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Canonical autophagy captures within specialized double-membrane organelles, termed autophagosomes, an array of cytoplasmic components destined for lysosomal degradation. An autophagosome is completed when the growing phagophore undergoes ESCRT-dependent membrane closure, a prerequisite for its subsequent fusion with endolysosomal organelles and degradation of the sequestered cargo. ATG9A, a key integral membrane protein of the autophagy pathway, is best known for its role in the formation and expansion of phagophores. Here, we report a hitherto unappreciated function of mammalian ATG9A in directing autophagosome closure. ATG9A partners with IQGAP1 and key ESCRT-III component CHMP2A to facilitate this final stage in autophagosome formation. Thus, ATG9A is a central hub governing all major aspects of autophagosome membrane biogenesis, from phagophore formation to its closure, and is a unique ATG factor with progressive functionalities affecting the physiological outputs of autophagy., (© 2025 Javed et al.)

ATG5 is one of the core autophagy proteins with additional functions such as noncanonical membrane atg8ylation, which among a growing number of biological outputs includes control of tuberculosis in animal models. Here, we show that ATG5 associates with retromer's core components VPS26, VPS29, and VPS35 and modulates retromer function. Knockout of ATG5 blocked trafficking of a key glucose transporter sorted by the retromer, GLUT1, to the plasma membrane. Knockouts of other genes essential for membrane atg8ylation, of which ATG5 is a component, affected GLUT1 sorting, indicating that membrane atg8ylation as a process affects retromer function and endosomal sorting. The contribution of membrane atg8ylation to retromer function in GLUT1 sorting was independent of canonical autophagy. These findings expand the scope of membrane atg8ylation to specific sorting processes in the cell dependent on the retromer and its known interactors., Competing Interests: MP, FW, ET, EH, YH, MS, MM, JJ, TD, RJ, BP, VD No competing interests declared, (© 2024, Paddar et al.)

Objectives: Intimate partner violence (IPV) has short- and long-term health effects, including physical injuries and traumatic brain injury, as well as sexual, reproductive, and mental health issues. However, accessing necessary health care is often challenging for IPV survivors and became even more difficult during the COVID-19 pandemic. We examined access to health care among those affected by IPV during the COVID-19 pandemic to better connect survivors to health and social support services., Methods: The Health Resources and Services Administration's Office of Women's Health partnered with its Bureau of Primary Health Care, the Administration for Children and Families' Office of Family Violence Prevention and Services, and the National Domestic Violence Hotline (Hotline) on the 2-year Survivor Health Connection Project. The Hotline administered 2 surveys to its contacts: a 2-question postinteraction survey from March 29, 2021, through September 30, 2022, and a longer focused survey in 2021 and 2022 that measured barriers and restrictions to accessing health care, telehealth safety, and interactions with health care providers., Results: Of 9918 respondents to the postinteraction survey, 6173 (62.2%) reported current health needs related to their abusive experience. Nearly half of 242 respondents to the 2021 survey (n = 106, 43.8%) indicated that the frequency or intensity of abuse increased during the COVID-19 pandemic, and 157 of 338 respondents to the 2022 survey (46.4%) reported that their partner had controlled and/or restricted their access to health care. Participants described barriers to accessing health care, including finances, health insurance coverage, and transportation., Conclusions: Findings illuminate opportunities to further support the health and social needs of those experiencing IPV, including continued coordination of efforts across health care and social service delivery partners., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant, CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin, which itself is bound by the sequence specific binding protein, CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromere's stiffness and the role of the CCAN. We degraded CENP-C and CENP-N containing auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching ∼3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms., Competing Interests: Conflicts of interest: The authors declare no competing financial interests.

The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin and bound by the sequence specific binding protein CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromere's stiffness and the role of the CCAN. We degraded CENP-C and CENP-N using auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching approximately 3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms.

ATG5 is one of the core autophagy proteins with additional functions such as noncanonical membrane atg8ylation, which among a growing number of biological outputs includes control of tuberculosis in animal models. Here we show that ATG5 associates with retromer's core components VPS26, VPS29 and VPS35 and modulates retromer function. Knockout of ATG5 blocked trafficking of a key glucose transporter sorted by the retromer, GLUT1, to the plasma membrane. Knockouts of other genes essential for membrane atg8ylation, of which ATG5 is a component, affected GLUT1 sorting, indicating that membrane atg8ylation as a process affects retromer function and endosomal sorting. The contribution of membrane atg8ylation to retromer function in GLUT1 sorting was independent of canonical autophagy. These findings expand the scope of membrane atg8ylation to specific sorting processes in the cell dependent on the retromer and its known interactors.

