Examining the Associations Between Substance Misuse and Suicide Bereavement.

Focusing on the understudied question of substance misuse among suicide bereaved adults we investigated patterns of binge drinking and non-prescribed drug use among a recently bereaved sample (n = 1,132). Comparing our respondents to the non-bereaved, those in the 2022 National Survey of Drug Use and Health (n = 71,369), we did not find heightened problematical substance misuses among our respondents. With t-tests and multiple regression analyses we examined whether binge drinkers and non-prescribed drug users had heightened levels of grief difficulties, PTSD, self-blaming and depression compared to others not bingeing or using non-prescribed drugs. Results showed binge drinkers had more of all these grieving problems when important confounding variables were also considered. Analysis of the demographic correlates of bingeing showed them dimly aware of their own additional grieving and substance misusing problems. Since 75% indicated being under the care of counseling professionals, this represents an important opportunity for psycho-educational helping.

Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Diverse Catalytic Reactions for the Stereoselective Synthesis of Cyclic Dinucleotide MK-1454.

As practitioners of organic chemistry strive to deliver efficient syntheses of the most complex natural products and drug candidates, further innovations in synthetic strategies are required to facilitate their efficient construction. These aspirational breakthroughs often go hand-in-hand with considerable reductions in cost and environmental impact. Enzyme-catalyzed reactions have become an impressive and necessary tool that offers benefits such as increased selectivity and waste limitation. These benefits are amplified when enzymatic processes are conducted in a cascade in combination with novel bond-forming strategies. In this article, we report a highly diastereoselective synthesis of MK-1454, a potent agonist of the stimulator of interferon gene (STING) signaling pathway. The synthesis begins with the asymmetric construction of two fluoride-bearing deoxynucleotides. The routes were designed for maximum convergency and selectivity, relying on the same benign electrophilic fluorinating reagent. From these complex subunits, four enzymes are used to construct the two bridging thiophosphates in a highly selective, high yielding cascade process. Critical to the success of this reaction was a thorough understanding of the role transition metals play in bond formation.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Suicide and Life Insurance.

Much has been written about the history of suicide and, notably, about societies that condemned both the act and the actor, resulting in a perpetuation of suicide being stigmatized in many cultures. One aspect of this perceived stigmatization involves exclusionary clauses in life insurance policies that reject paying benefits to survivor-beneficiaries of the decedent if the decedent has died by suicide within a prescribed time frame. From the perspective of the individual, life insurance is designed to protect the estate of a decedent from a significant financial burden. From the insurer's perspective, there are essentially 2 reasons for having a suicide exclusion clause: limiting risk and preventing or discouraging fraud. This column examines these rationales in light of the estimated few suicides that do occur during exclusionary clause time frames. Observations are made about the effect of these clauses on those impacted by the loss of a loved one who died by suicide within the exclusionary time frame. An examination of the perspectives of both the life insurance industry and the impacted survivors of suicide decedents raises questions about what are reasonable and appropriate exclusionary clause time frames that protect both the insurer and survivor-beneficiaries. The forensic expert consulting on such cases should be cognizant of these competing perspectives and engage in therapeutic assessment whenever possible, identifying opportunities to promote thoughtful suicide postvention.

A Comparison of Physician-Assisted/Death-With-Dignity-Act Death and Suicide Patterns in Older Adult Women and Men.

OBJECTIVE: To examine Oregon's Death-with-Dignity-Act (DWDA) death and suicide patterns among women age 65 and older, relative to patterns among same-age men, as a way to assess DWDA's impact on older adult women, a group considered vulnerable. DESIGN: Oregon's 1998-2018 DWDA- and suicide-mortality rates and confidence intervals were calculated. RESULTS: Between 1998 and 2018 women age 65 and older represented 46% of DWDA deaths and 16.3% of suicides in their age group. Among women age 65 and older DWDA and suicide mortality increased whereas among same-age men DWDA deaths increased and suicides declined. DWDA deaths were the most common form (52.7%) of self-initiated death for older adult women, and firearm suicides (65.7%) for older adult men. CONCLUSION: Legalization has a substantial impact on older adult women's engagement in self-initiated death. In Switzerland and in Oregon, where assisted suicide/medical-aid-in-dying (MAID) is legal and where assisted-suicide/MAID and suicide comparative-studies have been conducted, older adult women avoid self-initiated death except when physician-approved. Older adult women's substantial representation among assisted-suicide/MAID decedents, relative to suicide, may be a clue of their empowerment to determine the time of their death, when hastened-death assistance is permitted; or of their vulnerability to seeking a medicalized self-initiated death, when in need of care.

Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE.

Recent advances in biocatalysis and directed enzyme evolution has led to a variety of enzymatically-driven, elegant processes for active pharmaceutical ingredient (API) production. For biocatalytic processes, quantitation of any residual protein within a given API is of great importance to ensure process robustness and quality, pure pharmaceutical products. Typical analytical methods for analyzing residual enzymes within an API, such as enzyme-linked immunosorbent assays (ELISA), colorimetric assays, and liquid chromatographic techniques, are limited for determining only the concentration of known proteins and require harsh solvents with high API levels for analysis. For the first time, total residual protein content in a small molecule API was quantitated using image analysis applied to SDS-PAGE. Herein, a proposed methodology for residual protein detection, quantitation, and size-based speciation is presented, in which an orthogonal technique is offered to traditional analysis methods, such as ELISA. Results indicate that our application of the analytical methodology is able to reliably quantitate both protein standards and the total residual protein present within a final API, with good agreement as compared to traditional ELISA results. Further, speciation of the residual protein within the API provides key information concerning the individual residual proteins present, including their molecular weight, which can lead to improved process development efforts for residual protein rejection and control. This analytical methodology thus offers an alternative tool for easily identifying, quantitating, and speciating residual protein content in the presence of small molecule APIs, with potential for wide applicability across industry for biocatalytic or directed enzyme evolution efforts within process development.

Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.

Deep Dive into Machine Learning Models for Protein Engineering.

Protein redesign and engineering has become an important task in pharmaceutical research and development. Recent advances in technology have enabled efficient protein redesign by mimicking natural evolutionary mutation, selection, and amplification steps in the laboratory environment. For any given protein, the number of possible mutations is astronomical. It is impractical to synthesize all sequences or even to investigate all functionally interesting variants. Recently, there has been an increased interest in using machine learning to assist protein redesign, since prediction models can be used to virtually screen a large number of novel sequences. However, many state-of-the-art machine learning models, especially deep learning models, have not been extensively explored. Moreover, only a small selection of protein sequence descriptors has been considered. In this work, the performance of prediction models built using an array of machine learning methods and protein descriptor types, including two novel, single amino acid descriptors and one structure-based three-dimensional descriptor, is benchmarked. The predictions were evaluated on a diverse collection of public and proprietary data sets, using a variety of evaluation metrics. The results of this comparison suggest that Convolution Neural Network models built with amino acid property descriptors are the most widely applicable to the types of protein redesign problems faced in the pharmaceutical industry.

Hemoprotein-Catalyzed Cyclopropanation En Route to the Chiral Cyclopropanol Fragment of Grazoprevir.

Reactions that were once the exclusive province of synthetic catalysts can increasingly be addressed using biocatalysis. Through discovery of unnatural enzyme reactions, biochemists have significantly expanded the reach of enzymatic catalysis to include carbene transfer chemistries including olefin cyclopropanation. Here we describe hemoprotein cyclopropanation catalysts derived from thermophilic bacterial globins that react with diazoacetone and an unactivated olefin substrate to furnish a cyclopropyl ketone, a previously unreported reaction for enzyme catalysts. We further demonstrate that the resulting cyclopropyl ketone can be converted to a key cyclopropanol intermediate that occurs en route to the anti-hepatitis C drug grazoprevir.

Identifying the Social Demographic Correlates of Suicide Bereavement.

We investigated the demographic correlates associated with suicide bereavement among a representative sample of U.S. adults from the 2016 General Social Survey. A secondary aim of this study was to use this representative data platform to cross-check official data findings of U.S. completed suicides. Questions on suicide bereavement were administered to 1,432 GSS 2016 respondents and these were cross-tabulated with various demographic and social activity variables included in this omnibus survey to investigate whether suicide bereaved respondents shared any distinctive demographic characteristics. Findings showed that friends of the suicide deceased person outnumbered the deceased's first degree relatives by at least 2 to 1. We also observed older, White, Non-Hispanic, and native born women were over-represented among the suicide bereaved. The suicide bereaved were also less likely to live in the Pacific region, and to live in the nation's largest cities, and were more likely to come from homes where a gun was owned by someone in the household. More of the suicide bereaved reported themselves to be in poorer physical health and bereaved women were more likely to be Facebook subscribers. These findings are consistent with other data on U.S. suicide patterns and the greater likelihood of firearms being utilized in suicide deaths. These revealed demographic correlates of suicide offer valuable information to helping agencies seeking to reach potential clients among the suicide bereaved.

Suicide Bereavement and Differences in Religiosity.

