Human early-onset dementia caused by DAP12 deficiency reveals a unique signature of dysregulated microglia.

The TREM2-DAP12 receptor complex sustains microglia functions. Heterozygous hypofunctional TREM2 variants impair microglia, accelerating late-onset Alzheimer's disease. Homozygous inactivating variants of TREM2 or TYROBP-encoding DAP12 cause Nasu-Hakola disease (NHD), an early-onset dementia characterized by cerebral atrophy, myelin loss and gliosis. Mechanisms underpinning NHD are unknown. Here, single-nucleus RNA-sequencing analysis of brain specimens from DAP12-deficient NHD individuals revealed a unique microglia signature indicating heightened RUNX1, STAT3 and transforming growth factor-ß signaling pathways that mediate repair responses to injuries. This profile correlated with a wound healing signature in astrocytes and impaired myelination in oligodendrocytes, while pericyte profiles indicated vascular abnormalities. Conversely, single-nuclei signatures in mice lacking DAP12 signaling reflected very mild microglial defects that did not recapitulate NHD. We envision that DAP12 signaling in microglia attenuates wound healing pathways that, if left unchecked, interfere with microglial physiological functions, causing pathology in human. The identification of a dysregulated NHD microglia signature sparks potential therapeutic strategies aimed at resetting microglia signaling pathways.

The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

Non-syndromic congenital sideroblastic anaemia; phenotype, and genotype of 15 Indian patients.

Sideroblastic anaemias are a diverse group of congenital and acquired bone marrow failure disorders marked by the presence of ring sideroblasts, ineffective erythropoiesis, and systemic iron overload. Congenital Sideroblastic anaemia (CSA) is mainly caused by gene mutations associated with heme synthesis, iron-sulfur [Fe-S] cluster, and mitochondrial protein synthesis pathways. The most prevalent form of CSA is caused by mutations in the erythroid-specific -amino levulinate synthase (ALAS2) gene, which encodes the first enzyme in the heme synthesis pathway in red blood cells. The second most prevalent form of CSA is caused by a mutation in the Solute carrier family 25 member 38 (SLC25A38) gene, which codes for an erythroid-specific protein of the inner mitochondrial membrane. Additionally, 15-20 genes are altogether associated with CSA. In this study, we aim to identify the CSA patients, understand their genetics and establish genotype-phenotype correlation. We have identified fifteen cases of CSA using our targeted NGS (t-NGS) panel. The major clinical findings in our cohort were microcytic anaemia, ring sideroblasts, and dyserythropoiesis in the bone marrow. Currently, two patients are responsive to pyridoxine, while the rest are on blood transfusion support. We have identified ten variants in three different genes of CSA (ALAS2, SLC25A38 & HSPA9). Five patients harbour four hemizygous variants- p.Ala282Ser, p.Arg170Cys, p.Arg204Gln and exon 2 duplication in the ALAS2 gene. In seven patients, we have identified three homozygous mutations - p.Pro190Arg, p.Arg187Gln and p.Arg134Cys in the SLC25A38 gene. These mutations have been predominantly identified in the European population. Three patients revealed three heterozygous variants p. Thr463Ile, D326Tyr, and Arg284Trp in the HSPA9 gene. PyMoL was used to evaluate the functional effects of these variations and understand their effect on the structure of the protein. We believe that by combining a bone marrow examination with genetic sequencing, CSA patients can acquire a definitive diagnosis.

Simultaneous high-pitch multi-energy CT pulmonary angiography using a dual-source photon-counting-detector CT: A phantom experiment.

