Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

Large outbreak of Jimsonweed (Datura stramonium) poisoning due to consumption of contaminated humanitarian relief food: Uganda, March-April 2019.

BACKGROUND: Jimsonweed (Datura stramonium) contains toxic alkaloids that cause gastrointestinal and central nervous system symptoms when ingested. This can be lethal at high doses. The plant may grow together with leguminous crops, mixing with them during harvesting. On 13 March 2019, more than 200 case-patients were admitted to multiple health centres for acute gastrointestinal and neurologic symptoms. We investigated to determine the cause and magnitude of the outbreak and recommended evidence-based control and prevention measures. METHODS: We defined a suspected case as sudden onset of confusion, dizziness, convulsions, hallucinations, diarrhoea, or vomiting with no other medically plausible explanations in a resident of Napak or Amudat District from 1 March-30 April 2019. We reviewed medical records and canvassed all villages of the eight affected subcounties to identify cases. In a retrospective cohort study conducted in 17 villages that reported the earliest cases, we interviewed 211 residents about dietary history during 11-15 March. We used modified Poisson regression to assess suspected food exposures. Food samples underwent chemical (heavy metals, chemical contaminants, and toxins), proteomic, DNA, and microbiological testing in one national and three international laboratories. RESULTS: We identified 293 suspected cases; five (1.7%) died. Symptoms included confusion (62%), dizziness (38%), diarrhoea (22%), nausea/vomiting (18%), convulsions (12%), and hallucinations (8%). The outbreak started on 12 March, 2-12 h after Batch X of fortified corn-soy blend (CSB +) was distributed. In the retrospective cohort study, 66% of 134 persons who ate CSB + , compared with 2.2% of 75 who did not developed illness (RRadj = 22, 95% CI = 6.0-81). Samples of Batch X distributed 11-15 March contained 14 tropane alkaloids, including atropine (25-50 ppm) and scopolamine (1-10 ppm). Proteins of Solanaceae seeds and Jimsonweed DNA were identified. No other significant laboratory findings were observed. CONCLUSION: This was the largest documented outbreak caused by food contamination with tropane alkaloids. Implicated food was immediately withdrawn. Routine food safety and quality checks could prevent future outbreaks.

Comparative analysis of INLIGHT™-labeled enzymatically depolymerized heparin by reverse-phase chromatography and high-performance mass spectrometry.

Structural characterization of the microheterogeneity of heparin, heparan sulfate, and other glycosaminoglycans is a major analytical challenge. We present the use of a stable isotope-labeled hydrazide tag (INLIGHT™) with high-resolution/accurate mass (HRAM) reverse-phase LC-MS/MS, which was recently introduced for detailed study of N-glycan heterogeneity, to characterize heparinase-digested heparin (digHep) products without the use of semi-volatile ion pairing reagents. Using both full scan LC-MS and data-dependent LC-MS/MS, we identified 116 unique digHep species, a feat possible because of INLIGHT™ labeling. Of these, 83 digHep products were structurally identified, including the 12 standard disaccharides as well as 34 tetra- (DP4), 26 hexa- (DP6), 21 octa- (DP8), and 2 decasaccharides (DP10). Each of the 116 digHep species co-eluted with both light and heavy INLIGHT™ tags (L/Havg = 1.039 ± 0.163); thus enhancing confidence in their identification via MS and MS/MS. This work sets the foundation for INLIGHT™-based comparative analyses of different forms of heparin, heparan sulfate, and other GAGs with high quantitative precision using mainstay reverse-phase HRAM LC-MS/MS. Graphical Abstract Reducing end labeling strategy for mapping depolymerized heparin/heparan sulfate products by reverse-phase LC-MS/MS.

Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.

DNA alkylation with N-methylquinolinium quinone methide to N2-dG adducts resulting in extensive stops in primer extension with DNA polymerases and subsequent suppression of GFP expression in A549 cells.