Introduction: The PICK1 PDZ domain has been identified as a potential drug target for neurological disorders. After many years of effort, a few inhibitors, such as TAT-C5 and mPD5, have been discovered experimentally to bind to the PDZ domain with a relatively high binding affinity. With the rapid growth of computational research, there is an urgent need for more efficient computational methods to design viable ligands that target proteins., Method: Recently, a newly developed program called AfDesign (part of ColabDesign) at https:// github.com/sokrypton/ColabDesign), an open-source software built on AlphaFold, has been suggested to be capable of generating ligands that bind to targeted proteins, thus potentially facilitating the ligand development process. To evaluate the performance of this program, we explored its ability to target the PICK1 PDZ domain, given our current understanding of it. We found that the designated length of the ligand and the number of recycles play vital roles in generating ligands with optimal properties., Results: Utilizing AfDesign with a sequence length of 5 for the ligand produced the highest comparable ligands to that of prior identified ligands. Moreover, these designed ligands displayed significantly lower binding energy compared to manually created sequences., Conclusion: This work demonstrated that AfDesign can potentially be a powerful tool to facilitate the exploration of the ligand space for the purpose of targeting PDZ domains., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Epilepsy is a complex chronic brain disorder with diverse clinical features that can be caused by various triggering events, such as infections, head trauma, or stroke. During epileptogenesis, various abnormalities are observed, such as altered cellular homeostasis, imbalance of neurotransmitters, tissue changes, and the release of inflammatory mediators, which in combination lead to spontaneous recurrent seizures. Regulatory T cells (Tregs), a subtype of CD4 + Foxp3 + T cells, best known for their key function in immune suppression, also seem to play a role in attenuating neurodegeneration and suppressing pathological inflammation in several brain disease states. Considering that epilepsy is also highly associated with neuronal damage and neuroinflammation, modulation of Tregs may be an interesting way to modify the disease course of epilepsy and needs further investigation. In this review, we will describe the currently available information on Tregs in epilepsy., (© 2024 International League Against Epilepsy.)

This study sought to understand the effects of COVID-19, including movement-related restrictions such as shelter-in-place, quarantine, and isolation orders, on intimate partner violence (IPV) from the perspective of health care providers (HCPs) working at a public hospital in Atlanta, Georgia. From November 2020 to May 2021, we conducted 12 interviews. Three themes emerged: (1) HCPs perceived that COVID-19 movement-related restrictions likely exacerbated IPV; (2) HCPs encountered many practice-oriented and community barriers in IPV care provision during COVID-19; and (3) HCPs suggested process and partnership improvements for IPV response. These findings can inform future pandemic preparedness including improved communication, improved IPV screening and follow-up, and strengthened hospital-community partnerships., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Developmentally regulated Guanosine-5'-triphosphate-binding protein 1 (DRG1) is a highly conserved member of a class of GTPases implicated in translation. Although the expression of mammalian DRG1 is elevated in the central nervous system during development, and its function has been implicated in fundamental cellular processes, no pathogenic germline variants have yet been identified. Here, we characterize the clinical and biochemical consequences of DRG1 variants., Methods: We collate clinical information of 4 individuals with germline DRG1 variants and use in silico, in vitro, and cell-based studies to study the pathogenicity of these alleles., Results: We identified private germline DRG1 variants, including 3 stop-gained p.Gly54 ∗ , p.Arg140 ∗ , p.Lys263 ∗ , and a p.Asn248Phe missense variant. These alleles are recessively inherited in 4 affected individuals from 3 distinct families and cause a neurodevelopmental disorder with global developmental delay, primary microcephaly, short stature, and craniofacial anomalies. We show that these loss-of-function variants (1) severely disrupt DRG1 messenger RNA/protein stability in patient-derived fibroblasts, (2) impair its GTPase activity, and (3) compromise its binding to partner protein ZC3H15. Consistent with the importance of DRG1 in humans, targeted inactivation of mouse Drg1 resulted in preweaning lethality., Conclusion: Our work defines a new Mendelian disorder of DRG1 deficiency. This study highlights DRG1's importance for normal mammalian development and underscores the significance of translation factor GTPases in human physiology and homeostasis., Competing Interests: Conflict of Interest Maryam Najafi and Salem Alawbathani are employees of Centogene. All other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. The enzymes that install the post-translational modifications on these peptides have the potential to be useful catalysts in the production of natural-product-like compounds and can install non-proteogenic amino acids in peptides and proteins. However, engineering these enzymes has been somewhat limited, due in part to limited structural information on enzymes in the same families that nonetheless exhibit different substrate selectivities. Despite AlphaFold2's superior performance in single-chain protein structure prediction, its multimer version lacks accuracy and requires high-end GPUs, which are not typically available to most research groups. Additionally, the default parameters of AlphaFold2 may not be optimal for predicting complex structures like RiPP biosynthetic enzymes, due to their dynamic binding and substrate-modifying mechanisms. This study assessed the efficacy of the structure prediction program ColabFold (a variant of AlphaFold2) in modeling RiPP biosynthetic enzymes in both monomeric and dimeric forms. After extensive benchmarking, it was found that there were no statistically significant differences in the accuracy of the predicted structures, regardless of the various possible prediction parameters that were examined, and that with the default parameters, ColabFold was able to produce accurate models. We then generated additional structural predictions for select RiPP biosynthetic enzymes from multiple protein families and biosynthetic pathways. Our findings can serve as a reference for future enzyme engineering complemented by AlphaFold-related tools.

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes., (© 2023 The Authors.)

Although protein hydroxylation is a relatively poorly characterized posttranslational modification, it has received significant recent attention following seminal work uncovering its role in oxygen sensing and hypoxia biology. Although the fundamental importance of protein hydroxylases in biology is becoming clear, the biochemical targets and cellular functions often remain enigmatic. JMJD5 is a "JmjC-only" protein hydroxylase that is essential for murine embryonic development and viability. However, no germline variants in JmjC-only hydroxylases, including JMJD5, have yet been described that are associated with any human pathology. Here we demonstrate that biallelic germline JMJD5 pathogenic variants are deleterious to JMJD5 mRNA splicing, protein stability, and hydroxylase activity, resulting in a human developmental disorder characterized by severe failure to thrive, intellectual disability, and facial dysmorphism. We show that the underlying cellular phenotype is associated with increased DNA replication stress and that this is critically dependent on the protein hydroxylase activity of JMJD5. This work contributes to our growing understanding of the role and importance of protein hydroxylases in human development and disease.