PURPOSE: A dual-source CT system can be operated in a high-pitch helical mode to provide a temporal resolution of 66 ms, which reduces motion artifacts in CT pulmonary angiography (CTPA). It can also be operated in a multi-energy (ME) mode to provide iodine maps, beneficial in the evaluation of pulmonary embolism (PE). No energy-integrating detector (EID) CT can perform simultaneous ME and high-pitch acquisition. This phantom study aimed to evaluate the ability of a photon-counting-detector (PCD) CT to perform simultaneous high-pitch and ME imaging for CTPA. METHODS: A motion phantom was used to mimic the respiratory motion. Two tubes filled with iodine with intravascular thrombus mimicked by injecting glue within the tubes were placed along with 5, 10, and 15 mg/mL iodine samples, on a motion phantom at 20 and 30 revolutions per minute. Separate high-pitch and ME EID-CT scans and a single high-pitch ME PCD scan were acquired and virtual monoenergetic images and iodine maps reconstructed. Percent thrombus occlusion was measured and compared between static and moving images. RESULTS: When there was motion, EID-CT ME suffered from significant motion artifacts. The measured iodine concentrations with PCD-CT in high-pitch ME were more stable when there was a motion, with a lower standard deviation than EID-CT in ME mode. The estimated percent thrombus occlusion dropped significantly with applied motion on EID-CT, while PCD-CT high-pitch ME mode showed good agreement between measurements on static or moving images. CONCLUSION: PCD-CT with combined ME and high-pitch mode facilitates simultaneous accurate iodine quantification and assessment of intravascular occlusion.

Development of Centrifugal Partition Chromatography for the Purification of Antibody-Drug Conjugates.

Monoclonal antibody-drug conjugates (ADCs) are an expanding therapeutic class of biomolecules for which relatively few analytical and preparative separation options exist. Purification of ADCs with a specific drug antibody ratio is even more challenging. We report the first application of countercurrent separation (CCS) to this problem. An ADC mimic was successfully chromatographed using an aqueous two-phase system (ATPS) consisting of PEG 1000/sodium citrate pH 7.5/water, 17.75/17.75/64.50 (w/w/w). Notably, different partition coefficients (K) in this ATPS for the ADC mimic (0.09 < K < 0.16) and its monoclonal antibody backbone, IgG (0.16 < K < 0.27), were observed using CCS. Differential elution behavior of such high-molecular-weight biomolecules, 146,441 vs. ∼150,000 Da, using CCS has no precedent. The results provide a proof of concept for further exploration of the application of ATPSs and CCS to the separation of ADCs.

Rufomycins or Ilamycins: Naming Clarifications and Definitive Structural Assignments.

Rufomycin and ilamycin are synonymous for the same class of cyclopeptides, currently encompassing 33 structurally characterized isolates and 9 semisynthetic derivatives. Elucidation of new structures prioritized the consolidation of the names and established the structures of four diastereoisomeric rufomycins with a 2-piperidinone, named rufomycins 4-7, including full 1H/13C NMR assignments. The characteristic HSQC cross-peak for the CH-5, the hemiaminal carbon in amino acid #5, allows assignment of the stereocenters C-4 and C-5 within this ring. Semisynthetic derivatives (rufomycinSS 1, 2, and 3) were prepared from a rufomycins 4 and 6 mixture to validate the structural assignments. Based on the X-ray crystal structures of rufomycins 2 and 4, considering the NMR differences of rufomycins 7 vs 4-6 compared to rufomycinSS 1 vs 2 and 3, and taking into account that two major conformers, A and B, occur in both rufomycinSS 2 and 3, structural modeling was pursued. Collectively, this paper discusses the NMR spectroscopic differences of the stereoisomers and their possible 3D conformers and correlates these with the anti-Mycobacterium tuberculosis activity. In addition, a look at the history prioritizes names and numbering schemes for this group of antibiotics and leads to consolidated nomenclature for all currently known members, natural and semisynthetic derivatives, and serves to accommodate future discoveries.

Plain 1 H nuclear magnetic resonance analysis streamlines the quality control of antiviral favipiravir and congeneric World Health Organization essential medicines.

Favipiravir is an established antiviral that is currently being assessed as an investigational drug for the treatment of COVID-19. Favipiravir is strikingly similar to two molecules that the World Health Organization (WHO) lists as essential medicines, which also consist of a six-membered aromatic N-heterocycle bearing a carboxamide function: the anti-tuberculosis agent, pyrazinamide, and nicotinamide, also known as vitamin B3 . We demonstrate the utility of 1 H nuclear magnetic resonance (NMR) profiling, an emerging pharmacopoeial tool, for the highly specific identification, selective differentiation of congeners, and subsequent detection of drug falsification or adulteration of these medicines. The straightforward comparison of basic 1-D 1 H NMR spectra, obtained with benchtop or advanced NMR instruments alike, offers a rapid identity assay and works independently of physical reference materials. This approach accelerates and advances pharmaceutical quality control measures under situations of increased drug demand and altered economy, such as during a pandemic.