Quinone methides (QMs) are involved in the metabolism of many drugs and carcinogens as reactive intermediates to form covalent nucleobase adducts in DNA that associate with high mutagenicity. Recently, a plethora of synthetic QM DNA alkylating agents have been developed to form various nucleobase adducts as potential antitumor agents. However, the mutagenic potential of these synthetic QM alkylating agents has not been fully investigated. In this report, N-methylquinolinium QM was developed as a synthetic model to study biological consequences of the formation of nucleobase adducts in a DNA target. N-Methylquinolinium QM was generated in situ via an elimination process from a bis-quaternary ammonium precursor that was synthesized from a quinoline derivative. Alkylation with N-methylquinolinium QM on a DNA target produced mostly a stable N(2)-dG adduct as revealed by gel electrophoresis and DNA digestion assays and confirmed by mass and NMR analyses. The formation of N(2)-dG adducts of a DNA target was found to cause extensive stops in the primer extension with high fidelity DNA polymerase T7 and even low fidelity error prone Dpo4. The direct biological impact of a prealkylated green fluorescence protein plasmid with N-methylquinolinium QM was demonstrated as significant suppression of protein expression in A549 cells. Overall, our results suggested that nucleobase-QM adducts could potentially block nucleobase mismatch/translesion in the error-prone process to reduce the mutagenic potential if designed carefully.

Origin determination of the Eastern oyster (Crassostrea virginica) using a combination of whole-body compound-specific isotope analysis and heavy metal analysis.

Various samples of the Eastern oyster, Crassostrea virginica, were collected from five harvest bay areas in the Gulf of Mexico coastal waters of Florida (FL), Louisiana (LA) and Texas (TX). Cadmium and lead concentrations from the extracted whole-body soft tissues were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS), and bulk δ13C and δ15N isotope ratios and amino-acid-specific δ13C values were analyzed via isotope ratio mass-spectrometry (IRMS). The combined data was subjected to multivariate statistical analysis to assess whether oysters could be linked to their harvest area. Results indicate that discriminant analysis using the δ13C values of five amino acids-serine, glycine, valine, lysine and phenylalanine-could discriminate oysters from two adjacent harvesting in Florida with 90% success rate, using leave-one-out cross validation. The combination of trace elements and isotope ratios could also predict geographic provenance of oysters with a success rate superior to the isolated use of each technique. The combinatory approach proposed in this study is a proof-of-concept that compound specific stable isotope analysis is a potential tool for oyster fisheries managers, wildlife, and food safety enforcement officers, as well as to forensics and ecology research areas, although significantly more work would need to be completed to fully validate the approach and achieve more reliable statistical results.

Investigation into the Concentrations and Sources of Nitrates and Nitrites in Milk and Plant-Based Powders.

Milk powders in the United States (US) may contain nitrates and nitrites from several potential sources. These sources include the ingestion of nitrates and nitrites by dairy cows during grazing and drinking, nitric acid used during the sanitization of dairy equipment, and the production of nitrous oxides in directly heated spray dryers. Recently, milk powders manufactured in the US have been rejected during import to other countries because nitrite concentrations were greater than 2 mg/kg (ppm). To date, the concentrations of nitrates and nitrites in milk and plant-based powders in the US are unknown. In this study the nitrate and nitrite concentrations present in diverse milk powders were investigated including 81 milk powders from local and online retailers from 2015 to 2018. In addition, 71 commercial milk powders were obtained from blinded production facilities. Nitrate and nitrite concentrations were determined using ion chromatography with conductivity and UV detection. A subset of samples was analyzed for N-nitrosodimethylamine using gas chromatography-mass spectrometry. Carbon and nitrogen bulk isotope ratios analyzed using isotope ratio mass spectrometry were used to obtain some insights into the production method (organic vs conventional) and geographic source of the milk powder samples. Background nitrate concentrations in US-produced milk powder samples averaged 17 ± 12 mg/kg. Nitrite was detected at concentrations greater than 2 mg/kg in 5 out of 39 different brands of retail milk and plant-based powders. Of these brands, two were plant-based (soy and coconut) powders and the other three had consistently high nitrites. The analysis of milk powders using stable isotope analysis revealed further information about the cow's diet.

Interaction of trivalent antimony with a CCHC zinc finger domain: potential relevance to the mechanism of action of antimonial drugs.

Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the NCp7 protein of HIV-1, indicating that zinc finger proteins may be targets for antimony-based drugs and thus responsible for their important pharmacological actions.

New insights into the chemical structure and composition of the pentavalent antimonial drugs, meglumine antimonate and sodium stibogluconate.