Intrinsically disordered proteins (IDPs) contain more charged amino acids than folded proteins, resulting in a lack of hydrophobic core(s) and a tendency to adopt rapidly interconverting structures rather than well-defined structures. The structural heterogeneity of IDPs, encoded by the amino acid sequence, is closely related to their unique roles in biological pathways, which require them to interact with different binding partners. Recently, Robustelli and co-workers have demonstrated that a balanced all-atom force field can be used to sample heterogeneous structures of disordered proteins ( Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E4758-E4766). However, such a solution requires extensive computational resources, such as Anton supercomputers. Here, we propose a simple and effective solution to sample the conformational space of IDPs using a publicly available web server, namely, the UNited-RESidue (UNRES) web server. Our proposed solution requires no investment in computational resources and no prior knowledge of UNRES. UNRES Replica Exchange Molecular Dynamics (REMD) simulations were carried out on a set of eight disordered proteins at temperatures spanning from 270 to 430 K. Utilizing the latest UNRES force field designed for structured proteins, with proper selections of temperatures, we were able to produce comparable results to all-atom force fields as reported in work done by Robustelli and co-workers. In addition, NMR observables and the radius of gyration calculated from UNRES ensembles were directly compared with the experimental data to further evaluate the accuracy of the UNRES model at all temperatures. Our results suggest that carrying out the UNRES simulations at optimal temperatures using the UNRES web server can be a good alternative to sample heterogeneous structures of IDPs.

Objective: To evaluate and compare postoperative analgesic effects of grapiprant and carprofen in dogs undergoing ovariohysterectomy., Animals: 42 sexually intact female healthy dogs (< 35 kg and 0.5 to 7 years old) were enrolled., Procedures: In a masked, randomized, noninferiority clinical trial, dogs received either 2 mg/kg of grapiprant or 4.4 mg/kg of carprofen orally 2 hours prior to ovariohysterectomy. Postoperative pain was assessed using the Glasgow Composite Pain Scale-Short Form (GCPS-SF) at extubation and 2, 4, 6, 8, 18, and 24 hours postextubation and compared to baseline. After each pain scoring, mechanical nociceptive testing with von Frey monofilaments (vF) was performed to assess hyperalgesia. Hydromorphone (0.05 mg/kg, IM) was administered to any dog with a GCPS-SF of ≥ 5/24. The noninferiority limit (NI) for the GCPS-SF was Δ = 3. The NI for vF was Δ = -0.2. Following noninferiority, a mixed-effect ANOVA and post hoc comparisons were made with the Tukey correction method (P < .05)., Results: 3 dogs required rescue analgesia and were excluded from statistical analysis. Of the remaining 39 dogs, the upper CI for GCPS-SF was below the NI of 3 and the lower CI for vF was greater than the NI of -0.2, indicating noninferiority of grapiprant as compared to carprofen. There was no difference between treatment (P = .89) nor treatment by time (P = .62) for GCPS-SF. There was no difference between groups at any time point or over time when vF were used., Clinical Relevance: Our study results support the use of grapiprant as an analgesic alternative to carprofen in dogs undergoing ovariohysterectomy.

The unfolded state of proteins presents many challenges to elucidate the structural basis for biological function. This state is characterized by a large degree of structural heterogeneity which makes it difficult to generate structural models. However, recent experiments into the initial folding events of the 104-residue ribonuclease homologue onconase (ONC) were able to identify the regions in the protein that participate in the initial folding of this protein. Therefore, to gain additional structural insight into the unfolded state of proteins, this study utilized molecular dynamics simulations using the UNited-RESidue (UNRES) force field to evaluate whether there is a good agreement between the experimentally determined initial structures and the structures identified by computer simulations along a folding pathway. Indeed, these UNRES simulations accurately identified the two regions experimentally observed to form the initial native structure along the folding pathway of ONC. In addition, these regions are determined to be chain folding initiation sites (CFIS) according to methods developed previously. Subsequent self-organization maps (SOM) analysis has revealed key structural states involved in these early folding events.

The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

A number of experiments were done to further our understanding of the substrate utilization in button mushroom crops (Agaricus bisporus). An analysis of the degradation of dry matter of the substrate during a crop cycle revealed that for pin formation the upper 1/3rd layer is used, for the production of flush one all layers are involved and for flush two mainly the lower 1/3 layer is used. A reduction in substrate depth leads to a decrease in yield/m2 but an apparent increase in yield per tonne of substrate with a lower mushroom quality. A short daily interruption of the connection between the casing soil with the substrate results in a delay of the first flush. Interruptions with only part of the substrate did not lead to delay in production. Daily interruption of the connection with all or only part of the substrate leads to a shift in yield from flush one to flush two but the total yield remains unchanged. The mycelial biomass in the substrate increases from filling up to pinning, has a steeper increase during flush one, and is levelling off during flush two, indicating that in the period of venting and up to/including flush one, enzymes are secreted by growing hyphae generating nutrients to feed a fixed amount of mushroom biomass for two flushes. A sidewise extension of the substrate (without casing soil, thus not producing mushrooms) showed that the substrate at a distance more than somewhere between 20-50 cm away from the casing soil does not contribute to feeding mushrooms in the first two flushes. The observations are discussed with respect to relevant previous research., Competing Interests: The authors have declared that no competing interests exist.