Mechanosensitive Piezo1 ion channel protein (PIEZO1 gene): update and extended mutation analysis of hereditary xerocytosis in India.

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.

Thiophene-phenylquinazoline probe for selective ratiometric fluorescence and visual detection of Fe(iii) and turn-off fluorescence for I- and its applications.

A 2,5-bis(4-phenylquinazolin-2-yl)thiophene (BQT) probe is designed, synthesized and explored for selective ratiometric fluorescence and visual detection of Fe3+ and as a turn-off fluorescence probe for I- anion. BQT is colorless and has blue emission in CH3CN solution. BQT selectively complexes with Fe3+, turns its solution from colorless to greenish yellow and enables the ratiometric sensing of Fe3+ with limit of detection (LOD) and limit of quantitation (LOQ) of 2 × 10-8 M and 6.1 × 10-8 M, respectively. Binding constant of BQT with Fe3+ is found to be 4.1 × 10-4 M-1. BQT is also able to sense I- anion present in aqueous solution by selectively turning colorless to yellow and fluorescence quenching with a LOD of 1.7 × 10-7 M and LOQ of 5.2 × 10-7 M. BQT sensing ability is not influenced by the presence of other metal ions and anions in the vicinity. The BQT-Fe3+ complex is thoroughly characterized using MALDI-TOF, NMR and Job's plot. A reversibility experiment with EDTA suggests BQT is a reversible fluorescent chemosensor for Fe3+ ions. The spectroscopic data of BQT and its complexes are employed to construct a field test kit for qualitative analysis and INHIBIT logic gate.

Pharmaceutical cocrystal: a game changing approach for the administration of old drugs in new crystalline form.

Pharmaceutical cocrystals are still gaining the interest of the researchers due to their potential to alter physicochemical, mechanical, and pharmacokinetic properties of active pharmaceutical ingredients without negotiating therapeutic action. The diverse new applications of cocrystals, like taste masking, reduced toxicity, patenting opportunities, commercial potential, etc. act as driving force to the rising interest of the pharmaceutical industries. Initially, cocrystals from the view of regulatory authorities, design strategies, cocrystal preparation in brief with special emphasis on scalable and solvent-free hot melt extrusion method, and practical guide to characterization have been provided. The special focus has been given to the biopharmaceutical attributes of the cocrystal. Finally, challenges before and after cocrystal preparation are presented in this review along with some commercial examples of the cocrystals.

Synthesis and antimicrobial evaluation of piperic acid amides and their lower homologues.

Seven piperic acid amides along with their lower homologs (12) were synthesized using HATU-DIPEA coupling reagent. All the synthesized derivatives were evaluated for their antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and vancomycin-resistant P. aeruginosa. They were found to be more active on P. aeruginosa than on S. aureus. However, they did not exhibit potent activity on Vancomycin resistant P. aeruginosa. Among the tested compounds, methylenedioxycinnamic acid amide of anthranilic acid (MDCA-AA, 2a) was found to be most active against S. aureus with MIC of 3.125 µg/ml. The PAS and INH amides of piperic acid were screened against Mycobacterium tuberculosis H37Ra strain. They were found to be most active among all the tested compounds but were found to be less active than the standard drug, isoniazid.

Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Identification and characterization of reaction products of 5-hydroxytryptamine with methylglyoxal and glyoxal by liquid chromatography/tandem mass spectrometry.

RATIONALE: Methylglyoxal (MGO) and glyoxal (GO) are known to be at high levels in humans with diabetes. They react with amine-containing proteins and amino acids to form advanced glycation end products, however, their reactivity with other amine-containing metabolites, such as neurotransmitters, has not been explored. In this study, we aimed at studying the reactivity of 5-hydroxytryptamine (5-HT) with MGO or GO, which may alter the metabolic function of 5-HT. METHODS: Stock solutions of 5-HT, MGO and GO were made in PBS buffer at pH 7.4 and 5-HT was incubated with MGO or GO at different concentrations. The reactions were also performed at physiological concentrations. The reaction mixtures collected at different incubation times were analyzed by direct ESI-HRMS, LC/MS and LC/MS/MS to detect/characterize the products. Agilent 6545 Q-TOF and Agilent 6420 triple quadrupole mass spectrometers were used for the study, and LC separations were performed on a C18 column. RESULTS: The direct ESI-HRMS data of the reaction mixtures showed formation of three and four reaction products when 5-HT was reacted with MGO and GO, respectively. All the products showed dominant [M + H]+ ions. The products were characterized by HRMS, LC/MS/MS and literature reports on similar compounds. The products can easily be identified by LC/MS based on the accurate mass values together with retention time information. The MS/MS of the reaction products showed structure-indicative fragment ions. CONCLUSIONS: 5-HT reacts with one or two MGO/GO to form a set of reaction products. The reaction between 5-HT and MGO or GO was faster at higher concentrations of MGO/GO (<10 min), and the same products were found even at physiological concentrations (<48 h). The LC/MS/MS (SRM) method can be used to screen the reaction products when present at low levels.