The chemical structures of the pentavalent antimonial drugs, meglumine antimonate (MA) and sodium stibogluconate (SSG), were re-evaluated using electrospray ionization mass spectrometry (ESI-MS) and osmolarity measurements. Both MA and SSG were found to contain 1:1, 1:2, 2:2 and 2:3 Sb(V)-ligand complexes. ESI-MS analysis of MA showed negatively-charged 1:1 (m/z 364) and 2:2 (m/z 765) Sb(V)-meglumine complexes, supporting the predominance of zwitterionic species in solution. Our data are consistent with a structure for the 1:2 Sb(V)-meglumine, which differs from that previously postulated, with two positively-charged amino groups and one negatively-charged antimonate group. Instead of the commonly hypothesized structure for SSG, in which two Sb atoms are linked by an oxygen, an alternative structure is proposed, based on the ability of Sb(V)-gluconate complexes to polymerize. MA (or SSG) in concentrated aqueous solutions, such as of MA (or SSG) in its commercial form, is expected to consist mainly of a mixture of 2:2, 2:3 and 2:1 Sb(V)-ligand complexes, as suggested by the 2:1 Sb-to-particle ratio found by osmometry. 1:1 Sb(V)-ligand complexes in MA and SSG are expected to play an important pharmacological role, as suggested by the slow increase of osmolarity of MA solution upon dilution at 37 degrees C (half-time of 20min).

Au(iii) compounds as HIV nucleocapsid protein (NCp7)-nucleic acid antagonists.

The HIV nucleocapsid NCp7-SL2 RNA interaction is interrupted in the presence of a formally substitution-inert gold(dien)-nucleobase/N-heterocycle AuN4 compound where the N-heterocycle serves the dual purposes of a template for "non-covalent" molecular recognition of the essential tryptophan of the protein, mimicking the natural reaction and subsequent "fixation" by Au-Cys bond formation providing a chemotype for a new distinct class of nucleocapsid-nucleic acid antagonist.

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: piperidine-induced destabilization of the DNA duplex?

Strategies to produce single-stranded PCR amplicons for detection by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) were investigated using modified electrospray solutions and by thermally denaturing the duplex structures with a resistively heated electrospray ionization source. A synthetic 20-mer oligonucleotide annealed to its complementary strand was used as a model system for initial experiments. Electrospray solutions were altered by varying the relative proportion of aqueous phase in efforts to induce destabilization of the double helix. When the electrospray solution contains a 25% aqueous content, the 20-mer oligonucleotide is detected in its double-stranded form. Increasing the proportion of aqueous phase in the electrospray solution to 60% destabilized the double helix, resulting in the detection of only single-stranded species. This strategy was extended to an 82-bp polymerase chain reaction (PCR) product derived from the human tyrosine hydroxylase gene (HUMTH01). In efforts to destabilize the 82-bp PCR product, electrospray solutions reaching 70% aqueous content were necessary to promote the detection of only single-stranded amplicons. Implementation of the resistively heated transfer line and an electrospray solution in which the oligonucleotide is on the threshold of duplex stability allowed for double-stranded and single-stranded species to be generated from the same ESI solutions at both ambient and elevated transfer line temperatures, respectively, without disruption of the electrospray process. The volatile base piperidine, present at 20 mM concentrations in the electrospray solution, was found to play a critical role in the formation of single-stranded species at the higher aqueous percentages and a duplex destabilization mechanism has been proposed.

A new approach to glycan targeting: enzyme inhibition by oligosaccharide metalloshielding.

Metalloglycomics - the effects of defined coordination compounds on oligosaccharides and their structure and function - opens new areas for bioinorganic chemistry and expands its systematic study to the third major class of biomolecules after DNA/RNA and proteins.

Zinc finger peptide cleavage by a dinuclear platinum compound.

Electrospray ionisation mass spectrometry (ESI-MS) and {(1)H, (15)N HSQC} NMR spectroscopy has elucidated the binding of a dinuclear platinum compound to a zinc finger peptide with induced backbone cleavage. Cleavage is selective on the N-terminal side of the cysteine residue following incubation at neutral pH, and is further dependent on structure of dinuclear compound.

Interaction of arsenite with a zinc finger CCHC peptide: evidence for formation of an As-Zn-peptide mixed complex.

