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Context.— There has long been debate about whether and when there may be a shortage of pathologists in the United States. One way to assess this is to survey the hiring experiences of pathology practices. A 2018 survey revealed a strong demand for pathologists, with expectations of continued strength. This study updates that prior analysis using data from a 2021 survey of pathology practice leaders. Objective.— To assess the US pathologist job market and examine implications. Design.— We analyzed data from the 2021 College of American Pathologists Practice Leader Survey. This survey queried practice leaders, including regarding the hiring of pathologists, the level of experience being sought, success in filling positions, and expectations for hiring in the next 3 years. Results.— Among the 375 surveyed practice leaders (about one-third of all US pathology practices), 282 provided information about pathologist hiring in 2021. A total of 157 of these 282 practices (55.7%) sought to hire at least 1 pathologist in 2021, up from 116 of 256 practices (45.3%) in 2017; the mean number of pathologists hired per practice also increased. In 2021, a total of 175 of 385 positions (45.5%) were to fill new positions, compared with 95 of 249 positions (38.2%) in 2017. Most practice leaders were comfortable hiring pathologists with less than 2 years of posttraining experience. Practice leaders anticipated continued strong demand for hiring pathologists during the next 3 years. Conclusions.— Our analysis confirms that the demand in pathologist hiring is strong and much increased from 2017. We believe, in combination with other job market indicators, that demand may outstrip the supply of pathologists, which is limited by the number of trainees and has remained constant during the past 20 years.

Appalachian regions of Kentucky and Ohio are hotspots for colorectal cancer (CRC) mortality in the USA. Screening reduces CRC incidence and mortality; however, screening uptake is needed, especially in these underserved geographic areas. Implementation science offers strategies to address this challenge. The aim of the current study was to conduct multi-site, transdisciplinary research to evaluate and improve CRC screening processes using implementation science strategies. The study consists of two phases (Planning and Implementation). In the Planning Phase, a multilevel assessment of 12 health centers (HC) (one HC from each of the 12 Appalachian counties) was conducted by interviewing key informants, creating community profiles, identifying HC and community champions, and performing HC data inventories. Two designated pilot HCs chose CRC evidence-based interventions to adapt and implement at each level (i.e., patient, provider, HC, and community) with evaluation relative to two matched control HCs. During the Implementation Phase, study staff will repeat the rollout process in HC and community settings in a randomized, staggered fashion in the remaining eight counties/HCs. Evaluation will include analyses of electronic health record data and provider and county surveys. Rural HCs have been reluctant to participate in research because of concerns about capacity; however, this project should demonstrate that research does not need to be burdensome and can adapt to local needs and HC abilities. If effective, this approach could be disseminated to HC and community partners throughout Appalachia to encourage the uptake of effective interventions to reduce the burden of CRC.

Supplementary Figure 1. Anti-CD3 stimulation of primary human T-ALLs. Supplementary Figure 2. TCR stimulation by in vivo administration of agonistic monoclonal antibody in a preventive setting inhibits human TCR+ T-ALL development. Supplementary Figure 3. TCR signaling is essential to the anti-leukemic effect of OKT3. Supplementary Table 1. Immunophenotypic and oncogenic characteristics of T-ALL samples.

