How culturally unique are pandemic effects? Evaluating cultural similarities and differences in effects of age, biological sex, and political beliefs on COVID impacts.

Despite being bio-epidemiological phenomena, the causes and effects of pandemics are culturally influenced in ways that go beyond national boundaries. However, they are often studied in isolated pockets, and this fact makes it difficult to parse the unique influence of specific cultural psychologies. To help fill in this gap, the present study applies existing cultural theories via linear mixed modeling to test the influence of unique cultural factors in a multi-national sample (that moves beyond Western nations) on the effects of age, biological sex, and political beliefs on pandemic outcomes that include adverse financial impacts, adverse resource impacts, adverse psychological impacts, and the health impacts of COVID. Our study spanned 19 nations (participant N = 14,133) and involved translations into 9 languages. Linear mixed models revealed similarities across cultures, with both young persons and women reporting worse outcomes from COVID across the multi-national sample. However, these effects were generally qualified by culture-specific variance, and overall more evidence emerged for effects unique to each culture than effects similar across cultures. Follow-up analyses suggested this cultural variability was consistent with models of pre-existing inequalities and socioecological stressors exacerbating the effects of the pandemic. Collectively, this evidence highlights the importance of developing culturally flexible models for understanding the cross-cultural nature of pandemic psychology beyond typical WEIRD approaches.

Combined application of high resolution and tandem mass spectrometers to characterize methionine oxidation in a parathyroid hormone formulation.

Identification and monitoring of degradation products is a critical aspect of drug product stability programs. This process can present unique challenges when working with complex biopharmaceutical formulations that do not readily lend themselves to straightforward HPLC analysis. The therapeutic 34 amino acid parathyroid hormone fragment (PTH1-34) contains methionine (Met) residues at positions 8 and 18. Oxidation of these Met residues results in reduced biological activity and thus efficacy of the potential drug product. Here, we present an effective approach for the identification of PTH1-34 oxidation products in a drug product formulation in which the stability indicating method used non-MS compatible HPLC conditions to separate excipients, drug substance and degradation products. High resolution and tandem mass spectrometers were used in conjunction with cyanogen bromide (CNBr) mediated digestion to accurately identify the oxidation products observed in an alternative MS compatible HPLC method used for drug substance analysis. All anticipated CNBr digested peptide fragments, including both oxidized and nonoxidized peptide fragments, were positively identified using TOF MS without the need for additional enzymatic digestion. Once identified, the oxidation products generated were injected onto the original non-MS compatible HPLC drug product stability indicating method and the respective retention times were confirmed. This allowed the oxidative stability of different formulations to be effectively monitored during the solid state stability program and during variant selection.

Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human multiple myeloma xenograft and in monkeys.

PURPOSE: IMMU-110 is a drug immunoconjugate composed of doxorubicin conjugated to the humanized anti-CD74 monoclonal antibody, hLL1, at a doxorubicin/monoclonal antibody ratio of approximately 8:1 (mol/mol). CD74 is a rapidly internalizing molecule associated with HLA-DR, which has high expression by several tumor types. Here, we describe safety evaluations of IMMU-110 in mice and monkeys as well as efficacy studies in a xenograft model of the human multiple myeloma cell line, MC/CAR. EXPERIMENTAL DESIGN: In vitro binding of IMMU-110 was determined by a cell-based ELISA and cytotoxicity of IMMU-110 assayed with a tetrazolium assay. Pharmacokinetics and biodistribution of radiolabeled IMMU-110 were examined in tumor-free BALB/c mice, and the therapeutic effectiveness was evaluated in severe combined immunodeficient mice bearing MC/CAR cells. Acute toxicity of IMMU-110 was studied in CD74-positive cynomolgus monkeys (Macaca fascicularis). RESULTS: In vitro, IMMU-110 specifically binds to CD74 and is cytotoxic against MC/CAR cells. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile identical to that of unconjugated hLL1 monoclonal antibody, except for higher kidney uptake. Treatment with a single dose of IMMU-110 as low as 50 microg antibody/mouse (or 1.4 microg doxorubicin/mouse), 5 days postinjection of the multiple myeloma cells, resulted in cure of most mice. In mice, no host toxicity of IMMU-110 was observed at the highest protein dose tested (125 mg/kg). In cynomolgus monkeys, bone marrow toxicity was observed at 30 and 90 mg/kg doses. CONCLUSIONS: The excellent safety and efficacy profile of IMMU-110 supports clinical testing of this immunoconjugate in the treatment of CD74-positive B-cell malignancies.

Mutagenesis studies of protein farnesyltransferase implicate aspartate beta 352 as a magnesium ligand.