Background: The trauma from experiencing a loved one's suicide is often seen as an instigator of change in a person's religious life. Aims: We sought to examine whether suicide-bereaved adults were any differently disposed to religious participation and observances compared with the nonbereaved and whether religiously involved bereaved had any better mental health compared with religiously disaffiliated bereaved. Method: The 2016 General Social Survey (GSS) presented 11 new questions identifying suicide bereavement status. We examined how the bereaved (n = 516) compared with the nonbereaved (n = 916) in terms of their religious beliefs and participation. We also investigated whether suicide bereaved religiously committed adherents, who prayed at least weekly (n = 372), showed any better mental health compared with bereaved who were religiously disaffiliated (n = 102). Results: Initially, results showed the bereaved more inclined to pray and to believe in an afterlife compared with the nonbereaved. However, after sex differences were controlled for, most of the remaining differences between these contrasted groups faded. Limitations: Caution is advised regarding generalizations from these data to all subgroups of suicide bereaved due to the modest number of respondents in many subpopulations. The GSS does not include potentially important grief-related indices, and importantly, most of the current sample were friends of the deceased and not first-degree relatives. Conclusion: We discuss the implications of these findings and the need for further research on the interconnections between religiosity and suicide bereavement.

Suicide exposures and bereavement among American adults: Evidence from the 2016 General Social Survey.

BACKGROUND: We investigated lifetime suicide exposures and bereavement among a representative sample of American adults from the 2016 General Social Survey. METHODS: Questions on lifetime suicide exposures, bereavement and mental health status were administered to 1432 respondents. Suicide exposed and bereaved respondents were compared to non-exposed respondents on three different measures of mental health functioning with cross tabulations and means comparison tests. RESULTS: 51% of respondents had exposures to one suicide or more during their lifetimes, and 35% were deemed bereaved by suicide, having experienced moderate to severe emotional distress from their losses. Findings suggested more exposures and bereavements were associated with greater numbers of bad mental health days and more expectations of "having nervous breakdowns" but with no clear associations with CES-D scores. CONCLUSIONS: These findings suggest suicide exposures and bereavement are far more pervasive than commonly thought, with more than half of the population exposed and a third bereaved. Health professionals need to more actively assess for suicide exposures and bereavements, and be vigilant for significant impacts of suicide even when the suicide decedent is not a first degree family relative, helping to reduce the mental health distress presently associated with these experiences.

Molecular basis for the broad substrate selectivity of a peptide prenyltransferase.

The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.

Metabolic model for diversity-generating biosynthesis.

A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.

Highly Stereoselective Biocatalytic Synthesis of Key Cyclopropane Intermediate to Ticagrelor.

Extending the scope of biocatalysis to important non-natural reactions such as olefin cyclopropanation will open new opportunities for replacing multi-step chemical syntheses of pharmaceutical intermediates with efficient, clean, and highly selective enzyme-catalyzed processes. In this work, we engineered the truncated globin of Bacillus subtilis for the synthesis of a cyclopropane precursor to the antithrombotic agent ticagrelor. The engineered enzyme catalyzes the cyclopropanation of 3,4-difluorostyrene with ethyl diazoacetate on a preparative scale to give ethyl-(1R, 2R)-2-(3,4-difluorophenyl)-cyclopropanecarboxylate in 79% yield, with very high diastereoselectivity (>99% dr) and enantioselectivity (98% ee), enabling a single-step biocatalytic route to this pharmaceutical intermediate.

Enantioselective Enzyme-Catalyzed Aziridination Enabled by Active-Site Evolution of a Cytochrome P450.

One of the greatest challenges in protein design is creating new enzymes, something evolution does all the time, starting from existing ones. Borrowing from nature's evolutionary strategy, we have engineered a bacterial cytochrome P450 to catalyze highly enantioselective intermolecular aziridination, a synthetically useful reaction that has no natural biological counterpart. The new enzyme is fully genetically encoded, functions in vitro or in whole cells, and can be optimized rapidly to exhibit high enantioselectivity (up to 99% ee) and productivity (up to 1,000 catalytic turnovers) for intermolecular aziridination, demonstrated here with tosyl azide and substituted styrenes. This new aziridination activity highlights the remarkable ability of a natural enzyme to adapt and take on new functions. Once discovered in an evolvable enzyme, this non-natural activity was improved and its selectivity tuned through an evolutionary process of accumulating beneficial mutations.

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms.

Recognition sequences and substrate evolution in cyanobactin biosynthesis.

Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit "substrate evolution", where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme-substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.