Isowighteone (3’-isoprenyl genistein) is a prenylated stilbenoid derivative that exhibits neuroprotective, antibacterial, and anti-inflammatory properties. To establish a bioproduction system for this bioactive compound, hairy root cultures of pigeon pea ( Cajanus cajan (L.) Millsp.) were developed via Agrobacterium rhizogenes -mediated transformation. The cultures were co-treated with methyl jasmonate, cyclodextrin, hydrogen peroxide, and magnesium chloride to enhance the production of isowighteone. The amount of isowighteone that accumulated in the culture medium upon elicitation varied with the period of elicitation. Isowighteone was purified from extracts of the culture medium by semi-preparative HPLC, and its identity was confirmed by tandem mass spectrometry. After 144 h of elicitation in 12-day-old hairy root culture, the total yield of isowighteone was 8058.618 ± 445.78 μg/g DW, of which approximately 96% was found in the culture medium. The yield of isowighteone in the elicited hairy root culture was approximately 277-fold higher than in the non-elicited root culture. The difference between the phenotypes of the elicited and non-elicited pigeon pea hairy roots was studied using scanning electron microscopy. The non-elicited hairy roots had uniform surfaces whereas the elicited roots had non-uniform shapes. Pigeon pea hairy roots provide a sustainable platform for producing and studying the biosynthesis of isowighteone.

MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c -MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4-6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.

Bacterial blotch is one of the most economically important diseases of button 'mushroom. Knowledge of mechanisms of disease expression, inoculum thresholds, and disease management is limited to the most well-known pathogen, Pseudomonas tolaasii . Recent outbreaks in Europe have been attributed to ' P. gingeri ' and P. salomonii for ginger and brown blotch, respectively. Information about their identity, infection dynamics, and pathogenicity is largely lacking. The disease pressure in an experimental mushroom cultivation facility was evaluated for ' P. gingeri ' and P. salomonii over varying inoculation densities, casing soil types, environmental humidity, and cultivation cycles. The pathogen population structures in the casing soils were simultaneously tracked across the cropping cycle using highly specific and sensitive TaqMan-quantitative PCR assays. ' P. gingeri ' caused disease outbreaks at lower inoculum thresholds (10 4 CFU/g) in the soil than P. salomonii (10 5 CFU/g). Ginger blotch generically declined in later harvest cycles, although brown blotch did not. Casing soils were differentially suppressive to blotch diseases, based on their composition and supplementation. Endemic pathogen populations increased across the cultivation cycle although the inoculated pathogen populations were consistent between the first and second flush. In conclusion, ' P. gingeri ' and P. salomonii have unique infection and population dynamics that vary over soil types. Their endemic populations are also differently abundant in peat-based casing soils. This knowledge is essential for interpreting diagnostic results from screening mushroom farms and designing localized disease control strategies.

Cervical cancer is the leading cause of female cancer deaths in Sénégal which is ranked 17th in incidence globally, however, the screening rate there is very low. Nuanced gendered perceptions and health behaviors of both women and men play a significant role in women's health. Our study analyzed gender differences on perceptions of gender roles, discrimination, cancer attitudes, cancer stigma, and influences in healthcare decision making within our study population to inform ongoing cervical cancer prevention work in the rural region of Kédougou, Sénégal. We conducted a cross-sectional survey of 158 participants, 101 women and 57 men (ages 30-59) across nine non-probability-sampled communities from October 2018 through February 2019. Bivariate analysis was conducted to assess gender differences across all variables. We also conducted analyses to determine whether there were significant differences in beliefs and attitudes, by screening behavior and by education. We found significant gender differences regarding the perception of a woman's role (P < 0.001) and a man's role (P = 0.007) as well as in the everyday discrimination questions of "decreased respect by spouse" (P < 0.001). Regarding cancer stigma, among women, 18.00% disagreed and 10.00% strongly disagreed while among men, 3.6% disagreed and 1.8% strongly disagreed that "If I had cancer, I would want my family to know that I have it." When making decisions about one's healthcare, women are more likely than men to trust social contacts such as their spouse (46.5% vs 5.3%, p < 0.001) while men are more likely than women to trust health service personnel such as a nurse (50.9% vs 18.8%, p < 0.001). Furthermore, men and women were both more likely to state that men have the final decision regarding the healthcare decisions of women (p < 0.001). Our data reveal structural disadvantages for women within our study population as well as gender differences in the adapted everyday discrimination scale and cancer stigma scale. Higher rates of both personal and perceived cancer stigma among women has profound implications for how population and community level communication strategies for cancer prevention and control should be designed. Efforts to advance the goal of the elimination of cervical cancer should, in the short-term, seek to gain a more profound understanding of the ways that gender, language, and other social determinants impact negative social influences and other barriers addressable through interventions. Social and behavior change communication may be one approach that can focus both on education while seeking to leverage the social influences that exist in achieving immediate and long-term goals., Competing Interests: The authors have declared that no competing interests exist.

