Wnt-Frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt).

The establishment of planar cell polarity (PCP) in epithelial and mesenchymal cells is a critical, evolutionarily conserved process during development and organogenesis. Analyses in Drosophila and several vertebrate model organisms have contributed a wealth of information on the regulation of PCP. A key conserved pathway regulating PCP, the so-called core Wnt-Frizzled PCP (Fz/PCP) signaling pathway, was initially identified through genetic studies of Drosophila. PCP studies in vertebrates, most notably mouse and zebrafish, have identified novel factors in PCP signaling and have also defined cellular features requiring PCP signaling input. These studies have shifted focus to the role of Van Gogh (Vang)/Vangl genes in this molecular system. This review focuses on new insights into the core Fz/Vangl/PCP pathway and recent advances in Drosophila and vertebrate PCP studies. We attempt to integrate these within the existing core Fz/Vangl/PCP signaling framework.

Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.

Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. Here, we generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. We show that Tmem208 is broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, we identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, our study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity.

Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Cancer-associated fibroblasts influence Wnt/PCP signaling in gastric cancer cells by cytoneme-based dissemination of ROR2.

Cancer-associated fibroblasts (CAFs) are a crucial component in the tumor microenvironment influencing cancer progression. Besides shaping the extracellular matrix, these fibroblasts provide signaling factors to facilitate tumor survival and alter tumor behavior. In gastric cancer, one crucial signaling pathway influencing invasion and metastasis is the Wnt/Planar Cell Polarity (PCP) signaling. The crucial PCP ligand in this context is WNT5A, which is produced by the CAFs, and gastric cancer cells react upon this signal by enhanced polarized migration. Why gastric cancer cells respond to this signal is still unclear, as their expression level for the central WNT5A receptor, ROR2, is very low. Here, we show that CAFs display long and branched filopodia that form an extensive, complex network engulfing gastric cancer cells, such as the gastric cancer cell line AGS. CAFs have a significantly higher expression level of ROR2 than normal gastric fibroblasts and AGS cells. By high-resolution imaging, we observe a direct transfer of fluorescently tagged ROR2 from CAF to AGS cells by signaling filopodia, known as cytonemes. Surprisingly, we find that the transferred ROR2 complexes can activate Wnt/JNK signaling in AGS cells. Consistently, blockage of ROR2 function in the CAFs leads to reduced paracrine Wnt/JNK signaling, cell polarization, and migration of the receiving AGS cells. Complementary, enhanced migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These findings demonstrate a fresh role for cytoneme-mediated signaling in the tumor microenvironment. Cytonemes convey Wnt receptors from CAFs to gastric cancer cells, allowing them to respond to Wnt/PCP signals.

Pneumocystis jirovecii pneumonia in people living with HIV: a review.

Pneumocystis jirovecii is a ubiquitous opportunistic fungus that can cause life-threatening pneumonia. People with HIV (PWH) who have low CD4 counts are one of the populations at the greatest risk of Pneumocystis jirovecii pneumonia (PCP). While guidelines have approached the diagnosis, prophylaxis, and management of PCP, the numerous studies of PCP in PWH are dominated by the 1980s and 1990s. As such, most studies have included younger male populations, despite PCP affecting both sexes and a broad age range. Many studies have been small and observational in nature, with an overall lack of randomized controlled trials. In many jurisdictions, and especially in low- and middle-income countries, the diagnosis can be challenging due to lack of access to advanced and/or invasive diagnostics. Worldwide, most patients will be treated with 21 days of high-dose trimethoprim sulfamethoxazole, although both the dose and the duration are primarily based on historical practice. Whether treatment with a lower dose is as effective and less toxic is gaining interest based on observational studies. Similarly, a 21-day tapering regimen of prednisone is used for patients with more severe disease, yet other doses, other steroids, or shorter durations of treatment with corticosteroids have not been evaluated. Now with the widespread availability of antiretroviral therapy, improved and less invasive PCP diagnostic techniques, and interest in novel treatment strategies, this review consolidates the scientific body of literature on the diagnosis and management of PCP in PWH, as well as identifies areas in need of more study and thoughtfully designed clinical trials.

A vertebrate Vangl2 translational variant required for planar cell polarity.

First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues.

Alignment of the cell long axis by unidirectional tension acts cooperatively with Wnt signalling to establish planar cell polarity.