The interaction of arsenite with a Cys(3)His (CCHC) zinc finger model (34-51) HIV-1 nucleocapsid protein p7 (NCp7) peptide in the absence and presence of Zn(II) was studied using fluorescence spectroscopy, CD (circular dichroism) and ESI-MS (Electrospray Ionization Mass Spectrometry). We found that arsenic forms different complexes with the free peptide and the zinc finger peptide. In the former case the peptide conformation differed greatly from that of the zinc finger, whereas in the second case a mixed As-Zn-peptide complex was formed with partial preservation of zinc finger conformation. An apparent stability constant was estimated for the mixed As-Zn-peptide complex (K=2083 M(-1) and 442 M(-1) at 25°C and pHs 6 and 7, respectively). Our study also shows that the interaction of arsenic with the CCHC motif is facilitated by glutathione (GSH), through formation of a GS-As-peptide conjugate.

Excursions in polynuclear platinum DNA binding.

Polynuclear platinum agents are a structurally unique class of anti-cancer drugs, distinct from the cisplatin family. To describe the chemistry and biology of this class, it was necessary to challenge the accepted paradigms for the structure-activity relationships; design new chemotypes and delineate the structures and consequences of their DNA binding modes. This article summarizes the structural changes induced in DNA by both covalent (bond-forming) and non-covalent (ligand recognition) adducts. Solution (Nuclear Magnetic Resonance), solid state (crystallography) and gas-phase (Electrospray Ionization Mass Spectrometry) techniques have all been used to describe the new DNA structures along with molecular biological techniques. The combined approaches allow molecular description of hitherto unobserved adducts such as long-range major-groove interstrand crosslinks; directional isomers on DNA and a third class of ligand-DNA binding, the phosphate clamp. The phosphate recognition is distinct from ''classic'' minor-groove recognition or intercalation.

Zinc finger proteins as templates for metal ion exchange: Substitution effects on the C-finger of HIV nucleocapsid NCp7 using M(chelate) species (M=Pt, Pd, Au).

The interactions of monofunctional [MCl(chelate)] compounds (M=Pt(II), Pd(II) or Au(III) and chelate=diethylenetriamine, dien or 2,2',2''-terpyridine, terpy) with the C-terminal finger of the HIV nucleocapsid NCp7 zinc finger (ZF) were studied by mass spectrometry and circular dichroism spectroscopy. In the case of [M(dien)] species, Pt(II) and Pd(II) behaved in a similar fashion with evidence of adducts caused by displacement of Pt-Cl or Pd-Cl by zinc-bound thiolate. Labilization, presumably under the influence of the strong trans influence of thiolate, resulted in loss of ligand (dien) as well as zinc ejection and formation of species with only Pd(II) or Pt(II) bound to the finger. For both Au(III) compounds the reactions were very fast and only "gold fingers" with no ancillary ligands were observed. For all terpyridine compounds ligand scrambling and metal exchange occurred with formation of [Zn(terpy)](2+). The results conform well to those proposed from the study of model Zn compounds such as N,N'-bis(2-mercapto-ethyl)-1,4-diazacycloheptanezinc(II), [Zn(bme-dach)](2). The possible structures of the adducts formed are discussed and, for Pt(II) and Pd(II), the evidence for possible expansion of the zinc coordination sphere from four- to five-coordinate is discussed. This observation reinforces the possibility of change in geometry for zinc in biology, even in common "structural" sites in metalloenzymes. The results further show that the extent and rate of zinc displacement by inorganic compounds can be modulated by the nature (metal, ligands) of the reacting compound.

Reactions of palladium and gold complexes with zinc-thiolate chelates using electrospray mass spectrometry and X-ray diffraction: molecular identification of [Pd(bme-dach)], [Au(bme-dach]+ and [ZnCl(bme-dach)]2Pd.