Background: The onus of sex trafficking victim identification currently falls to professionals in youth-serving organizations. Accurate identification of domestic minor sex trafficking victims (DMST) and subsequent delivery of services can be impeded by common myths related to human trafficking., Objective: Determine if human service professionals understand the difference between myths and facts pertaining to sex trafficking and if their knowledge affects subsequent service provision., Participants and Setting: Professionals (n = 69) represented youth-serving organizations from a large metropolitan city in the Southwestern United States., Methods: Data was collected in a cross-sectional survey from March 2016 to April 2016., Results: A minority of professionals working in youth-serving organizations believed DMST myths, though administrators most commonly believed the myth that force, fraud, or coercion are required in DMST. Most professionals could not provide an accurate number of victims identified and felt their organizations would benefit from further training. A statistically significant relationship (p < .05) was observed between the belief that "elements of physical force, restraint, bondage, and/or violence" must be present in DMST and participants' ability to identify victims served by their organizations in a given year. Organizations reported that they failed to provide services requested by DMST victims., Conclusions: The persistence of DMST myths likely hampers victim identification and tailored service delivery. Cross-sector collaborations are needed to ensure victims' needs are met. Trainings are needed that cover the legal definitions of domestic minor sex trafficking and victim identification and train on accurate identifiers. Training initiatives should include survivors to ensure practices are trauma-informed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hydroxylation is a novel protein modification catalyzed by a family of oxygenases that depend on fundamental nutrients and metabolites for activity. Protein hydroxylases have been implicated in a variety of key cellular processes that play important roles in both normal homeostasis and pathogenesis. Here, in this review, we summarize the current literature on a highly conserved sub-family of oxygenases that catalyze protein histidyl hydroxylation. We discuss the evidence supporting the biochemical assignment of these emerging enzymes as ribosomal protein hydroxylases, and provide an overview of their role in immunology, bone development, and cancer.

Degradation of lignin by fungi enhances availability of cellulose and hemicellulose in plant waste and thereby increases the amount of carbon source available to these microorganisms. The button mushroom Agaricus bisporus degrades only about half of the lignin in compost and about 40% of the carbohydrates remain unutilized during mushroom cultivation. Here it was assessed whether over-expression of the manganese peroxidase gene mnp1 improves lignin degradation and, as a consequence, carbohydrate breakdown by A. bisporus. Transformants expressing mnp1 under the control of actin regulatory sequences produced MnP activity in malt extract medium, while the parental strain A15 did not. MnP activity was increased 0.3- and 3-fold at casing and after the 2nd flush of a semi-commercial cultivation, respectively, when compared to strain A15. Pyrolysis-GC-MS showed that overexpression of MnP decreased phenylmethane and phenylethane type lignin relative to the phenylpropane type after the 2nd flush. However, it neither affected the syringyl/guaiacyl derived residue ratio nor the ratio of oxidized to non-oxidized lignin residues. Moreover, the carbohydrate content and accessibility was not affected in compost. Notably, the capacity of compost extract to consume the MnP co-factor H 2 O 2 was 4- to 8-fold higher than its production. This may well explain why over-expression of mnp1 did not improve carbohydrate degradation in compost. In fact, availability of H 2 O 2 may limit lignin degradation by wild-type A. bisporus.

The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation. Its inactivation results in a strain that is arrested at the stage of aggregate formation. In this study, the c2h2 orthologue of Agaricus bisporus was over-expressed in this white button mushroom forming basidiomycete using Agrobacterium-mediated transformation. Morphology, cap expansion rate, and total number and biomass of mushrooms were not affected by over-expression of c2h2. However, yield per day of the c2h2 over-expression strains peaked 1 day earlier. These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development, making its encoding gene a target for breeding of commercial mushroom strains.

Measurement feedback systems (MFS) are a class of health information technology (HIT) that function as an implementation support strategy for integrating measurement based care or routine outcome monitoring into clinical practice. Although many MFS have been developed, little is known about their functions. This paper reports findings from an application of health information technology-academic and commercial evaluation (HIT-ACE), a systematic and consolidated evaluation method, to MFS designed for use in behavioral healthcare settings. Forty-nine MFS were identified and subjected to systematic characteristic and capability coding. Results are presented with respect to the representation of characteristics and capabilities across MFS.

The current study evaluated why and how school mental health clinicians use standardized assessment tools in their work with youth and families. Quantitative and qualitative (focus group) data were collected prior to and following a training and consultation sequence as part of a trial program to assess school clinician's (n = 15) experiences administering standardized tools to youth on their caseloads (n = 191). Findings indicated that, although assessment use was initially somewhat low, clinicians used measures to conduct initial assessments with the bulk of their caseloads (average = 62.2%) during the implementation period. Clinicians also reported on factors influencing their use of assessments at the client, provider, and system levels; perceived functions of assessment; student responses to assessment use; and use of additional sources of clinically-relevant information (primarily educational data) for the purposes of assessment and progress monitoring. Implications for the contextual appropriateness of standardized assessment and training in assessment tools are discussed.

To maximize impact across the broad spectrum of mental health needs exhibited by youth in school settings, interventions must be designed to be effective, efficient, and demonstrate good fit with the educational context. The current paper reports on the second phase of an iterative development process for a short-term, "Tier 2" intervention for use by school-based mental health providers - the Brief Intervention for School Clinicians (BRISC) - using mixed qualitative and quantitative analyses to evaluate feasibility, acceptability, and appropriateness while emphasizing student experiences. This phase was intended to yield information to drive further protocol refinement and testing across subsequent phases. We describe the rationale for, development of, and formative testing of the BRISC intervention. Results suggest that BRISC may be feasible to deliver, acceptable to students, and appropriate to the school context. In particular, the BRISC process appears to be effective in enhancing student engagement in the intervention and identifying and addressing individualized student needs. These findings and directions for further enhancing BRISC's potential for positive impact highlight how treatment development may benefit from initial, small-scale evaluations focused both on client and implementation outcomes.