Planar cell polarity (PCP) is the aligned cell polarity within a tissue plane. Mechanical signals are known to act as a global cue for PCP, yet their exact role is still unclear. In this study, we focused on PCP in the posterior neuroectoderm of Xenopus laevis and investigated how mechanical signals regulate polarity. We reveal that the neuroectoderm is under a greater tension in the anterior-posterior direction and that perturbation of this tension causes PCP disappearance. We show that application of uniaxial stretch to explant tissues can control the orientation of PCP and that cells sense the tissue stretch indirectly through a change in their shape, rather than directly through detection of anisotropic tension. Furthermore, we reveal that PCP is most strongly established when the orientation of tissue stretch coincides with that of diffusion of locally expressed Wnt ligands, suggesting a cooperative relationship between these two PCP regulators.

Sequential action of JNK genes establishes the embryonic left-right axis.

The establishment of the left-right axis is crucial for the placement, morphogenesis and function of internal organs. Left-right specification is proposed to be dependent on cilia-driven fluid flow in the embryonic node. Planar cell polarity (PCP) signalling is crucial for patterning of nodal cilia, yet downstream effectors driving this process remain elusive. We have examined the role of the JNK gene family, a proposed downstream component of PCP signalling, in the development and function of the zebrafish node. We show jnk1 and jnk2 specify length of nodal cilia, generate flow in the node and restrict southpaw to the left lateral plate mesoderm. Moreover, loss of asymmetric southpaw expression does not result in disturbances to asymmetric organ placement, supporting a model in which nodal flow may be dispensable for organ laterality. Later, jnk3 is required to restrict pitx2c expression to the left side and permit correct endodermal organ placement. This work uncovers multiple roles for the JNK gene family acting at different points during left-right axis establishment. It highlights extensive redundancy and indicates JNK activity is distinct from the PCP signalling pathway.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

Vertebrate Wnt5a - At the crossroads of cellular signalling.

Wnt signalling is an essential pathway in embryogenesis, differentiation, cell motility, development, and adult tissue homeostasis in vertebrates. The Wnt signalling network can activate several downstream pathways such as the ß-catenin-dependent TCF/LEF transcription, the Wnt/planar cell polarity (PCP) pathway, and the Wnt/Calcium pathway. Wnt5a is a vertebrate Wnt ligand that is most often associated with the Wnt/PCP signalling pathway. Wnt5a/PCP signalling has a well-described role in embryogenesis via binding to a receptor complex of Frizzled and its co-receptors to initiate downstream activation of the c-Jun N-terminal kinase (JNK) signalling cascade and the Rho and Rac GTPases, Rho-Kinase (ROCK). This activation results in the cytoskeletal remodelling required for cell polarity, migration, and subsequently, tissue re-arrangement and organ formation. This review will focus on more recent work that has revealed new roles for Wnt5a ligands and consequently, an emerging broader function. This is partly due to our growing understanding of the crosstalk between the Wnt/PCP pathway with both the Wnt/ß-catenin pathway and other signalling pathways, and in part due to the identification of novel atypical receptors for Wnt5a that demonstrate a far broader role for this ligand.

Planar cell polarity (PCP) proteins support spermatogenesis through cytoskeletal organization in the testis.

Few reports are found in the literature regarding the role of planar cell polarity (PCP) in supporting spermatogenesis in the testis. Yet morphological studies reported decades earlier have illustrated the directional alignment of polarized developing spermatids, most notably step 17-19 spermatids in stage V-early VIII tubules in the testis, across the plane of the epithelium in seminiferous tubules of adult rats. Such morphological features have unequivocally demonstrated the presence of PCP in developing spermatids, analogous to the PCP noted in hair cells of the cochlea in mammals. Emerging evidence in recent years has shown that Sertoli and germ cells express numerous PCP proteins, mostly notably, the core PCP proteins, PCP effectors and PCP signaling proteins. In this review, we discuss recent findings in the field regarding the two core PCP protein complexes, namely the Van Gogh-like 2 (Vangl2)/Prickle (Pk) complex and the Frizzled (Fzd)/Dishevelled (Dvl) complex. These findings have illustrated that these PCP proteins exert their regulatory role to support spermatogenesis through changes in the organization of actin and microtubule (MT) cytoskeletons in Sertoli cells. For instance, these PCP proteins confer PCP to developing spermatids. As such, developing haploid spermatids can be aligned and orderly packed within the limited space of the seminiferous tubules in the testes for the production of sperm via spermatogenesis. Thus, each adult male in the mouse, rat or human can produce an upward of 30, 50 or 300 million spermatozoa on a daily basis, respectively, throughout the adulthood. We also highlight critical areas of research that deserve attention in future studies. We also provide a hypothetical model by which PCP proteins support spermatogenesis based on recent studies in the testis. It is conceivable that the hypothetical model shown here will be updated as more data become available in future years, but this information can serve as the framework by investigators to unravel the role of PCP in spermatogenesis.

