells and mice.

Nicotinamide adenine dinucleotide (NAD+ ) homeostasis is constantly compromised due to degradation by NAD+ -dependent enzymes. NAD+ replenishment by supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can alleviate this imbalance. However, NMN and NR are limited by their mild effect on the cellular NAD+ pool and the need of high doses. Here, we report a synthesis method of a reduced form of NMN (NMNH), and identify this molecule as a new NAD+ precursor for the first time. We show that NMNH increases NAD+ levels to a much higher extent and faster than NMN or NR, and that it is metabolized through a different, NRK and NAMPT-independent, pathway. We also demonstrate that NMNH reduces damage and accelerates repair in renal tubular epithelial cells upon hypoxia/reoxygenation injury. Finally, we find that NMNH administration in mice causes a rapid and sustained NAD+ surge in whole blood, which is accompanied by increased NAD+ levels in liver, kidney, muscle, brain, brown adipose tissue, and heart, but not in white adipose tissue. Together, our data highlight NMNH as a new NAD+ precursor with therapeutic potential for acute kidney injury, confirm the existence of a novel pathway for the recycling of reduced NAD+ precursors and establish NMNH as a member of the new family of reduced NAD+ precursors.

Safety evaluation after acute and sub-chronic oral administration of high purity nicotinamide mononucleotide (NMN-C®) in Sprague-Dawley rats.

ß-nicotinamide mononucleotide (NMN) is a natural molecule intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Preclinical evidences point to the beneficial effect of NMN administration on several age-related conditions. The present work aimed at studying mutagenicity, and genotoxicity, acute oral toxicity and subchronic oral toxicity of a high purity synthetic form of NMN (NMN-C®) following the OECD guidelines. In the experimental conditions tested, NMN-C® was not mutagenic or genotoxic. Acute toxicity assay revealed that at an oral limit dose of 2666 mg/kg, NMN-C® did not lead to any mortality or treatment-related adverse signs. Over a 90-day sub-chronic period of repeated oral administration of NMN-C® at doses of 375, 750 and 1500 mg/kg/d followed by a 28-day treatment-free recovery period, NMN-C® appeared to be safe and did not promote toxic effects as seen from body weight change, food and water consumption, feed conversion efficiency, biochemical and blood parameters as well as organ toxicity and histological examinations of main organs. In conclusion, we provide the first data highlighting the safety of short to intermediate term (sub-chronic) oral administration of NMN and our experimental results allowed to determine a No-Observable Adverse Effect Level (NOAEL) for NMN-C® to be ≥ 1500 mg/kg/d.

Structural and Functional Characterization of NadR from Lactococcus lactis.

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a 'sink' for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains.

Bacteria Boost Mammalian Host NAD Metabolism by Engaging the Deamidated Biosynthesis Pathway.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD+ salvage pathway in the nucleus.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

Nicotinamide mononucleotide and related metabolites induce disease resistance against fungal phytopathogens in Arabidopsis and barley.

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), is known to act as a functional molecule in animals, whereas its function in plants is largely unknown. In this study, we found that NMN accumulated in barley cultivars resistant to phytopathogenic fungal Fusarium species. Although NMN does not possess antifungal activity, pretreatment with NMN and related metabolites enhanced disease resistance to Fusarium graminearum in Arabidopsis leaves and flowers and in barley spikes. The NMN-induced Fusarium resistance was accompanied by activation of the salicylic acid-mediated signalling pathway and repression of the jasmonic acid/ethylene-dependent signalling pathways in Arabidopsis. Since NMN-induced disease resistance was also observed in the SA-deficient sid2 mutant, an SA-independent signalling pathway also regulated the enhanced resistance induced by NMN. Compared with NMN, NAD and NADP, nicotinamide pretreatment had minor effects on resistance to F. graminearum. Constitutive expression of the NMNAT gene, which encodes a rate-limiting enzyme for NAD biosynthesis, resulted in enhanced disease resistance in Arabidopsis. Thus, modifying the content of NAD-related metabolites can be used to optimize the defence signalling pathways activated in response to F. graminearum and facilitates the control of disease injury and mycotoxin accumulation in plants.

Synthesis of an oligonucleotide with a nicotinamide mononucleotide residue and its molecular recognition in DNA helices.