Stormwater drainage and other water systems are vulnerable to changes in rainfall and runoff and need to be adapted to climate change. This paper studies impacts of rainfall variability and changing return periods of rainfall extremes on cost-effective adaptation of water systems to climate change given a predefined system performance target, for example a flood risk standard. Rainfall variability causes system performance estimates to be volatile. These estimates may be used to recurrently evaluate system performance. This paper presents a model for this setting, and develops a solution method to identify cost-effective investments in stormwater drainage adaptations. Runoff and water levels are simulated with rainfall from stationary rainfall distributions, and time series of annual rainfall maxima are simulated for a climate scenario. Cost-effective investment strategies are determined by dynamic programming. The method is applied to study the choice of volume for a storage basin in a Dutch polder. We find that 'white noise', i.e. trend-free variability of rainfall, might cause earlier re-investment than expected under projected changes in rainfall. The risk of early re-investment may be reduced by increasing initial investment. This can be cost-effective if the investment involves fixed costs. Increasing initial investments, therefore, not only increases water system robustness to structural changes in rainfall, but could also offer insurance against additional costs that would occur if system performance is underestimated and re-investment becomes inevitable., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study analyzed the prevalence of metabolic syndrome (MetS) and evaluated the factors associated with MetS. Subjects numbered 2,213 adults who participated in the health screening survey. Results show that the prevalence of MetS, Risk-MetS, and Non-MetS was 28.2%, 44.9%, and 26.9% respectively. Factors influencing variables were age, gender, history of hypertension and diabetes mellitus, abdominal obesity, high-density lipoprotein cholesterol, triglyceride, blood pressure, fasting blood glucose, alcohol consumption, and level of physical activity. Community health nurses should plan to develop MetS management programs that target Mets or Risk-MetS groups to promote health.

The Internet has emerged as an important tool for the delivery of health promotion and disease prevention interventions. Our community-based participatory research (CBPR) partnership developed and piloted CyBER/testing, a culturally congruent intervention designed to promote HIV testing among men who have sex with men (MSM) within existing Internet chat rooms. Using a quasi-experimental, single-group study design, cross-sectional data were collected from chat room participants, known as "chatters," at pretest (n = 346) and posttest (n = 315). Extant profile data also were collected to describe the demographics of the online population. The intervention significantly increased self-reported HIV testing among chatters overall, increasing rates from 44.5% at pretest to nearly 60% at posttest (p < .001). Furthermore, chatters who reported having both male and female sexual partners had nearly 6 times the odds of reporting HIV testing at posttest. Findings suggest that chat room-based HIV testing intervention may increase testing among MSM who may be difficult to reach in traditional physical spaces.

It remains unclear which classroom experiences, if any, foster critical think ability. We measured the effectiveness of interdisciplinary, case-based learning on the critical-thinking ability of graduate students enrolled in allied health care programs. We designed a voluntary classroom experience to examine the effectiveness of case studies used in an interdisciplinary setting to increase critical-thinking ability. Two groups of students were measured for their critical thinking ability using an online assessment both before and after their respective classroom experiences. One group of 14 graduate students from 4 different allied health care programs (interdisciplinary, ID) discussed complex interdisciplinary case studies and answered multiple-choice type questions formed around the cases. The second group was composed of graduate students (n = 28) from a single disciple enrolled in a clinical anatomy course (discipline specific, DS). They discussed complex case studies specific to their discipline and answered multiple-choice questions formed around the cases. There was no overall change in critical-thinking scores from the pre- to post-test in either group (delta scores: ID 1.5 ± 5.3, DS -1.7 ± 5.7). However, ID students scoring below the median on the pretest improved significantly (paired t-test, pre 50.7 ± 3.8, post 54.2 ± 1.7, p = 0.02). The interdisciplinary learning experience improved critical-thinking ability in students with the least proficiency. As case studies have long been used to advance deeper learning, these data provide evidence for a broader impact of cases when used in an interdisciplinary setting, especially for those students coming in with the least ability.

Humans tend to prefer walking patterns that minimize energetic cost, but must also maintain stability to avoid falling over. The relative importance of these two goals in determining the preferred gait pattern is not currently clear. We investigated the relationship between energetic cost and stability during downhill walking, a context in which gravitational energy will assist propulsion but may also reduce stability. We hypothesized that humans will not minimize energetic cost when walking downhill, but will instead prefer a gait pattern that increases stability. Simulations of a dynamic walking model were used to determine whether stable downhill gaits could be achieved using a simple control strategy. Experimentally, twelve healthy subjects walked downhill at 1.25 m/s (0, 0.05, 0.10, and 0.15 gradients). For each slope, subjects performed normal and relaxed trials, in which they were instructed to reduce muscle activity and allow gravity to maximally assist their gait. We quantified energetic cost, stride timing, and leg muscle activity. In our model simulations, increase in slope reduced the required actuation but also decreased stability. Experimental subjects behaved more like the model when using the relaxed rather than the normal walking strategy; the relaxed strategy decreased energetic cost at the steeper slopes but increased stride period variability, an indicator of instability. These results indicate that subjects do not take optimal advantage of the propulsion provided by gravity to decrease energetic cost, but instead prefer a more stable and more costly gait pattern., (2010 Elsevier Ltd. All rights reserved.)

Molecular techniques are playing an ever-increasing role in all areas of anatomic and clinical pathology. The field is currently in need of well-trained technologists in this area of the clinical laboratory who are situated to bridge the current state of practice and the continuing developments in high complexity testing. For the close term, use of "home-brew" and analyte specific reagents (ASR)-based tests will require well-trained personnel with strong biomedical science backgrounds and a thorough understanding of technologies used in assay development. Here, we discuss the selection and evaluation of molecular diagnostic training preceptor sites and tasks indicated for trainees that most meet the needs of the newest facet of the laboratory. We present evaluation tools developed over the course of four years of clinical education used to assess practical performance of trainees in a molecular diagnostic pathology laboratory and conclude with considerations for future training of laboratory technologists.