Bridging the gap between non-canonical and canonical Wnt signaling through Vangl2.

Non-canonical Wnt signaling (encompassing Wnt/PCP and WntCa2+) has a dual identity in the literature. One stream of research investigates its role in antagonizing canonical Wnt/ß-catenin signaling in cancer, typically through Ca2+, while the other stream investigates its effect on polarity in development, typically through Vangl2. Rarely do these topics intersect or overlap. What has become clear is that Wnt5a can mobilize intracellular calcium stores to inhibit Wnt/ß-catenin in cancer cells but there is no evidence that Vangl2 is involved in this process. Conversely, Wnt5a can independently activate Vangl2 to affect polarity and migration but the role of calcium in this process is also limited. Further, Vangl2 has also been implicated in inhibiting Wnt/ß-catenin signaling in development. The consensus is that a cell can differentiate between canonical and non-canonical Wnt signaling when presented with a choice, always choosing non-canonical at the expense of canonical Wnt signaling. However, these are rare events in vivo. Given the shared resources between non-canonical and canonical Wnt signaling it is perplexing that there is not more in vivo evidence for cross talk between these two pathways. In this review we discuss the intersection of non-canonical Wnt, with a focus on Wnt/PCP, and Wnt/ß-catenin signaling in an attempt to shed some light on pathways that rarely meet at a crossroads in vivo.

Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis.

BACKGROUND: This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations. METHODS: A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined. RESULTS: Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients. CONCLUSIONS: On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested.

The alveolar fibroproliferative response in moderate to severe COVID-19-related acute respiratory distress syndrome and 1-yr follow-up.

COVID-19-related acute respiratory distress syndrome (ARDS) can lead to long-term pulmonary fibrotic lesions. Alveolar fibroproliferative response (FPR) is a key factor in the development of pulmonary fibrosis. N-terminal peptide of procollagen III (NT-PCP-III) is a validated biomarker for activated FPR in ARDS. This study aimed to assess the association between dynamic changes in alveolar FPR and long-term outcomes, as well as mortality in COVID-19 ARDS patients. We conducted a prospective cohort study of 154 COVID-19 ARDS patients. We collected bronchoalveolar lavage (BAL) and blood samples for measurement of 17 pulmonary fibrosis biomarkers, including NT-PCP-III. We assessed pulmonary function and chest computed tomography (CT) at 3 and 12 mo after hospital discharge. We performed joint modeling to assess the association between longitudinal changes in biomarker levels and mortality at day 90 after starting mechanical ventilation. 154 patients with 284 BAL samples were analyzed. Of all patients, 40% survived to day 90, of whom 54 completed the follow-up procedure. A longitudinal increase in NT-PCP-III was associated with increased mortality (HR 2.89, 95% CI: 2.55-3.28; P < 0.001). Forced vital capacity and diffusion for carbon monoxide were impaired at 3 mo but improved significantly at one year after hospital discharge (P = 0.03 and P = 0.004, respectively). There was no strong evidence linking alveolar FPR during hospitalization and signs of pulmonary fibrosis in pulmonary function or chest CT images during 1-yr follow-up. In COVID-19 ARDS patients, alveolar FPR during hospitalization was associated with higher mortality but not with the presence of long-term fibrotic lung sequelae within survivors.NEW & NOTEWORTHY This is the first prospective study on the longitudinal alveolar fibroproliferative response in COVID-19 ARDS and its relationship with mortality and long-term follow-up. We used the largest cohort of COVID-19 ARDS patients who had consecutive bronchoalveolar lavages and measured 17 pulmonary fibroproliferative biomarkers. We found that a higher fibroproliferative response during admission was associated with increased mortality, but not correlated with long-term fibrotic lung sequelae in survivors.

QuantifyPolarity, a new tool-kit for measuring planar polarized protein distributions and cell properties in developing tissues.

