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Communication between parents and professionals in the NICU is a necessary part of collaborative decision making in the provision of family-centered care. Decisions with ethical components, those regarding treatment plans or neonatal research enrollment, need to be made conjointly with parents and health care professionals. This article reviews the present state of knowledge of how parents' input can be facilitated in regard to decisions made about their children. Research studies involving decisions made with ethical components in the NICU since the advent of the Baby Doe regulations reveal parents' frustration with communication practices, their need for control of information, and the trust in their children's health care providers that is required to best facilitate their input into ethical decisions made about their children. [ABSTRACT FROM AUTHOR]

Small molecule probes exist for only ∼2% of human proteins because most lack functional binding pockets or cannot be assayed for high-throughput screening. Selective translation modulation circumvents canonical druggability and assay development constraints by using in vitro transcription-translation (IVTT) as a universal biochemical screening assay. We developed an IVTT activity assay by fusing a GFP reporter to various target gene sequences and screened the target sequences for inhibitors in microfluidic picoliter-scale droplets using a 5,348-member translation inhibitor DNA-encoded library (DEL). Screening a proof-of-concept PCSK9-GFP reporter yielded many hits; 6/7 hits inhibited PCSK9-GFP IVTT (IC 50 1-20 μM), and the lead hit reduced PCSK9 levels in HepG2 cells. Preliminary selectivity was informed by counterscreening the DEL against a frameshift mutant PCSK9-GFP reporter. A plug-and-play approach to assay development and screening was demonstrated by scouting the DEL for activity using reporter genes of targets with difficult-to-assay or even unknown function (RPL27, KRAS G12D , MST1, USO1). This microfluidic IVTT DEL screening platform could scale probe discovery to the human proteome and perhaps more broadly across the tree of life., Competing Interests: The authors declare the following competing financial interest(s): M.G.P., P.A.P., F.R.W., and B.O.B. are employees and shareholders of Initial Therapeutics. B.M.P. and K.F.M. are paid consultants for and shareholders of Initial Therapeutics. R.N.P. is a paid consultant of Initial Therapeutics., (© 2024 The Authors. Published by American Chemical Society.)

As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-L-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi-Dunitz angle of 76-115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers., (© 2023. The Author(s).)

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement., Competing Interests: ZW, FW, RM, OA, AS, JB, JC No competing interests declared, (© 2020, Watson et al.)

By transplanting identity elements into E. coli tRNA fMet , we have engineered an orthogonal initiator tRNA (itRNA Ty2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA Ty2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA fMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better in vivo β-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo , it is inactive in vitro , and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.

The drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small number of proteins by an unknown mechanism. In structures of PF846-stalled human ribosome nascent chain complexes, PF846 binds in the ribosome exit tunnel in a eukaryotic-specific pocket formed by 28S ribosomal RNA, and alters the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state of translocation, in which the peptidyl-transfer RNA 3'-CCA end is improperly docked in the peptidyl transferase center. Selections of messenger RNAs from mRNA libraries using translation extracts reveal that PF846 can stall translation elongation, arrest termination or even enhance translation, depending on nascent chain sequence context. These results illuminate how a small molecule selectively targets translation by the human ribosome, and provides a foundation for developing small molecules that modulate the production of proteins of therapeutic interest.

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

Ethics consultation is a familiar concept to clinicians, and there are site-specific guidelines detailing procedures for both obtaining and performing these consults. Evaluative data about clinician experiences with ethics consults are becoming more extensive but information about family experiences, especially parent perceptions, of the same is lacking. Without a better understanding of those family experiences, an evidence base for ethics consultations cannot be built. This manuscript describes the reasons for obtaining this necessary information, details prior research designed to obtain knowledge about family experiences with pediatric ethics consultations, and suggests mechanisms for acquiring it.

This study analyzes the expectations that incoming students and faculty bring to accelerated pre-licensure education programs for second-degree students. Although research supports the congruence of expectations between students and faculty as essential to learning, anecdotal evidence and single case reports suggest there may be important discrepancies in expectations of second-degree students and their faculty. Data are intended to support curriculum review, refinement, and innovation in these programs., (Copyright 2010, SLACK Incorporated.)

