Unique characteristics of the J-domain proximal regions of Hsp70 cochaperone Apj1 in prion propagation/elimination and its overlap with Sis1 function.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s. The Class A JDP Apj1 of the yeast cytosol has an unusually complex region between the N-terminal J-domain and the substrate binding region-often called the Grich or GF region in Class A and B JDPs because of its typical abundance of glycine. The N-terminal 161-residue Apj1 fragment is known to be sufficient for Apj1 function in prion curing, driven by the overexpression of Hsp104. Further analyzing the N-terminal segment of Apj1, we found that a 90-residue fragment that includes the 70-residue J-domain and the adjacent 12-residue glutamine/alanine (Q/A) segment is sufficient for curing. Furthermore, the 121-residue fragment that includes the Grich region was sufficient to not only sustain the growth of cells lacking the essential Class B JDP Sis1 but also enabled the maintenance of several prions normally dependent on Sis1 for propagation. A J-domain from another cytosolic JDP could substitute for the Sis1-related functions but not for Apj1 in prion curing. Together, these results separate the functions of JDPs in prion biology and underscore the diverse functionality of multi-domain cytosolic JDPs in yeast.

Chaperone function in Fe-S protein biogenesis: Three possible scenarios.

Among the six known iron­sulfur (FeS) cluster biogenesis machineries that function across all domains of life only one involves a molecular chaperone system. This machinery, called ISC for 'iron sulfur cluster', functions in bacteria and in mitochondria of eukaryotes including humans. The chaperone system - a dedicated J-domain protein co-chaperone termed Hsc20 and its Hsp70 partner - is essential for proper ISC machinery function, interacting with the scaffold protein IscU which serves as a platform for cluster assembly and subsequent transfer onto recipient apo-proteins. Despite many years of research, surprisingly little is known about the specific role(s) that the chaperones play in the ISC machinery. Here we review three non-exclusive scenarios that range from involvement of the chaperones in the cluster transfer to regulation of the cellular levels of IscU itself.

Comparative structural and functional analysis of the glycine-rich regions of Class A and B J-domain protein cochaperones of Hsp70.

J-domain proteins are critical Hsp70 co-chaperones. A and B types have a poorly understood glycine-rich region (Grich) adjacent to their N-terminal J-domain (Jdom). We analyzed the ability of Jdom/Grich segments of yeast Class B Sis1 and a suppressor variant of Class A, Ydj1, to rescue the inviability of sis1-∆. In each, we identified a cluster of Grich residues required for rescue. Both contain conserved hydrophobic and acidic residues and are predicted to form helices. While, as expected, the Sis1 segment docks on its J-domain, that of Ydj1 does not. However, data suggest both interact with Hsp70. We speculate that the Grich-Hsp70 interaction of Classes A and B J-domain proteins can fine tune the activity of Hsp70, thus being particularly important for the function of Class B.

NAC and Zuotin/Hsp70 chaperone systems coexist at the ribosome tunnel exit in vivo.

The area surrounding the tunnel exit of the 60S ribosomal subunit is a hub for proteins involved in maturation and folding of emerging nascent polypeptide chains. How different factors vie for positioning at the tunnel exit in the complex cellular environment is not well understood. We used in vivo site-specific cross-linking to approach this question, focusing on two abundant factors-the nascent chain-associated complex (NAC) and the Hsp70 chaperone system that includes the J-domain protein co-chaperone Zuotin. We found that NAC and Zuotin can cross-link to each other at the ribosome, even when translation initiation is inhibited. Positions yielding NAC-Zuotin cross-links indicate that when both are present the central globular domain of NAC is modestly shifted from the mutually exclusive position observed in cryogenic electron microscopy analysis. Cross-linking results also suggest that, even in NAC's presence, Hsp70 can situate in a manner conducive for productive nascent chain interaction-with the peptide binding site at the tunnel exit and the J-domain of Zuotin appropriately positioned to drive stabilization of nascent chain binding. Overall, our results are consistent with the idea that, in vivo, the NAC and Hsp70 systems can productively position on the ribosome simultaneously.

Health and social care needs of people living with dementia: a qualitative study of dementia support in the Victorian region of Gippsland, Australia.