The coordination of cells or structures within the plane of a tissue is known as planar polarization. It is often governed by the asymmetric distribution of planar polarity proteins within cells. A number of quantitative methods have been developed to provide a readout of planar polarized protein distributions. However, previous planar polarity quantification methods can be affected by variation in cell geometry. Hence, we developed a novel planar polarity quantification method based on Principal Component Analysis (PCA) that is shape insensitive. Here, we compare this method with other state-of-the-art methods on simulated models and biological datasets. We found that the PCA method performs robustly in quantifying planar polarity independently of variation in cell geometry and other image conditions. We designed a user-friendly graphical user interface called QuantifyPolarity, equipped with three polarity methods for automated quantification of polarity. QuantifyPolarity also provides tools to quantify cell morphology and packing geometry, allowing the relationship of these characteristics to planar polarization to be investigated. This tool enables experimentalists with no prior computational expertise to perform high-throughput cell polarity and shape analysis automatically and efficiently.

Understanding the role of the NMDA receptor subunit, GluN2D, in mediating NMDA receptor antagonist-induced behavioral disruptions in male and female mice.

Noncompetitive NMDA receptor (NMDAR) antagonists like phencyclidine (PCP) and ketamine cause psychosis-like symptoms in healthy humans, exacerbate schizophrenia symptoms in people with the disorder, and disrupt a range of schizophrenia-relevant behaviors in rodents, including hyperlocomotion. This is negated in mice lacking the GluN2D subunit of the NMDAR, suggesting the GluN2D subunit mediates the hyperlocomotor effects of these drugs. However, the role of GluN2D in mediating other schizophrenia-relevant NMDAR antagonist-induced behavioral disturbances, and in both sexes, is unclear. This study aimed to investigate the role of the GluN2D subunit in mediating schizophrenia-relevant behaviors induced by a range of NMDA receptor antagonists. Using both male and female GluN2D knockout (KO) mice, we examined the effects of the NMDAR antagonist's PCP, the S-ketamine enantiomer (S-ket), and the ketamine metabolite R-norketamine (R-norket) on locomotor activity, anxiety-related behavior, and recognition and short-term spatial memory. GluN2D-KO mice showed a blunted locomotor response to R-norket, S-ket, and PCP, a phenotype present in both sexes. GluN2D-KO mice of both sexes showed an anxious phenotype and S-ket, R-norket, and PCP showed anxiolytic effects that were dependent on sex and genotype. S-ket disrupted spatial recognition memory in females and novel object recognition memory in both sexes, independent of genotype. This datum identifies a role for the GluN2D subunit in sex-specific effects of NMDAR antagonists and on the differential effects of the R- and S-ket enantiomers.

The primary care provider (PCP)-cancer specialist relationship: A systematic review and mixed-methods meta-synthesis.

Although they are critical to models of coordinated care, the relationship and communication between primary care providers (PCPs) and cancer specialists throughout the cancer continuum are poorly understood. By using predefined search terms, the authors conducted a systematic review of the literature in 3 databases to examine the relationship and communication between PCPs and cancer specialists. Among 301 articles identified, 35 met all inclusion criteria and were reviewed in-depth. Findings from qualitative, quantitative, and disaggregated mixed-methods studies were integrated using meta-synthesis. Six themes were identified and incorporated into a preliminary conceptual model of the PCP-cancer specialist relationship: 1) poor and delayed communication between PCPs and cancer specialists, 2) cancer specialists' endorsement of a specialist-based model of care, 3) PCPs' belief that they play an important role in the cancer continuum, 4) PCPs' willingness to participate in the cancer continuum, 5) cancer specialists' and PCPs' uncertainty regarding the PCP's oncology knowledge/experience, and 6) discrepancies between PCPs and cancer specialists regarding roles. These data indicate a pervasive need for improved communication, delineation, and coordination of responsibilities between PCPs and cancer specialists. Future interventions aimed at these deficiencies may improve patient and physician satisfaction and cancer care coordination. CA Cancer J Clin 2017;67:156-169. © 2016 American Cancer Society.

Features and global impact of invasive fungal infections caused by Pneumocystis jirovecii: A systematic review to inform the World Health Organization fungal priority pathogens list.