It would be useful to researchers and bioethicists to know more about parents' decision processes and emotional state during the time they are deciding whether to enroll their infant in a clinical trial. The aim of this research study was to discover whether parents who had been previously asked to enroll their neonates in clinical trials would have found concurrent research about their decision-making overly burdensome. Twenty-seven parents of critically ill neonates who had been approached for their child's research participation in a clinical trial were asked what they believed about the potential burden or value of being interviewed during the time of research decision-making about their infant's participation. For this qualitative descriptive study, interviews were audio-recorded, transcribed, and analyzed using content analysis techniques. Participants considered concurrent research acceptable for them but potentially problematic for others. Theories of risk preferences and social comparison might explain these findings.

Objective: This study examined parental beliefs about participating in clinical trials involving greater than minimal risk to their neonate, and explored their views of their experiences., Study Design: In this qualitative descriptive study, parents in the neonatal intensive care unit (NICU) who had been approached for permission for their neonates to be enrolled in research were asked to describe their decisions about their consent for or disagreement to their neonate's research participation. A total of 27 parents from three different hospital NICUs in the Mid-Atlantic region of the United States participated. Transcribed interviews were analyzed using qualitative content analysis., Results: Participant decisions developed through a dynamic process of meaning-making based on their beliefs about themselves and their neonates. The processes involved making sense of the chaos that they perceived in the environment and their own vulnerability, through taking control of their situation.

Objectives: When prospective informed consent is not feasible, clinical research that presents more than minimal risk can proceed only after a community consultation and public disclosure process and the granting of exception from informed consent from the federal government. The applicability of exception from informed consent to pediatric resuscitation research has not been described. The objectives of this study were 1) to perform a community consultation and public disclosure process specific to a trial of induced hypothermia immediately after pediatric cardiac arrest and 2) to determine the applicability of exception from informed consent to randomized, controlled trials of emergency interventions after resuscitation from inpatient pediatric cardiac arrest., Methods: Focus groups, information sheets with options for written responses, posted notices, e-mails, and telephone conversations with parents of critically ill children and hospital staff were conducted at a tertiary care children's hospital. Data were stored, organized, and retrieved using NVivo qualitative analysis software (QSR International)., Results: In focus groups (n = 8), parents (n = 23) and hospital staff (n = 33) concluded that prospective informed consent is not feasible for a trial of induced hypothermia after inpatient pediatric cardiac arrest. Focus group participants endorsed exception from informed consent for a trial of induced hypothermia but only if study information is easily available prospectively and if all parents have an explicit opportunity to decline participation in a verbal conversation before study enrollment. Separate from and without knowledge of the focus group results, 7 (100%) of 7 parents of past or current patients and 21 (50%) of 42 hospital staff who provided written opinions endorsed exception from informed consent for this study. Five (12%) of 42 hospital staff opposed, and 16 (38%) of 42 were neutral. In telephone conversations, 14 (70%) of 20 parents of children who were previously resuscitated from cardiac arrest endorsed exception from informed consent for this study, 3 (15%) of 20 opposed, and 3 (15%) of 20 were unsure., Conclusions: Community consultation for inpatient resuscitation research can be conducted in a children's hospital, with hospital staff and parents of patients as the relevant community. Exception from informed consent is necessary and appropriate for a randomized trial of induced hypothermia begun within 30 minutes after pediatric cardiac arrest. A process in which families are informed prospectively and have a pre-enrollment option to decline participation will likely be acceptable to families, health care providers, and the institution.

The inhibitory effect of somatostatin on insulin, glucagon and growth hormone secretion was studied in 5 patients with diabetes mellitus. In three maturity onset diabetics, somatostatin infusion abolished the insulin rise induced by breakfast and oral glucose, and in 2 of them, inhibited the basal insulin secretion by 50% seen during control studies. Concomitantly, there was a marked and prompt reduction of glucagon levels (50%) with a sustained effect. The plasma glucose levels were either unchanged or slightly increased. Following the somatostatin infusion, there was a prompt rebound increase in both insulin and glucagon levels with a relatively stable plasma glucose concentration. In contrast, a drastic reduction of plasma glucose in face of a relatively small fall in plasma glucagon in response to somatostatin infusion was observed in 2 insulin-dependent diabetics. In all patients, the episodic release of growth hormone seen during the control day was abolished during somatostatin infusion.