Notch signaling without the APH-2/nicastrin subunit of gamma secretase in Caenorhabditis elegans germline stem cells.

The final step in Notch signaling activation is the transmembrane cleavage of Notch receptor by γ secretase. Thus far, genetic and biochemical evidence indicates that four subunits are essential for γ secretase activity in vivo: presenilin (the catalytic core), APH-1, PEN-2, and APH-2/nicastrin. Although some γ secretase activity has been detected in APH-2/nicastrin-deficient mammalian cell lines, the lack of biological relevance for this activity has left the quaternary γ secretase model unchallenged. Here, we provide the first example of in vivo Notch signal transduction without APH-2/nicastrin. The surprising dispensability of APH-2/nicastrin is observed in Caenorhabditis elegans germline stem cells (GSCs) and contrasts with its essential role in previously described C. elegans Notch signaling events. Depletion of GLP-1/Notch, presenilin, APH-1, or PEN-2 causes a striking loss of GSCs. In contrast, aph-2/nicastrin mutants maintain GSCs and exhibit robust and localized expression of the downstream Notch target sygl-1. Interestingly, APH-2/nicastrin is normally expressed in GSCs and becomes essential under conditions of compromised Notch function. Further insight is provided by reconstituting the C. elegans γ secretase complex in yeast, where we find that APH-2/nicastrin increases but is not essential for γ secretase activity. Together, our results are most consistent with a revised model of γ secretase in which the APH-2/nicastrin subunit has a modulatory, rather than obligatory role. We propose that a trimeric presenilin-APH-1-PEN-2 γ secretase complex can provide a low level of γ secretase activity, and that cellular context determines whether or not APH-2/nicastrin is essential for effective Notch signal transduction.

Overdose education and naloxone distribution program design informed by people who use drugs and naloxone distributors.

People who use drugs (PWUD) are the most directly affected by the overdose epidemic. However, they are not often targets of overdose education and naloxone distribution (OEND) programs. Instead, these programs target friends or family members of people prescribed opioids or general community members. This study aimed to understand the perspectives of PWUD and community naloxone distributors on OEND program design. We used a community-based participatory research model to elucidate participant perspectives on what OEND programs should look like in the context of each individual's specific risk environment. We conducted semi-structured in-depth interviews with PWUD and naloxone distributors (n = 30) in New Brunswick and Newark, New Jersey between February and November of 2022. We analyzed interviews using thematic analysis and identified the following themes: increasing naloxone knowledge, peer-based naloxone access, increasing PWUD-informed OEND program design, and desired broader OEND program scope. All Participants knew what naloxone was and emphasized that naloxone needed to be ubiquitous in the community. Participants prioritized peer-based distribution, integrating distribution into community organizations, and addressing psychosocial issues related to naloxone administration and drug use. In summary, PWUD and community naloxone distributors emphasized peer-led community naloxone distribution that prioritized novel ways for PWUD to access naloxone. OEND program design should prioritize PWUD's perspectives and direct community naloxone distribution.

Examining the Role of Quality of Institutionalized Healthcare on Maternal Mortality in the Dominican Republic.

The main study objective was to determine the extent to which the quality of institutionalized healthcare, sociodemographic factors of obstetric patients, and institutional factors affect maternal mortality in the Dominican Republic. COM-Poisson distribution and the Pearson correlation coefficient were used to determine the relationship of predictor factors (i.e., hospital bed rate, vaginal birth rate, teenage mother birth rate, single mother birth rate, unemployment rate, infant mortality rate, and sex of child rate) in influencing maternal mortality rate. The factors hospital bed rate, teenage mother birth rate, and unemployment rate were not correlated with maternal mortality. Maternal mortality increased as vaginal birth rates and infant death rates increased whereas it decreased as single mother birth rates increased. Further research to explore alternate response variables, such as maternal near-misses or severe maternal morbidity is warranted. Additionally, the link found between infant death and maternal mortality presents an opportunity for collaboration among medical specialists to develop multi-faceted solutions to combat adverse maternal and infant health outcomes in the DR.

Tiny Earth: A Big Idea for STEM Education and Antibiotic Discovery.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery.

Psychological Impact of Abortion due to Fetal Anomaly: A Review of Published Research.