Supplementary Methods, Figure Legends, Table Legend

We conducted a retrospective/prospective worldwide study on patients with neuroendocrine neoplasms (NENs) and a molecularly proven SARS-CoV-2 positivity. Preliminary results regarding 85 patients of the INTENSIVE study have been published in 2021. Now we are reporting the 2-year analysis.Here, we are reporting data from consecutive patients enrolled between 1 June 2020, and 31 May 2022. Among the 118 contacted centers, 25 were active to enroll and 19 actively recruiting at the time of data cut-off for a total of 280 patients enrolled. SARS-CoV-2 positivity occurred in 47.5% of patients in 2020, 35.1% in 2021, and 17.4% in 2022. The median age for COVID-19 diagnosis was 60 years. Well-differentiated tumors, non-functioning, metastatic stage, and gastroenteropancreatic (GEP) primary sites represented most of the NENs. COVID-19-related pneumonia occurred in 22.8% of the total, with 61.3% of them requiring hospitalization; 11 patients (3.9%) needed sub-intensive or intensive care unit therapies and 14 patients died (5%), in 11 cases (3.9%) directly related to COVID-19. Diabetes mellitus and age at COVID-19 diagnosis > 70 years were significantly associated with COVID-19 mortality, whereas thoracic primary site with COVID-19 morbidity. A significant decrease in both hospitalization and pneumonia occurred in 2022 vs 2020. In our largest series of NEN patients with COVID-19, the NEN population is similar to the general population of patients with NEN regardless of COVID-19. However, older age, non-GEP primary sites and diabetes mellitus should be carefully considered for increased COVID-19 morbidity and mortality. Relevant information could be derived by integrating our results with NENs patients included in other cancer patients with COVID-19 registries.

Nuclear actin has been implicated in dynamic chromatin rearrangements in diverse eukaryotes. In mammalian cells, it is required to reposition double-strand DNA breaks to enable homologous recombination repair and to enhance transcription by facilitating RNA Pol II recruitment to gene promoters. In the yeast Saccharomyces cerevisiae, nuclear actin modulates interphase chromosome dynamics and is required to reposition the induced INO1 gene to the nuclear periphery. Here, we have investigated the role of actin in driving intergenic interactions between Heat Shock Factor 1 (Hsf1)-regulated Heat Shock Protein (HSP) genes in budding yeast. These genes, dispersed on multiple chromosomes, dramatically reposition following exposure of cells to acute thermal stress, leading to their clustering within dynamic biomolecular condensates. Using an auxin-induced degradation strategy, we found that conditional depletion of nucleators of either linear or branched F-actin (Bni1/Bnr1 and Arp2, respectively) had little or no effect on heat shock-induced HSP gene coalescence or transcription. In addition, we found that pretreatment of cells with latrunculin A, an inhibitor of both filamentous and monomeric actin, failed to affect intergenic interactions between activated HSP genes and their heat shock-induced intragenic looping and folding. Moreover, latrunculin A pretreatment had little effect on HSP gene expression at either RNA or protein levels. In notable contrast, we confirmed that repositioning of activated INO1 to the nuclear periphery and its proper expression do require actin. Collectively, our work suggests that transcriptional activation and 3D genome restructuring of thermally induced, Hsf1-regulated genes can occur in the absence of actin.

To assess the efficacy and safety of 177Lu-DOTATATE in patients with somatostatin receptor (SSR) positive lung neuroendocrine tumor (NET). Methods: This is a retrospective review of the outcome of patients with typical carcinoid (TC) and atypical carcinoid (AC), treated with 177Lu-DOTATATE at two ENETS Centres of Excellence. Morphological imaging (RECIST 1.1) and 68Ga-DOTATATE PET/CT responses were assessed at 3 months after completion of 177Lu-DOTATATE. Concordance between two response assessment methods was evaluated by Kappa statistics. Progression-free survival (PFS) and overall survival (OS) was estimated by Kaplan-Meier analysis and compared by Log-rank test. Treatment-related adverse events (AEs) were graded based on CTCAE version 5. Results: Of 48 patients (median age, 63 years, 13 female), 43 (90%) had AC and 5 (10%) TC. Almost all patients (47, 98%) were treated due to progression. Majority (40, 83%) received somatostatin analogs and 10 patients (20%) had prior everolimus, chemotherapy or both. All patients had high SSR expression (≥ modified Krenning score 3) on pre-treatment 68Ga-DOTATATE PET/CT. Patients received a median 4 (range 1-4) cycles of 177Lu-DOTATATE (33% with concurrent radiosensitizing chemotherapy) to a median cumulative activity of 27GBq (range 6-43GBq). At median follow-up of 42 months, the median PFS and OS were 23 months (95% CI 18-28 months) and 59 months (95% CI 50-not reached [NR]), respectively. Of 40 patients with RECIST-measurable disease and 39 patients with available 68Ga-DOTATATE PET/CT response categories were: partial response, 20% (95% CI 10-35%) and 44% (95% CI 30-59%); stable disease, 68% (95% CI 52-80%) and 44% (95% CI 30-59%) and progressive disease 12% (95% CI 5-27%) by both, respectively. There was a moderate concordance between response categories by RECIST and 68Ga-DOTATATE PET/CT, weighted Kappa of 0.51 (95% CI 0.21-0.68). Of patients with stable disease by RECIST, those with partial response on 68Ga-DOTATATE PET/CT had longer OS compared to those with no response, NR vs 52 months (95% CI 28-64), HR 0.2 (95% CI 0.1-0.6), p 0.001. Most grade 3/4 AEs were reversible and the most common was lymphopenia (14%) with no incidence of myelodysplasia/leukemia. Conclusion: In patients with advanced progressive lung NET and satisfactory SSR expression, 177Lu-DOTATATE is effective and safe with a high disease control rate and encouraging PFS and OS.