INTRODUCTION: There is a lack of information about the experiences of people living with dementia and their carers, especially in rural and regional areas. Understanding these experiences helps to identify gaps and unmet needs within the health system and improve quality of care and outcomes for people living with dementia. The aim of this study was to improve our knowledge of dementia support needs. This included access to health and social care services and supports for people living with dementia and those who provide informal or formal support to someone living with dementia. METHODS: Interviews were conducted with 26 participants from the Gippsland region of Victoria, Australia with knowledge of dementia care. Purposive sampling engaged people with lived experience, carers/family members and health professionals delivering dementia care and social services. Discussions centred around participants' experiences of support services, the diagnosis process and what they thought was needed to improve the services and supports offered. Thematic analysis of the data was undertaken using the framework method. RESULTS: The interview data indicated that the needs of many people living with dementia and their carers were not currently being met. The themes were limited access to services and supports, including primary and specialist care, often impacted by lack of knowledge of care options, difficulty navigating the system and funding models as a barrier, leading to delays in getting a diagnosis and accessing specialist services; lack of holistic care to enable people living with dementia to 'live well'; and stigma impacted by a lack of knowledge of dementia among professionals and in the community. Relationship-centred care was described as a way to improve the lives of people living with dementia. CONCLUSION: Key areas for improvement include increasing community awareness of dementia and available local services, more support to obtain an early dementia diagnosis, increased help to navigate the system, especially immediately after diagnosis, and easier access to appropriate home support services when they are needed. Other recommendations include person-centred care across settings - supported by funding models, more education and communication skills training for health professionals and care staff - and greater support for and increased recognition of carers.

Analysis of Reconstituted Tripartite Complex Supports Avidity-based Recruitment of Hsp70 by Substrate Bound J-domain Protein.

Hsp70 are ubiquitous, versatile molecular chaperones that cyclically interact with substrate protein(s). The initial step requires synergistic interaction of a substrate and a J-domain protein (JDP) cochaperone, via its J-domain, with Hsp70 to stimulate hydrolysis of its bound ATP. This hydrolysis drives conformational changes in Hsp70 that stabilize substrate binding. However, because of the transient nature of substrate and JDP interactions, this key step is not well understood. Here we leverage a well characterized Hsp70 system specialized for iron-sulfur cluster biogenesis, which like many systems, has a JDP that binds substrate on its own. Utilizing an ATPase-deficient Hsp70 variant, we isolated a Hsp70-JDP-substrate tripartite complex. Complex formation and stability depended on residues previously identified as essential for bipartite interactions: JDP-substrate, Hsp70-substrate and J-domain-Hsp70. Computational docking based on the established J-domain-Hsp70(ATP) interaction placed the substrate close to its predicted position in the peptide-binding cleft, with the JDP having the same architecture as when in a bipartite complex with substrate. Together, our results indicate that the structurally rigid JDP-substrate complex recruits Hsp70(ATP) via precise positioning of J-domain and substrate at their respective interaction sites - resulting in functionally high affinity (i.e., avidity). The exceptionally high avidity observed for this specialized system may be unusual because of the rigid architecture of its JDP and the additional JDP-Hsp70 interaction site uncovered in this study. However, functionally important avidity driven by JDP-substrate interactions is likely sufficient to explain synergistic ATPase stimulation and efficient substrate trapping in many Hsp70 systems.

PTSD service dogs foster resilience among veterans and military families.

Military deployment and reintegration challenges permeate the lives and relationships of Veterans, their spouses, and their families. Among these challenges, 23% of post-9/11 Veterans have been diagnosed with posttraumatic stress disorder (PTSD). Psychiatric service dogs have been found to help clinically alleviate PTSD symptoms when used as a complementary intervention. However, minimal research exists that explores the role of the service dog as a mechanism for cultivating resilience within the military family system. Researchers utilized a qualitative, constant comparative approach to analyze self-reported experiences of 101 individuals, including Veterans (n = 67) and their spouses (n = 34). Analyzed through the framework of the Theory of Resilience and Relational Load (Afifi et al., 2016), findings suggest complex communication processes that facilitate relational and family adaptation. These processes encompassed (a) the role of the service dog in building emotional reserves, (b) relational load introduced when caring for the service dog, and (c) the service dog's facilitation of relational maintenance behaviors among family members that contributed to communal orientation. Based on the results of this qualitative analysis, researchers suggest educational interventions where service dog trainers and mental health practitioners can incorporate relational maintenance strategies and family-focused approaches to integrating service dogs as military family members.

J-Domain Proteins Orchestrate the Multifunctionality of Hsp70s in Mitochondria: Insights from Mechanistic and Evolutionary Analyses.