Over the past several decades, prenatal testing for fetal anomalies has become standard across the developed world. Most women who learn of a severe anomaly during pregnancy elect to terminate. The authors reviewed ten studies which examined the psychological sequelae of pregnant women following prenatal diagnoses of severe life limiting conditions. Based on the available data, the authors found evidence that women who abort due to a poor prenatal diagnosis are at higher risk of post-traumatic stress and depression than women who continue with pregnancy. They also found that current practice is to make reactive recommendations for post-abortion care, suggesting a presumption in favor of abortion, leaving alternatives like perinatal hospice inadequately explored. This review critiques that approach, recommending that doctors and patients need to have the best possible information before a decision is made.

The Complete Genome Sequence of Proteus mirabilis Strain BB2000 Reveals Differences from the P. mirabilis Reference Strain.

We announce the complete genome sequence for Proteus mirabilis strain BB2000, a model system for self recognition. This opportunistic pathogen contains a single, circular chromosome (3,846,754 bp). Comparisons between this genome and that of strain HI4320 reveal genetic variations corresponding to previously unknown physiological and self-recognition differences.

Two independent pathways for self-recognition in Proteus mirabilis are linked by type VI-dependent export.

UNLABELLED: Swarming colonies of the bacterium Proteus mirabilis are capable of self-recognition and territorial behavior. Swarms of independent P. mirabilis isolates can recognize each other as foreign and establish a visible boundary where they meet; in contrast, genetically identical swarms merge. The ids genes, which encode self-identity proteins, are necessary but not sufficient for this territorial behavior. Here we have identified two new gene clusters: one (idr) encodes rhs-related products, and another (tss) encodes a putative type VI secretion (T6S) apparatus. The Ids and Idr proteins function independently of each other in extracellular transport and in territorial behaviors; however, these self-recognition systems are linked via this type VI secretion system. The T6S system is required for export of select Ids and Idr proteins. Our results provide a mechanistic and physiological basis for the fundamental behaviors of self-recognition and territoriality in a bacterial model system. IMPORTANCE: Our results support a model in which self-recognition in P. mirabilis is achieved by the combined action of two independent pathways linked by a shared machinery for export of encoded self-recognition elements. These proteins together form a mechanistic network for self-recognition that can serve as a foundation for examining the prevalent biological phenomena of territorial behaviors and self-recognition in a simple, bacterial model system.

Response to second generation antipsychotics in youth with comorbid bipolar disorder and autism spectrum disorder.

OBJECTIVE: To assess the impact of comorbid autism spectrum disorders (ASD) on the response to second-generation antipsychotics (SGA) in pediatric bipolar disorder (BPD). METHODS: Secondary analysis of identically designed 8-week open-label trials of SGA monotherapy (risperidone, olanzapine, quetiapine, ziprasidone, or aripiprazole) in youth with BPD. RESULTS: Of the 151 BPD subjects 15% (n= 23) met criteria for comorbid ASD. There were no differences in the rate of antimanic response (YMRS change ≥30% or CGI-Improvement ≤2: 65% vs. 69%; P= 0.7) in the presence of comorbid ASD. CONCLUSION: No difference observed in the rate of antimanic response or tolerability to SGA monotherapy in the presence of ASD comorbidity.

Quantifying the dynamics of bacterial secondary metabolites by spectral multiphoton microscopy.

Phenazines, a group of fluorescent small molecules produced by the bacterium Pseudomonas aeruginosa, play a role in maintaining cellular redox homeostasis. Phenazines have been challenging to study in vivo due to their redox activity, presence both intra- and extracellularly, and their diverse chemical properties. Here, we describe a noninvasive in vivo optical technique to monitor phenazine concentrations within bacterial cells using time-lapsed spectral multiphoton fluorescence microscopy. This technique enables simultaneous monitoring of multiple weakly fluorescent molecules (phenazines, siderophores, NAD(P)H) expressed by bacteria in culture. This work provides the first in vivo measurements of reduced phenazine concentration as well as the first description of the temporal dynamics of the phenazine-NAD(P)H redox system in Pseudomonas aeruginosa, illuminating an unanticipated role for 1-hydroxyphenazine. Similar approaches could be used to study the abundance and redox dynamics of a wide range of small molecules within bacteria, both as single cells and in communities.