While isolating and characterizing cervical mucin glycoproteins, oviductin (Muc9) was identified in the rabbit endocervix. Following tissue homogenization, endocervical proteins were fractionated by exclusion chromatography (Sepharose CL-4B). High molecular weight components of the void volume were resolved by density gradient centrifugation using cesium bromide and dissociative conditions (4 M guanidinium chloride). High density fractions (rho = 1.40 - 1.56 g/ml) were deglycosylated with anhydrous trifluoromethane sulfonic acid and sent to Harvard Microchemistry where in situ digestion and tryptic peptide separation were performed. Out of an HPLC map, microsequence (KLIMGFPTYGR) from peak 51 was 100% identical to mouse oviductin, and microsequence (KSTGHNFPLP) from peak 70 was 90% identical to hamster oviductin. Temporal expression of oviductin transcripts (2.4-kilobase) was negligible during the first three months of postnatal cervical differentiation. Transcripts were minimally detectable in the cervices of 4-month-old juveniles. Strong expression in the endocervices of adults was eliminated by ovariectomy and restored by estrogen treatment. The presence of oviductin in the rabbit endocervix indicates this glycoprotein may have multiple functions, and it can no longer be considered oviduct-specific.

Prolactin enhances progesterone-dependent transcription of the rabbit uteroglobin gene. RUSH transcription factors are implicated in the signal transduction pathway. The RUSH acronym identifies key features of these nuclear phosphoproteins, that is, RING-finger motif, binds the uteroglobin promoter, structurally related to the SWI/SNF family of transcription factors, and helicase-like. Cloned by recognition site screening, RUSH proteins bind to an 85-bp region (-170/-85) of the uteroglobin promoter that was subsequently identified as a novel prolactin-responsive region by promoter deletion analysis. Gel shift and linker-scanning assays further reduced the RUSH target site to -160/-110. A hexameric core of MCWTDK was identified as the RUSH-specific DNA-binding site (-126/-121) by CASTing. This site overlaps authentic HNF3 beta and OCT-1 binding sites. A unique Type IV P-type ATPase that is embedded in the inner nuclear membrane binds the RING domain of RUSH. The conformationally flexible loop portion of this RING-finger binding protein (RFBP) extends into the nucleoplasm to contact euchromatin. The physical association of RFBP with transcriptionally active chromatin supports the speculation that RFBP targets RUSH transcription factors to the active uteroglobin promoter.

Objective: To determine if head-injured patients with premorbid nasal colonization with Staphylococcus aureus are at increased risk for S. aureus infection., Design: Patients admitted over a 2-yr period were enrolled if they met the following criteria: Injury Severity Score > or = 9, intensive care unit (ICU) admission, hospitalization in another hospital < 24 hrs, no recent use of antibiotics., Setting: Acute care trauma facility., Patients: Any patient sustaining acute, blunt, or penetrating injury and meeting the enrollement criteria were eligible., Interventions: Swab cultures of both internal nares were performed within 72 hrs of readmission and cultured for S. Aureus. Patients were prospectively monitored for S. Aureus infections until discharge., Measurements and Main Results: Admission nasal cultures were positive (NC+) for S. aureus in 144 of the 776 patients cultured. Forty of the 144 NC+ patients had isolated head (37) or high cervical spine (3) injury, and 11 of that group (27.5%) developed S. aureus infections. The remaining 104 patients positive for S. aureus on admission had no head injury (74) or head combined with torso and extremity injuries (30). S. aureus infection was diagnosed in 11 of the 104 patients (10.6%). The difference in incidence of infections is significant (p <.01), as is the difference in incidence of pneumonia (20% vs. 3.8%, respectively [p <.01]). Organisms causing pneumonia were often the same organisms isolated from the nares on admission., Conclusions: Nasal colonization with S. aureus at the time of severe head injury increases the risk of S. aureus pneumonia during hospitalization. Prophylactic measures against S. aureus pneumonia may help reduce the length and cost of hospitalization.

In Delaware, asthma affects almost 14,000 children. The American Lung Association of Delaware and the University of Delaware surveyed school nurses to identify the needs of children with asthma and the services and accommodations available for these children. Researchers developed a survey instrument that was mailed to all Delaware schools (N = 324). The response rate was 38.6% (n = 125). According to respondents, a variety of protocols were in place regarding the administration of asthma medications. Respondents also reported that several measures had been taken to modify the school environment to improve air quality and reduce asthma triggers. Most respondents (77%) indicated they did not have asthma education programs in their schools. Findings from this study sparked development of a multidisciplinary Delaware Asthma Committee, an Asthma Education Center, and a statewide system for communicating with the parents of children with asthma.