Mitochondrial J-domain protein (JDP) co-chaperones orchestrate the function of their Hsp70 chaperone partner(s) in critical organellar processes that are essential for cell function. These include folding, refolding, and import of mitochondrial proteins, maintenance of mitochondrial DNA, and biogenesis of iron-sulfur cluster(s) (FeS), prosthetic groups needed for function of mitochondrial and cytosolic proteins. Consistent with the organelle's endosymbiotic origin, mitochondrial Hsp70 and the JDPs' functioning in protein folding and FeS biogenesis clearly descended from bacteria, while the origin of the JDP involved in protein import is less evident. Regardless of their origin, all mitochondrial JDP/Hsp70 systems evolved unique features that allowed them to perform mitochondria-specific functions. Their modes of functional diversification and specialization illustrate the versatility of JDP/Hsp70 systems and inform our understanding of system functioning in other cellular compartments.

Behavioral Measures to Assess Adolescent Sexual Communication with Partners: A Scoping Review and Call for Further Studies.

Sexual communication with partners is important for adolescents' sexual and socioemotional well-being. Behavioral assessments of partner sexual communication capture the complex and nuanced process of communication and are commonly used with adults, yet the existing literature among adolescents overwhelmingly relies on self-report measures. In the current paper, we reviewed the literature on adolescent partner sexual communication, identifying 14 studies including 2,043 participants (M age = 16) that used behavioral assessments (i.e., dyadic observations, role-plays with confederates, role-plays with vignettes). We also identify key gaps in the current literature: First, only one study recruited couples; studies that assessed dyadic interactions largely relied on confederates. Second, assessments often assumed that participants engaged in heterosexual sex, and no studies specifically recruited LGBTQ+ adolescents. Third, behavioral tasks often involved assumptions of participants' sexual goals (e.g., desire to refuse sex) and focused almost exclusively on sexual refusal and condom negotiation. Additionally, coding schemes lacked standardization and micro-analytic strategies (e.g., coding change over time). Finally, observational methods have been almost exclusively used to assess intervention efficacy, rather than to understand associations between behaviorally-assessed communication skills and sexual outcomes or self-reported communication in basic research. We discuss recommendations for future research, including regular use of behavioral observation methods with diverse samples, to triangulate across multiple methodologies and identify correspondence between behavioral and self-report measures.

Interaction of client-the scaffold on which FeS clusters are build-with J-domain protein Hsc20 and its evolving Hsp70 partners.

In cells molecular chaperone systems consisting of Hsp70 and its obligatory J-domain protein (JDP) co-chaperones transiently interact with a myriad of client proteins-with JDPs typically recruiting their partner Hsp70 to interact with particular clients. The fundamentals of this cyclical interactions between JDP/Hsp70 systems and clients are well established. Much less is known about other aspects of JDP/Hsp70 system function, including how such systems evolved over time. Here we discuss the JDP/Hsp70 system involved in the biogenesis of iron-sulfur (FeS) clusters. Interaction between the client protein, the scaffold on which clusters are built, and its specialized JDP Hsc20 has stayed constant. However, the system's Hsp70 has changed at least twice. In some species Hsc20's Hsp70 partner interacts only with the scaffold, in others it has many JDP partners in addition to Hsc20 and interacts with many client proteins. Analysis of this switching of Hsp70 partners has provided insight into the insulation of JDP/Hsp70 systems from one another that can occur when more than one Hsp70 is present in a cellular compartment, as well as how competition among JDPs is balanced when an Hsp70 partner is shared amongst a number of JDPs. Of particularly broad relevance, even though the scaffold's interactions with Hsc20 and Hsp70 are functionally critical for the biogenesis of FeS cluster-containing proteins, it is the modulation of the Hsc20-Hsp70 interaction per se that allows Hsc20 to function with such different Hsp70 partners.

Sexual Communication between Adolescent Partners: A Scoping Review and Directions for Future Research.

Sexual communication between adolescent partners is an important component of sexual health and wellbeing. Over 40 years of research on adolescent sexual communication has yielded rich information, yet there remain gaps in our understanding of the communication process. The purpose of this scoping review was to synthesize the body of research on adolescent sexual communication to identify how communication has been conceptualized, how researchers have measured communication, and what theoretical frameworks have been applied across the literature. We identified 198 assessments of sexual communication across 119 quantitative studies. This work included 127,489 adolescents (Mage = 15.97) from 15 countries (81.5% U.S.-based). Most studies relied on self-reports (93.4%) and surveyed only one member of a couple (97.5%). The definition of sexual communication was highly varied across the literature: in half of assessments (52.0%) sexual communication was operationalized as a behavior-the verbal or nonverbal exchange of messages about sex-whereas the remaining half of assessments captured social-cognitive aspects of communication (e.g., communication self-efficacy, fear/anxiety). There was also a tendency for investigators to create their own idiosyncratic instruments: half of studies (48.9%) used instruments created by the research team with limited or no discussion of reliability/validity. Regarding the topic of communication, a third of assessments (33.8%) focused exclusively on condom communication and another quarter (24.0%) focused on other safer-sex issues (e.g., STDs, abstinence). Notably absent were studies focused on communication surrounding consent or sexual pleasure. Also absent was a guiding conceptual model or theory that could unify this body of work. Overall, results highlight gaps and inconsistencies in how partner sexual communication has been conceptualized, measured, and theorized about in previous work. We provide several recommendations for future theory-building efforts as well as rigorous, multimethod empirical investigations of adolescent sexual communication that would further our understanding of this important aspect of adolescent sexual wellbeing.