Differential Cationization of Fatty Acids with Monovalent Cations Studied by ESI-MS/MS and Computational Approach.

RATIONALE: The intercellular and intracellular transport of charged species (Na+ /K+ ) entail interaction of the ions with neutral organic molecules and formation of adduct ions. The rate of transport of the ions across the cell membrane(s) may depend on the stability of the adduct ions, which in turn rely on structural aspects of the organic molecules that interact with the ions. METHODS: Positive ion ESI mass spectra were recorded for the solutions containing fatty acids (FAs) and monovalent cations (X=Li+ , Na+ , K+ , Rb+ and Cs+ ). Product ion spectra of the [FA+X]+ ions were recorded at different collision energies. Theoretical studies were exploited under both gas phase and solvent phase to investigate the structural effects of the fatty acids during cationization. Stability of [FA+X]+ adduct ions were further estimated by means of AIM topological analyses and interaction energy (IE) values. RESULTS: Positive ion ESI-MS analyses of the solution of FAs and X+ ions showed preferential binding of the K+ ions to FAs. The K+ ion binding increased with the increase in number of double bonds of FAs, while decreased with increase in the number of carbons of FAs. Dissociation curves of [FA+X]+ ions indicated the relative stability order of the [FA+X]+ ions and it was in line with the observed trends in ESI-MS. The solvent phase computational studies divulged the mode of binding and the binding efficiencies of different FAs with monovalent cations. CONCLUSIONS: Among the studied monovalent cations, the cationization of FAs follow the order K+ >>Na+ >Li+ >Rb+ >Cs+ . The docosahexaenoic acid showed high efficiency in binding with K+ ion. The K+ ion binding efficiency of FAs depends on the number of double bonds in unsaturated FAs and the carbon chain length in saturated FAs. The cationization trends of FAs obtained from the ESI-MS, ESI-MS/MS analyses were in good agreement with solvent phase computational studies.

Response bias and response monitoring: Evidence from healthy older adults and patients with mild Alzheimer's disease.

Patients with Alzheimer's disease (AD) often exhibit an abnormally liberal response bias in recognition memory tests, responding "old" more frequently than "new." Investigations have shown patients can to shift to a more conservative response bias when given instructions. We examined if patients with mild AD could alter their response patterns when the ratio of old items is manipulated without explicit instruction. Healthy older adults and AD patients studied lists of words and then were tested in three old/new ratio conditions (30%, 50%, or 70% old items). A subset of participants provided estimates of how many old and new items they saw in the memory test. We demonstrated that both groups were able to change their response patterns without the aid of explicit instructions. Importantly, AD patients were more likely to estimate seeing greater numbers of old than new items, whereas the reverse was observed for older adults. Elevated estimates of old items in AD patients suggest their liberal response bias may be attributed to their reliance on familiarity. We conclude that the liberal response bias observed in AD patients is attributable to their believing that more of the test items are old and not due to impaired meta-memorial monitoring abilities.

Tracheoesophageal Fistula following Endotracheal Intubation for Organophosphorus Poisoning.

Tracheoesophageal fistula (TEF) is an abnormal communication between the trachea and esophagus. Iatrogenic TEF can be due to endotracheal intubation, rigid bronchoscopy or tracheostomy. Tracheostomy tube cuff volumes and pressures require constant monitoring to avoid tracheal injury. Acquired TEF which occurs after prolonged intubation, usually develops after 15-200 days of mechanical ventilation. We report a case of a large TEF secondary to endotracheal intubation for organophosphorus poison-induced respiratory failure. Patient presented with dysphagia and recurrent aspiration pneumonia after extubation. She underwent trachea-esophageal fistulectomy and closure with a sternocleidomastoid muscle flap.