Protein farnesyltransferase (FTase) catalyzes the addition of a farnesyl chain onto the sulfur of a C-terminal cysteine of a protein substrate. Magnesium ions enhance farnesylation catalyzed by FTase by several hundred-fold, with a KMg value of 4 mM. The magnesium ion is proposed to coordinate the diphosphate leaving group of farnesyldiphosphate (FPP) to stabilize the developing charge in the farnesylation transition state. Here we further investigate the magnesium binding site using mutagenesis and biochemical studies. Free FPP binds Mg2+ with a Kd of 120 microM. The 10-fold weaker affinity for Mg2+ observed for the FTase.FPP.peptide ternary complex is probably caused by the positive charges in the diphosphate binding pocket of FTase. Furthermore, mutation of aspartate beta 352 to alanine (D beta 352A) or lysine (D beta 352K) in FTase drastically alters the Mg2+ dependence of FTase catalysis without dramatically affecting the rate constant of farnesylation minus magnesium or the binding affinity of either substrate. In D beta 352A FTase, the KMg increases 28-fold to 110 +/- 30 mM, and the farnesylation rate constant at saturating Mg2+ decreases 27-fold to 0.30 +/- 0.05 s-1. Substitution of a lysine for Asp-beta 352 removes the magnesium activation of farnesylation catalyzed by FTase but does not significantly enhance the rate constant for farnesylation in the absence of Mg2+. In wild type FTase, Mg2+ can be replaced by Mn2+ with a 2-fold lower KMn (2 mM). These results suggest both that Mg2+ coordinates the side chain carboxylate of Asp-beta 352 and that the role of magnesium in the reaction includes positioning the FPP prior to catalysis.

Structural characterization of the zinc site in protein farnesyltransferase.

X-ray absorption spectroscopy has been used to determine the structure of the Zn site in protein farnesyltransferase. Extended X-ray absorption fine structure (EXAFS) data are consistent with a Zn site that is ligated to three low-Z (oxygen or nitrogen) ligands and one cysteine sulfur, as predicted from the crystal structures that are available for farnesyltransferase. However, in contrast with the crystallographic results the EXAFS data do not show evidence for significant distortions in the Zn-ligand distances. The average Zn-(N/O) and Zn-S distances are 2.04 and 2.31 A, respectively. Addition of a farnesyl diphosphate analogue causes no detectable change in the structure of the Zn site. However, addition of peptide substrate causes a change in ligation from ZnS(N/O)(3) to ZnS(2)(N/O)(2), consistent with ligation of the C-terminal cysteine to the Zn. There is no significant change in Zn-ligand distances when a substrate binds, demonstrating that the Zn remains four-coordinate. Addition of both peptide and farnesyl diphosphate to give the product complex causes the Zn to return to ZnS(N/O)(3) ligation, indicating that the product thioether is not tightly coordinated to the Zn. These spectroscopic experiments provide insight into the catalytic mechanism of FTase.

Kinetic studies of protein farnesyltransferase mutants establish active substrate conformation.

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon farnesyl moiety from farnesyl diphosphate (FPP) to a cysteine residue near the C-terminus of a protein substrate. Several crystal structures of inactive FTase.FPP.peptide complexes indicate that K164alpha interacts with the alpha-phosphate and that H248beta and Y300beta form hydrogen bonds with the beta-phosphate of FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601-16611]. Mutations K164Aalpha, H248Abeta, and Y300Fbeta were prepared and analyzed by single turnover kinetics and ligand binding studies. These mutations do not significantly affect the enzyme affinity for FPP but do decrease the farnesylation rate constant by 30-, 10-, and 500-fold, respectively. These mutations have little effect on the pH and magnesium dependence of the farnesylation rate constant, demonstrating that the side chains of K164alpha, Y300beta, and H248beta do not function either as general acid-base catalysts or as magnesium ligands. Mutation of H248beta and Y300beta, but not K164alpha, decreases the farnesylation rate constant using farnesyl monophosphate (FMP). These data suggest that, contrary to the conclusions derived from analysis of the static crystal structures, the transition state for farnesylation is stabilized by interactions between the alpha-phosphate of the isoprenoid substrate and the side chains of Y300beta and H248beta. These results suggest an active substrate conformation for FTase wherein the C1 carbon of the FPP substrate moves toward the zinc-bound thiolate of the protein substrate to react, resulting in a rearrangement of the diphosphate group relative to its ground state position in the binding pocket.

Photoaffinity analogues of farnesyl pyrophosphate transferable by protein farnesyl transferase.

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized analogues (3-6) of farnesyl pyrophosphate (FPP) to probe the range of modifications possible to the FPP skeleton which allow for efficient transfer by FTase. Photoaffinity analogues of FPP (5, 6) were prepared by substituting perfluorophenyl azide functional groups for the omega-terminal isoprene of FPP. Substituted anilines replace the omega-terminal isoprene in analogues 3 and 4. Compounds 3-5 were prepared by reductive amination of the appropriate anilines with 8-oxo-geranyl acetate, followed by ester hydrolysis, chlorination, and pyrophosphorylation. Additional substitution of three methylenes for the beta-isoprene of FPP gave photoprobe 6 in nine steps. Preparation of the analogues required TiCl(4)-mediated imine formation prior to NaBH(OAc)(3) reduction for anilines with a pK(a) < 1. The azide moiety was not affected by Ph(3)PCl(2) conversion of allylic alcohols 13-16 into corresponding chlorides 17-20. Analogues 3-6 are efficiently transferred to target N-dansyl-GCVLS peptide substrate by mammalian FTase. Comparison of analogue structures and kinetics of transfer to those of FPP reveals that ring fluorination and para substituents have little effect on the affinity of the analogue pyrophosphate for FTase and its transfer efficiency. These results are also supported with models of the analogue binding modes in the active site of FTase. The transferable azide photoprobe 5 photoinactivates FTase. Transferable analogues 5 and 6 allow the formation of appropriately posttranslationally modified photoreactive peptide probes of isoprene function.