Incidence of microplastic fiber ingestion by Common Terns (Sterna hirundo) and Roseate Terns (S. dougallii) breeding in the Northwestern Atlantic.

Ingestion of microplastics has been documented across marine species, but exposure remains sparsely described in many seabird species. We assess microplastic (between 0.2 and 5.0 mm) ingestion in two Northwestern Atantic - breeding species for which exposure to microplastics is entirely or largely undescribed: Common Terns (Sterna hirundo) and Roseate Terns (S. dougallii). Common Tern microplastic load did not vary between life stages (p = 0.590); microplastic load did differ in Common Tern adults breeding at two of three colonies explored (p = 0.002), with no other regional differences observed. Roseate Terns ingested significantly more microplastics than Common Terns (p = 0.007). Our results show that microplastic ingestion by terns varies regionally and interspecifically, but not by life stage, trends potentially explained by dietary differences. We provide the first quantification of microplastic fiber ingestion by terns in the Northwestern Atlantic and identify trophic dynamics related to microplastic ingestion, representing an important step toward understanding the risk of the pollutant to terns across regions, as well as toward the use of terns as potential bioindicators of microplastics.

During FeS cluster biogenesis, ferredoxin and frataxin use overlapping binding sites on yeast cysteine desulfurase Nfs1.

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron-sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins. Nfs1 functions both as a sulfur donor for cluster assembly and as a binding platform for other proteins functioning in the process. These include not only the dedicated scaffold protein (Isu1) on which FeS clusters are synthesized but also accessory FeS cluster biogenesis proteins frataxin (Yfh1) and ferredoxin (Yah1). Yfh1 has been shown to activate cysteine desulfurase enzymatic activity, whereas Yah1 supplies electrons for the persulfide reduction. While Yfh1 interaction with Nfs1 is well understood, the Yah1-Nfs1 interaction is not. Here, based on the results of biochemical experiments involving purified WT and variant proteins, we report that in Saccharomyces cerevisiae, Yah1 and Yfh1 share an evolutionary conserved interaction site on Nfs1. Consistent with this notion, Yah1 and Yfh1 can each displace the other from Nfs1 but are inefficient competitors when a variant with an altered interaction site is used. Thus, the binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system. Our findings are consistent with the generally accepted scenario that the mitochondrial FeS cluster assembly system was inherited from bacterial ancestors of mitochondria.

Essentiality of Sis1, a J-domain protein Hsp70 cochaperone, can be overcome by Tti1, a specialized PIKK chaperone.

J-domain protein cochaperones drive much of the functional diversity of Hsp70-based chaperone systems. Sis1 is the only essential J-domain protein of the cytosol/nucleus of Saccharomyces cerevisiae. Why it is required for cell growth is not understood, nor how critical its role is in regulation of heat shock transcription factor 1 (Hsf1). We report that single-residue substitutions in Tti1, a component of the heterotrimeric TTT complex, a specialized chaperone system for phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, allow growth of cells lacking Sis1. Upon depletion of Sis1, cells become hypersensitive to rapamycin, a specific inhibitor of TORC1 kinase. In addition, levels of the three essential PIKKs (Mec1, Tra1, and Tor2), as well as Tor1, decrease upon Sis1 depletion. Overexpression of Tti1 allows growth without an increase in the other subunits of the TTT complex, Tel2 and Tti2, suggesting that it can function independent of the complex. Cells lacking Sis1, with viability supported by Tti1 suppressor, substantially up-regulate some, but not all, heat shock elements activated by Hsf1. Together, our results suggest that Sis1 is required as a cochaperone of Hsp70 for the folding/maintenance of PIKKs, making Sis1 an essential gene, and its requirement for Hsf1 regulation is more nuanced than generally appreciated.

Pathway of Hsp70 interactions at the ribosome.

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Optimizing detection of postoperative leaks on upper gastrointestinal fluoroscopy: a step-by-step guide.