The reaction between the complexes [MCl(L)]Cl(x) (L = 2,2',2''-terpyridine, terpy and dien, diethylenetriamine; M = Pd, x = 1; M = Au, x = 2) and [Zn(bme-dach)](2), an N(2)S(2)-Zn-thiolate bridged dimer used to mimic zinc finger protein sites, was studied by Electrospray Ionisation Mass Spectrometry and the structures of some of the products confirmed by X-ray crystallography. All reactions investigated in this work gave heteronuclear (Zn-thiolate)-metal products, the predominant species being the trinuclear dithiolate-bridged aggregate {[Zn(bme-dach)](2)M}(n+) (M = Pd, Au). X-Ray diffraction studies verified the molecular structure of [{ZnCl(bme-dach)}(2)Pd], and further confirmed that the zinc within the [Zn(bme-dach)](2) unit was retained within the N(2)S(2) binding site. The Zn-bound thiolates form stable thiolate bridges to Pd(2+) in a stair-step shape, held together by a planar PdS(4) center. In addition, both zinc atoms maintained penta-coordinate coordination with apical chloride ligands rather than the more commonly observed tetrahedral geometry. Further, [Pd(bme-dach)] was directly synthesized for X-ray structural characterization of the metal exchanged product observed in mass spectrometry experiments. In the case of Au compounds, the reactions were very fast and the products were similar for both [AuCl(L)]Cl(2) (L = terpy and dien) starting materials. In addition to the multimetallic Zn,Au,Zn aggregate formation, the predominant species from the reaction between [Zn(bme-dach)](2) and both Au compounds was the [Au(bme-dach](+) cation observable via ESI-MS, suggesting Zn/Au metal exchange immediately after mixing the compounds. The direct synthesis of [Au(bme-dach)]BPh(4) confirmed the molecular structure of this species through X-ray crystallography. The reactivity profile of Pd(2+) and Au(3+) species is compared with previous studies using the isostructural Pt compounds and the biological relevance of the results discussed.

Transferring the concept of multinuclearity to ruthenium complexes for improvement of anticancer activity.

Multinuclear platinum anticancer complexes are a proven option to overcome resistance of established anticancer compounds. Transferring this concept to ruthenium complexes led to the synthesis of dinuclear Ru(II)-arene compounds containing a bis(pyridinone)alkane ligand linker. A pronounced influence of the spacer length on the in vitro anticancer activity was found, which is correlated to the lipophilicity of the complexes. IC(50) values in the same dimension as for established platinum drugs were found in human tumor cell lines. No cross-resistance to oxoplatin, a cisplatin prodrug, was observed for the most active complex in three resistant cell lines; in fact, a 10-fold reversal of sensitivity in two of the oxoplatin-resistant lines was found. (Bio)analytical characterization of the representative examples showed that the ruthenium complexes hydrolyze rapidly, forming predominantly diaqua species that exhibit affinity toward transferrin and DNA, indicating that both proteins and nucleobases are potential targets.

Selective N1-alkylation of 2'-deoxyguanosine with a quinolinyl quinone methide.

Nucleobase modification by quinone methides (QMs) has been extensively studied in the past decade, and multiple QM adducts were observed. For 2'-deoxyguanosine (dG), the N (2)-dG alkylation adduct was favored under aqueous buffered conditions over other N1-dG, N7-dG, and N7-guanine adducts. We report in this communication that the N1-dG adduct was selectively formed with a quinolinyl QM in 30% aqueous DMF and 10 mM phosphate buffer (pH 7.0) as a favored dG alkylation product. The quinolinyl QM was formed through the fluoride-induced desilylation and elimination of acetate, and the structure of the N1-dG adduct was fully established by one- and two-dimensional NMR analyses. In addition, the concentration of salt played a significant role in N1-dG adduct formation. Further HPLC analysis indicated that the addition of salt decreased the rate of QM formation from the acetate intermediate, although an in-depth mechanistic study is needed.

Pre-association of polynuclear platinum anticancer agents on a protein, human serum albumin. Implications for drug design.

The interactions of polynuclear platinum complexes with human serum albumin were studied. The compounds examined were the "non-covalent" analogs of the trinuclear BBR3464 as well as the dinuclear spermidine-bridged compounds differing in only the presence or absence of a central -NH(2)-(+) (BBR3571 and analogs). Thus, closely-related compounds could be compared. Evidence for pre-association, presumably through electrostatic and hydrogen-bonding, was obtained from fluorescence and circular dichroism spectroscopy and Electrospray Ionization Mass Spectrometry (ESI-MS). In the case of those compounds containing Pt-Cl bonds, further reaction took place presumably through displacement by sulfur nucleophiles. The implications for protein pre-association and plasma stability of polynuclear platinum compounds are discussed.