This systematic review evaluates the current global impact of invasive infections caused by Pneumocystis jirovecii (principally pneumonia: PJP), and was carried out to inform the World Health Organization Fungal Priority Pathogens List. PubMed and Web of Science were used to find studies reporting mortality, inpatient care, complications/sequelae, antifungal susceptibility/resistance, preventability, annual incidence, global distribution, and emergence in the past 10 years, published from January 2011 to February 2021. Reported mortality is highly variable, depending on the patient population: In studies of persons with HIV, mortality was reported at 5%-30%, while in studies of persons without HIV, mortality ranged from 4% to 76%. Risk factors for disease principally include immunosuppression from HIV, but other types of immunosuppression are increasingly recognised, including solid organ and haematopoietic stem cell transplantation, autoimmune and inflammatory disease, and chemotherapy for cancer. Although prophylaxis is available and generally effective, burdensome side effects may lead to discontinuation. After a period of decline associated with improvement in access to HIV treatment, new risk groups of immunosuppressed patients with PJP are increasingly identified, including solid organ transplant patients.

The ENGAGE study: a 3-arm randomized hybrid type 1 effectiveness and implementation study of an in-home, collaborative PCP model of remote telegenetic services to increase uptake of cancer genetic services in childhood cancer survivors.

BACKGROUND: Germline cancer genetic testing has become a standard evidence-based practice, with established risk reduction and screening guidelines for genetic carriers. Access to genetic services is limited in many places, which leaves many genetic carriers unidentified and at risk for late diagnosis of cancers and poor outcomes. This poses a problem for childhood cancer survivors, as this is a population with an increased risk for subsequent malignant neoplasms (SMN) due to cancer therapy or inherited cancer predisposition. The ENGaging and Activating cancer survivors in Genetic services (ENGAGE) study evaluates the effectiveness of an in-home, collaborative PCP model of remote telegenetic services to increase uptake of cancer genetic testing in childhood cancer survivors compared to usual care options for genetic testing. METHODS: The ENGAGE study is a 3-arm randomized hybrid type 1 effectiveness and implementation study within the Childhood Cancer Survivor Study population which tests a clinical intervention while gathering information on its delivery during the effectiveness trial and its potential for future implementation among 360 participants. Participants are randomized into three arms. Those randomized to Arm A receive genetic services via videoconferencing, those in Arm B receive these services by phone, and those randomized to Arm C will receive usual care services. DISCUSSION: With many barriers to accessing genetic services, innovative delivery models are needed to address this gap and increase uptake of genetic services. The ENGAGE study evaluates the effectiveness of an adapted model of remote delivery of genetic services to increase the uptake of recommended genetic testing in childhood cancer survivors. This study assesses the uptake in remote genetic services and identify barriers to uptake to inform future recommendations and a theoretically-informed process evaluation which can inform modifications to enhance dissemination beyond this study population and to realize the benefits of precision medicine. TRIAL REGISTRATION: This protocol was registered at clinicaltrials.gov (NCT04455698) on July 2, 2020.

Morphological and molecular markers of mouse area CA2 along the proximodistal and dorsoventral hippocampal axes.

Hippocampal area CA2 is a molecularly and functionally distinct region of the hippocampus that has classically been defined as the area with large pyramidal neurons lacking input from the dentate gyrus and the thorny excrescences (TEs) characteristic of CA3 neurons. A modern definition of CA2, however, makes use of the expression of several molecular markers that distinguish it from neighboring CA3 and CA1. Using immunohistochemistry, we sought to characterize the staining patterns of commonly used CA2 markers along the dorsal-ventral hippocampal axis and determine how these markers align along the proximodistal axis. We used a region of CA2 that stained for both Regulator of G-protein Signaling 14 (RGS14) and Purkinje Cell Protein 4 (PCP4; "double-labeled zone" [DLZ]) as a reference. Here, we report that certain commonly used CA2 molecular markers may be better suited for drawing distinct boundaries between CA2/3 and CA2/1. For example, RGS14+ and STEP+ neurons showed minimal to no extension into area CA1 while areas stained with VGluT2 and Wisteria Floribunda agglutinin were consistently smaller than the DLZ/CA2 borders by ~100 µ on the CA1 or CA3 sides respectively. In addition, these patterns are dependent on position along the dorsal-ventral hippocampal axis such that PCP4 labeling often extended beyond the distal border of the DLZ into CA1. Finally, we found that, consistent with previous findings, mossy fibers innervate a subset of RGS14 positive neurons (~65%-70%) and that mossy fiber bouton number and relative size in CA2 are less than that of boutons in CA3. Unexpectedly, we did find evidence of some complex spines on apical dendrites in CA2, though much fewer in number than in CA3. Our results indicate that certain molecular markers may be better suited than others when defining the proximal and distal borders of area CA2 and that the presence or absence of complex spines alone may not be suitable as a distinguishing feature differentiating CA3 from CA2 neurons.