Postoperative leaks after gastrointestinal surgery are important to identify to decrease patient morbidity and mortality. Fluoroscopic studies are commonly employed to detect postoperative leak. While the literature addresses the sensitivity and specificity of these examinations, there is generally a lack of description of the fluoroscopic technique itself and there may be variability between radiologists in how these studies are performed. It is important to balance a standardized fluoroscopy protocol while tailoring the exam for each surgical and patient situation. Here we will briefly review common postoperative anatomy in the upper gastrointestinal tract, propose fluoroscopic techniques to improve postoperative leak detection, and illustrate teaching points with clinical cases.

Complications of liver transplant.

Liver transplantation has become a definitive treatment for patients with end-stage liver disease and those meeting Milan criteria for hepatocellular carcinoma. The morbidity and mortality associated with liver transplantation continues to decrease thanks to refinements in surgical technique, immunosuppression, and imaging. In particular, imaging plays a vital role by facilitating early detection of post-operative complications and enabling prompt treatment. Post-operative complications that lead to graft failure and patient morbidity/mortality can be generally categorized as vascular, biliary, parenchymal, and malignant. Vascular complications include stenosis and thrombosis of the hepatic artery, portal vein, and inferior vena cava; hepatic artery pseudoaneurysm; arteriovenous fistula; and celiac stenosis. Biliary abnormalities include strictures, bile leak, obstruction, recurrent disease, and infection. While imaging is not primarily utilized to diagnose allograft rejection, it plays an important role in excluding mechanical causes of graft dysfunction that can mimic rejection. Ultrasound is routinely performed as the first-line imaging evaluation for the detection and follow-up of early and delayed complications. Cholangiography and magnetic resonance cholangiopancreatography are useful in detecting and characterizing biliary complications. Computed tomography is often used to further evaluate abnormal findings on ultrasound or for the characterization of post-operative fluid collections. The aim of this review is to discuss and illustrate the imaging findings of complications associated with liver transplantation and their role in facilitating treatment.

Patient-Centered Communication (PCC) in Equine Assisted Mental Health.

Experiencing trauma can lead to a variety of chronic and acute symptoms, including post- traumatic stress disorder (PTSD), anxiety, depression, substance abuse, and poor social skills. Given the variety of causes for trauma incorporating individualized treatment options is important for efficacy. Equine assisted mental health (EAMH) - a team approach incorporating equines, clients, and practitioners - has been successful in treating those who have experienced trauma, including veterans and individuals with PTSD, at-risk youth, victims of sexual violence, and children who have been neglected. Although researchers and practitioners understand some about how EAMH treatment results in positive outcomes for these individuals, little is known about the communicative processes that support them. The current study included 19 in-depth interviews with EAMH therapists and practitioners to explore the role of equine communication (i.e., congruence, ongoing positive regard, and empathy) as a communicative process that is integral to the facilitation of EAMH as individualized therapeutic treatment. Using tenets of patient-centered communication (PCC) and principles of client-centered therapy, implications for human-horse communication in therapeutic contexts and client-centered care are discussed.

Two-step mechanism of J-domain action in driving Hsp70 function.

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface-thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition-via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage.

Biochemical Convergence of Mitochondrial Hsp70 System Specialized in Iron-Sulfur Cluster Biogenesis.

Mitochondria play a central role in the biogenesis of iron-sulfur cluster(s) (FeS), protein cofactors needed for many cellular activities. After assembly on scaffold protein Isu, the cluster is transferred onto a recipient apo-protein. Transfer requires Isu interaction with an Hsp70 chaperone system that includes a dedicated J-domain protein co-chaperone (Hsc20). Hsc20 stimulates Hsp70's ATPase activity, thus stabilizing the critical Isu-Hsp70 interaction. While most eukaryotes utilize a multifunctional mitochondrial (mt)Hsp70, yeast employ another Hsp70 (Ssq1), a product of mtHsp70 gene duplication. Ssq1 became specialized in FeS biogenesis, recapitulating the process in bacteria, where specialized Hsp70 HscA cooperates exclusively with an ortholog of Hsc20. While it is well established that Ssq1 and HscA converged functionally for FeS transfer, whether these two Hsp70s possess similar biochemical properties was not known. Here, we show that overall HscA and Ssq1 biochemical properties are very similar, despite subtle differences being apparent - the ATPase activity of HscA is stimulated to a somewhat higher levels by Isu and Hsc20, while Ssq1 has a higher affinity for Isu and for Hsc20. HscA/Ssq1 are a unique example of biochemical convergence of distantly related Hsp70s, with practical implications, crossover experimental results can be combined, facilitating understanding of the FeS transfer process.