Recruitment of SMC by ParB-parS organizes the origin region and promotes efficient chromosome segregation.

Organization and segregation of replicated chromosomes are essential processes during cell division in all organisms. Similar to eukaryotes, bacteria possess centromere-like DNA sequences (parS) that cluster at the origin of replication and the structural maintenance of chromosomes (SMC) complexes for faithful chromosome segregation. In Bacillus subtilis, parS sites are bound by the partitioning protein Spo0J (ParB), and we show here that Spo0J recruits the SMC complex to the origin. We demonstrate that the SMC complex colocalizes with Spo0J at the origin and that insertion of parS sites near the replication terminus targets SMC to this position leading to defects in chromosome organization and segregation. Consistent with these findings, the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. We propose a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chromosome segregation.

The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis.

The FtsK/SpoIIIE family of ATP-dependent DNA transporters mediates proper chromosome segregation in dividing bacteria. In sporulating Bacillus subtilis cells, SpoIIIE translocates much of the circular chromosome from the mother cell into the forespore, but the molecular mechanism remains unclear. Using a new assay to monitor DNA transport, we demonstrate that the two arms of the chromosome are simultaneously pumped into the forespore. Up to 70 molecules of SpoIIIE are recruited to the site of DNA translocation and assemble into complexes that could contain 12 subunits. The fusion of the septal membranes during cytokinesis precedes DNA translocation and does not require SpoIIIE, as suggested by analysis of lipid dynamics, serial thin-section electron microscopy, and cell separation by protoplasting. These data support a model for DNA transport in which the transmembrane segments of FtsK/SpoIIIE form linked DNA-conducting channels across the two lipid bilayers of the septum.

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor.

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [3H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in Kd and EC50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.

Characterization of 5-HT3 receptor mutations identified in schizophrenic patients.

The 5-HT3 receptor is a member of the Cys-loop family of ligand-gated ion channels, proteins that have been implicated in the pathology of several neurological disorders. In this study, we examine two mutations (R344H and P391R) that have been previously identified in individuals diagnosed with schizophrenia. These mutations are located in the M3-M4 loop of the 5-HT3 receptor and their occurrence presents the possibility that they contribute toward the etiology of this disorder. Radioligand binding with the 5-HT receptor antagonist, [3H]granisetron, revealed no significant difference in receptor affinity or density between mutant and wild-type receptors when expressed in HEK293 cells. However, comparison of EC50 values using whole-cell patch clamp for wild-type (1.68 microM +/- 0.01, n = 38), R344H (1.70 microM +/- 0.02, n = 18), and P391R (2.73 microM +/- 0.01, n = 8) receptors revealed a significant increase in the EC50 of the P391R mutant. Analysis of Hill co-efficients, and activation and desensitization rate constants showed no significant difference between wild-type and mutant receptors. These data suggest that the P391R, but not the R344H, mutation may play a role in the pathology of schizophrenia.

Mapping of a ligand-binding site for the human thromboxane A2 receptor protein.

The human thromboxane A(2) (TP) receptor, a member of the G protein-coupled receptor superfamily, consists of seven transmembrane segments. Attempts to elucidate the specific segment(s) that define the receptor ligand-binding pocket have produced less than definitive and sometimes conflicting results. On this basis, the present work identified an amino acid sequence of the TP receptor that is directly involved in ligand binding. Mapping of this domain was confirmed by two separate approaches: photoaffinity labeling and site-specific antibodies. The newly synthesized, biotinylated photoaffinity probe, SQBAzide, was first shown to specifically label TP receptor protein. Sequential digestion of this protein with CNBr/trypsin revealed photolabeling of a 2.9-kDa peptide. Using anti-peptide antibodies directed against different regions of the receptor protein, it was established that this peptide represents the predicted cleavage product for CNBr/trypsin and corresponds to amino acids Arg(174)-Met(202) of the receptor protein. Furthermore, antibody screening revealed that inhibition of the amino acid region Cys(183)-Asp(193) was critical for radioligand binding and platelet aggregation, whereas inhibition of Gly(172)-Cys(183) was not. Collectively these findings provide evidence that ligands interact with amino acids contained within the C-terminal portion of the third extracellular domain (ED3) of the receptor protein. This information should be of significant value in the study of TP receptor structure and signaling.