Insights into the binding sites of sulforaphane on insulin studied by electrospray ionization mass spectrometry.

RATIONALE: Sulforaphane (SFN) is a natural isothiocyanate, known to reduce the risk of cancer and also aortic damage and diabetic cardiomyopathy induced by type 2 diabetes, etc. A more detailed knowledge on the direct interaction of SFN with insulin and its binding sites is necessary for better understanding the role of SFN on diabetes. METHODS: Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and in-source fragmentation experiments were performed on a Thermo Exactive orbitrap mass spectrometer. The solution of insulin and SFN was incubated and analyzed by mass spectrometry. Isotopic distribution pattern, accurate mass values and theoretical product ions were used to analyze the mass spectrometry data. The nature of binding of SFN and its binding sites with insulin were evaluated by LC/MS data. RESULTS: ESI-MS analysis of the incubated solution of insulin and SFN showed 1:1 and 1:2 complexes of [Insulin-SFN]. LC/MS analysis revealed that the [Insulin+SFN] complexes were due to covalent binding of SFN at two different sites. The in-source fragmentation experiments revealed that the SFN is binding to the NH2 groups of N-terminal amino acids of A and B chains of insulin. Further study of SFN with insulin reduced with dithiothreitol (DTT) showed exclusive modification of cysteines with SFN. CONCLUSIONS: The interaction of SFN was studied with insulin using ESI-MS. SFN is found to bind covalently with the free NH2 group of the N-terminal of the A and B chains of insulin. However, when insulin is reduced SFN preferably binds to SH groups of cysteines. Hence, the present study helps in the understanding of the binding sites of SFN on insulin.

Modeling of RNA nanotubes using molecular dynamics simulation.

In this study, we construct novel RNA nanoclusters, RNA nanotubes made of several nanorings up to the size of 20 nm, utilizing the molecular dynamics simulation, and study their structural properties [i.e., the root mean square deviation, the radius of gyration and the radial distribution function (RDF)] in physiological solutions that can be used for drug delivery into the human body. The patterns of energy and temperature variations of the systems are also discussed. Furthermore, we study the concentration of ions around the tube as a function of time at a particular temperature. We have found that when the temperature increases, the number of ions increases within a certain distance of the tube. We report that the number of ions within this distance around the tubes decreases in quenched runs. This indicates that some ions evaporate with decrease in temperature, as has been observed in the case of the nanoring. RDF plots also demonstrate a similar trend with temperature, as was found in the case of RNA nanorings.

Sulforaphane interaction with amyloid beta 1-40 peptide studied by electrospray ionization mass spectrometry.

RATIONALE: Aggregation of amyloid beta 1-40 (Aß) in the brain causes Alzheimer's disease (AD) and several small molecules are known to inhibit the aggregation process. Sulforaphane (SFN) is a natural isothiocyanate which is known to prevent various neurodegenerative processes. However, its interaction with Aß is yet to be explored. Such studies could provide new mechanistic insights for its neuroprotective properties. METHODS: Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and in-source fragmentation experiments were performed on an Orbitrap mass spectrometer. The solution of Aß and SFN was incubated and analyzed by mass spectrometry. Isotopic distribution patterns, accurate mass values and theoretical product ions were used to analyze the mass spectrometry data. The nature of binding of SFN and its binding sites with Aß were evaluated by LC/MS and trypsin digestion experiments. RESULTS: ESI-MS analysis of the incubated solution of Aß and SFN showed a 1:1 complex of [Aß+SFN]. LC/MS analysis revealed that the solution contains three different [Aß+SFN] complexes due to covalent binding of SFN to Aß at three different sites. The in-source fragmentation experiments revealed that SFN is binding to free NH(2) groups (N-terminal amino acid and lysines) in Aß. Trypsin digestion experiments further confirmed the SFN binding sites in Aß. CONCLUSIONS: The interaction of SFN, an anticancer agent, with Aß was studied using ESI-MS. SFN is found to bind covalently and specifically with the free NH(2) group of N-terminal aspartic acid and the ε-amino group of lysine at positions 16 and 28. Aggregation assay studies showed a lesser inclination of Aß to aggregate when SFN is present. Hence the present study helps in understanding the mechanism of the action of SFN on the Aß peptide.