Nicotinamide adenine dinucleotide (NAD) is a pivotal redox cofactor of primary metabolism. Its redox reactivity is based on the nicotinamide mononucleotide (NMN) moiety. We investigated whether NMN(+) can engage in pairing interactions, when incorporated into an oligonucleotide. Here we describe the incorporation of NMN(+) at the 3'-terminus of an oligodeoxynucleotide via a phosphoramidate coupling in solution. The stability of duplexes and triplexes with the NMN(+)-containing strand was measured in UV-melting curves. While the melting points of duplexes with different bases facing the nicotinamide were similar, triplex stabilities varied greatly between different base combinations, suggesting specific pairing. The most stable triplexes were found when a guanine and an adenine were facing the NMN(+) residue. Their triplex melting points were higher than those of the corresponding triplexes with a thymidine residue at the same position. These results show that NMN(+) residues can be recognized selectively in DNA helices and are thus compatible with the molecular recognition in nucleic acids.

METALLOPROTEINS. A tethered niacin-derived pincer complex with a nickel-carbon bond in lactate racemase.

Lactic acid racemization is involved in lactate metabolism and cell wall assembly of many microorganisms. Lactate racemase (Lar) requires nickel, but the nickel-binding site and the role of three accessory proteins required for its activation remain enigmatic. We combined mass spectrometry and x-ray crystallography to show that Lar from Lactobacillus plantarum possesses an organometallic nickel-containing prosthetic group. A nicotinic acid mononucleotide derivative is tethered to Lys(184) and forms a tridentate pincer complex that coordinates nickel through one metal-carbon and two metal-sulfur bonds, with His(200) as another ligand. Although similar complexes have been previously synthesized, there was no prior evidence for the existence of pincer cofactors in enzymes. The wide distribution of the accessory proteins without Lar suggests that it may play a role in other enzymes.

PARPs and ADP-Ribosylation: 50 Years and Counting.

Over 50 years ago, the discovery of poly(ADP-ribose) (PAR) set a new field of science in motion-the field of poly(ADP-ribosyl) transferases (PARPs) and ADP-ribosylation. The field is still flourishing today. The diversity of biological processes now known to require PARPs and ADP-ribosylation was practically unimaginable even two decades ago. From an initial focus on DNA damage detection and repair in response to genotoxic stresses, the field has expanded to include the regulation of chromatin structure, gene expression, and RNA processing in a wide range of biological systems, including reproduction, development, aging, stem cells, inflammation, metabolism, and cancer. This special focus issue of Molecular Cell includes a collection of three Reviews, three Perspectives, and a SnapShot, which together summarize the current state of the field and suggest where it may be headed.

Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity.

OBJECTIVE: Niacin (nicotinic acid) as a monotherapy can reduce vascular disease risk, but its mechanism of action remains controversial, and may not be dependent on systemic lipid modifying effects. Niacin has recently been shown to improve endothelial function and vascular regeneration, independent of correcting dyslipidemia, in rodent models of vascular injury and metabolic disease. As a potential biosynthetic precursor for NAD(+), niacin could elicit these vascular benefits through NAD(+)-dependent, sirtuin (SIRT) mediated responses. Alternatively, niacin may act through its receptor, GPR109A, to promote endothelial function, though endothelial cells are not known to express this receptor. We hypothesized that niacin directly improves endothelial cell function during exposure to lipotoxic conditions and sought to determine the potential mechanism(s) involved. METHODS AND RESULTS: Angiogenic function in excess palmitate was assessed by tube formation following treatment of human microvascular endothelial cells (HMVEC) with either a relatively low concentration of niacin (10 µM), or nicotinamide mononucleotide (NMN) (1 µM), a direct NAD(+) precursor. Although both niacin and NMN improved HMVEC tube formation during palmitate overload, only NMN increased cellular NAD(+) and SIRT1 activity. We further observed that HMVEC express GRP109A. Activation of this receptor with either acifran or MK-1903 recapitulated niacin-induced improvements in HMVEC tube formation, while GPR109A siRNA diminished the effect of niacin. CONCLUSION: Niacin, at a low concentration, improves HMVEC angiogenic function under lipotoxic conditions, likely independent of NAD(+) biosynthesis and SIRT1 activation, but rather through niacin receptor activation.

Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.

Characterization of bacterial NMN deamidase as a Ser/Lys hydrolase expands diversity of serine amidohydrolases.

NMN deamidase (PncC) is a bacterial enzyme involved in NAD biosynthesis. We have previously demonstrated that PncC is structurally distinct from other known amidohydrolases. Here, we extended PncC characterization by mutating all potential catalytic residues and assessing their individual roles in catalysis through kinetic analyses. Inspection of these residues' spatial arrangement in the active site, allowed us to conclude that PncC is a serine-amidohydrolase, employing a Ser/Lys dyad for catalysis. Analysis of the PncC structure in complex with a modeled NMN substrate supported our conclusion, and enabled us to propose the catalytic mechanism.

Investigation of NADH binding, hydride transfer, and NAD(+) dissociation during NADH oxidation by mitochondrial complex I using modified nicotinamide nucleotides.

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. During catalysis, NADH oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. Alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen and redox-cycling molecules to produce reactive oxygen species. Here, we study two steps in the mechanism of NADH oxidation by complex I. First, molecular fragments of NAD(H), tested as flavin-site inhibitors or substrates, reveal that the adenosine moiety is crucial for binding. Nicotinamide-containing fragments that lack the adenosine do not bind, and ADP-ribose binds more strongly than NAD(+), suggesting that the nicotinamide is detrimental to binding. Second, the primary kinetic isotope effects from deuterated nicotinamide nucleotides confirm that hydride transfer is from the pro-S position and reveal that hydride transfer, along with NAD(+) dissociation, is partially rate-limiting. Thus, the transition state energies are balanced so that no single step in NADH oxidation is completely rate-limiting. Only at very low NADH concentrations does weak NADH binding limit NADH:ubiquinone oxidoreduction, and at the high nucleotide concentrations of the mitochondrial matrix, weak nucleotide binding constants assist product dissociation. Using fast nucleotide reactions and a balance between the nucleotide binding constants and concentrations, complex I combines fast and energy-conserving NADH oxidation with minimal superoxide production from the nucleotide-free site.

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide.

Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Šresolution. The Ss-QAPRTase-NAMN cocrystals belonged to space group P321, with unit-cell parameters a=119.1, b=119.1, c=93.7 Å, γ=120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å3 Da(-1) and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.

Comparison of the formation of nicotinic acid conjugates in leaves of different plant species.

There are three metabolic fates of nicotinic acid in plants: (1) nicotinic acid mononucleotide formation for NAD synthesis by the so-called salvage pathway of pyridine nucleotide biosynthesis; (2) nicotinic acid N-glucoside formation; and (3) trigonelline (N-methylnicotinic acid) formation. In the present study, the metabolism of [carbonyl-(14)C]nicotinamide was investigated in leaves of 23 wild plant species. All species readily converted nicotinamide to nicotinic acid, and only a fraction of nicotinic acid was utilised for NAD and NADP synthesis. The remaining nicotinic acid is converted to the nicotinic acid conjugates. Only one plant species, Cycas revoluta, produced both nicotinic acid N-glucoside and trigonelline; the other 22 species produced one or other of the conjugates. The nicotinic acid N-glucoside-forming plants are Cyathea lepifera, Arenga trewmula var. englri, Barringtonia racemosa, Ilex paraguariensis, Angelica japonica, Scaevola taccada and Farfugium japonicum. In contrast, trigonelline is formed in C. lepifera, Ginkgo biloba, Pinus luchuensis, Casuarina equisetifolia, Alocasia odora, Pandanus odoratissimus, Hylocereus undatus, Kalanchoe pinnata, Kalanchoe tubiflora, Populus alba, Garcinia subelliptica, Oxalis corymbosa, Leucaena leucocephala, Vigna marina, Hibiscus tiliaceus and Melicope triphylla. The diversity of nicotinic acid conjugate formation in plants is discussed using these results and our previous investigation involving a few model plants, various crops and ferns. Nicotinic acid N-glucoside formation was restricted mostly to ferns and selected orders of angiosperms, whereas other plants produce trigonelline. In most cases the formation of both nicotinic acid conjugates is incompatible, but some exceptions have been found.