Staphylococcus aureus is a major cause of nosocomial infections. During the period from March 1992 to March 1994, the patients admitted to the intensive care unit of the University of Maryland Shock Trauma Center were monitored for the development of S. aureus infections. Among the 776 patients eligible for the study, 60 (7.7%) patients developed 65 incidents of nosocomial S. aureus infections. Of the clinical isolates, 43.1% possessed a polysaccharide type 5 capsule, 44.6% possessed a type 8 capsule, and the remaining 12.3% had capsules that were not typed by the type 5 or type 8 antibodies. Six antibiogram types were noted among the infection-related isolates, with the majority of the types being resistant only to penicillin and ampicillin. It was noted that the majority of cases of pneumonia were caused by relatively susceptible strains, while resistant strains were isolated from patients with bacteremia and other infections. Only 16 (6.3%) of the isolates were found to be methicillin-resistant S. aureus (MRSA). DNA fingerprinting by pulsed-field gel electrophoresis showed 36 different patterns, with characteristic patterns being found for MRSA strains and the strains with different capsular types. Clonal relationships were established, and the origins of the infection-related isolates in each patient were determined. We conclude that (i) nosocomial infection-related isolates from the shock trauma patients did not belong to a single clone, although the predominance of a methicillin-resistant genotype was noted, (ii) most infection-related S. aureus isolates were relatively susceptible to antibiotics, but a MRSA strain was endemic, and (iii) for practical purposes, the combination of the results of capsular and antibiogram typing can be used as a useful epidemiological marker.

We measured the antibody response in 10 victims of acute blunt trauma and penetrating trauma who were immunized against Klebsiella pneumoniae and Pseudomonas species within 72 hours of injury. The two vaccines, which were previously shown to be safe and immunogenic in uninjured humans, were a 24-valent K. pneumoniae capsular polysaccharide vaccine and an eight-valent Pseudomonas O-polysaccharide-toxin A conjugate vaccine. The patients were between 18 and 44 years of age, had Injury Severity Scores that ranged between 9 and 34, and did not have chronic infections or malignancies. On days 14 and 28 after immunization, all patients had a response of greater than fourfold to at least six of the nine Pseudomonas vaccine antigens. Half of the patients responded to eight of the nine antigens. Nine patients responded to at least 18 of 24 Klebsiella antigens, and seven patients responded to 22 of the 24 antigens. No important side effects were attributed to the vaccines. The results of this preliminary study indicate that active immunization against potential pathogens is possible in victims of acute trauma.

We evaluated the presence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and rapid plasma reagin (RPR) among patients admitted to our trauma unit from April 15 to June 30, 1993. Of 984 patients tested, we found 255 (26%) had evidence of exposure to one or more of these agents: HIV, 4%; HBV, 20%; HCV, 14%; and RPR, 1%. Thirty-eight percent of patients had more than one positive serology, 75% of the HIV patients, 49% of the HBV patients, and 66% of the HCV patients. There was no difference between penetrating and nonpenetrating trauma with respect to any of the viruses. The risk factors for HIV-positive patients were non-White race, positive drug screen, positive alcohol screen, and city resident. Risk factors for HBV patients were non-White race, positive drug screen, and city resident. Risk factors for HBC patients were male sex, non-White race, positive alcohol screen, positive drug screen, and city resident. The risk of blood-borne infections in this group of patients is substantial.

The formation of myometrial gap junctions coincides with onset of labor in many mammalian species, including humans. The assembly of gap junction protein into functional gap junction plaques is a final step in a cascade that begins with estrogen-dependent expression of the connexin43 (cx43) gene and continues with synthesis of cx43 in the rough endoplasmic reticulum (RER) and transport to the Golgi, followed by its trafficking to the plasma membrane and its assembly into functional gap junctions. Moreover, in several models of preterm labor in rats, precocious synthesis, trafficking, and assembly of cx43 follow an increase in the estrogen:progesterone ratio. The actions of these steroids on cx43 expression, gap junction formation, and labor led us to consider whether or not the cascade of cx43 expression and gap junction assembly typical of preterm and term labor would be disrupted by manipulations that inhibit labor through experimental reduction of the estrogen:progesterone ratio. Ovariectomized and non-ovariectomized pregnant rats were treated with minimal doses of progesterone or the anti-estrogenic compound ICI 182780 over a time course sufficient to inhibit labor. We found that cx43-positive gap junction formation was prevented in all animals treated with ICI 182780 or progesterone but that the mechanism by which this disruption occurred was different in anti-estrogen- and progesterone-treated animals. We found that ICI 182780 significantly inhibited the typical rise in myometrial cx43 concentrations normally observed just before labor. In contrast, it was surprising to find that significant cx43 was synthesized in myometrium of progesterone-treated intact and ovariectomized animals even though labor was inhibited. However, we found that the trafficking of myometrial cx43 from the Golgi and assembly into gap junctions at the plasma membrane were suppressed in these progesterone-treated animals, providing further support for the hypothesis that it is not synthesis of cx43 per se but trafficking of cx43 to the plasma membrane and its assembly into gap junctions that are required for effective synchronized myometrial contractions typical of labor.

The Celay-system offers an interesting alternative to existing systems for esthetic posterior restorations with ceramics. The basic idea of the concept is to prepare, either directly on the patient or indirectly on a plaster model, a pro-inlay using a light-curable precision material. The modelling also covers the occlusal surface. This pro-inlay is then used with the help of a milling center to machine a precise replica out of a ceramic block. The copying process is carried out via three-dimensional mechanical scanning and milling using eight axes of freedom. In this way additional corrections on the ceramic within the mouth can almost completely be avoided. The system is designed to be used as a direct chairside method by the dentist or as an indirect method by the dental technician. It is a promising but unproved restoration, more research and long-term clinical evaluation is recommended.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.

One aspect of biofeedback treatment with chronically incapacitated persons concerns their resistance to getting better. In effect, the removal of their symptom is equivalent to undercutting a characteristic way of relating to themselves and to the world, and hence, is opposed. Even though many biofeedback procedures are impressively effective, the therapist must watch for another symptom reemerging in the place of the older somatic complaint, or for psychologically regressive attempts to preserve one's character defenses.