Objective: Accelerated atherosclerosis in diabetes constitutes an ongoing challenge despite optimal medical therapies. This study aimed to identify evolutionarily conserved lesion-based regulatory signaling networks in diabetic versus nondiabetic conditions during the development of atherosclerosis in an initial translational effort to provide insights for targets. Approach and Results: Serial 3-mm coronary artery segments of hypercholesterolemic Yorkshire swine and diabetic-hypercholesterolemic swine were characterized as mild, moderate, or severe phenotypic manifestations of coronary atherosclerosis based on histopathologic examination. Lesional RNA sequencing was performed (n=3–8 lesions per group) corresponding to increasing phenotypic severity. Differentially expressed genes, transcription factors, upstream regulators, and hubs were validated using the NanoString technology and a human atherosclerotic specimen cohort. Despite similar stage histopathologic characterization of lesions, genome-wide transcriptomics revealed gene sets and nodal signaling pathways uniquely expressed in diabetic lesions including signaling pathways for Th17, IL (interleukin)-17F, TWEAK (TNF [tumor necrosis factor]-related weak inducer of apoptosis), CD27, and PI3K/Akt. In contrast, pathways of nondiabetic lesions involved TREM-1 and Th1 and Th2 responses during the initiation stage, whereas networks for mitochondrial dysfunction, oxidative phosphorylation, and lipid metabolism emerged with progression. RNA sequencing data were validated in a human atherosclerosis specimen cohort using machine learning algorithms. F8 , MAPKAPK3 , and ITGB1 emerged as powerful genes for clustering diabetic versus nondiabetic lesions and for separating different degrees of atherosclerosis progression. Conclusions: This study identifies evolutionarily conserved gene signatures and signaling pathways in a stage-specific manner that successfully distinguishes diabetes- and non–diabetes-associated atherosclerosis. These findings establish new molecular insights and therapeutic opportunities to address accelerated atherosclerotic lesion formation in diabetes.

There has been little rigorous assessment of burnout among pathologists and pathology trainees. Given this relative dearth of relevant literature on pathologist burnout, this report aims to raise awareness of the issue among those working in and around this specialty. Our results are based on a survey given in conjunction with the American Board of Pathology's (ABPath) biennial Continuing Certification (CC) reporting of activities required of diplomates to maintain certification. The survey was voluntary, open to all diplomates participating in CC, and conducted over two consecutive years (2019 and 2020), with alternate years comprising different sets of diplomates. The data are based on 1256 respondents (820 from 2019 to 436 from 2020). The three highest aggregate reported rates of burnout (reported as experienced nearly all of the time, most of the time, or part of the time) occurred when respondents were in their first year of residency training (41.1%) and when they were in (47.6%) and beyond (46.6%) their first three years of practice. We considered this high-low-high, or U-shaped distribution in recollected burnout over time among pathologists a notable finding and investigated its distribution among respondents. Conversely at every point in their training and practice, from half to three-quarters of respondents reported never or infrequently experiencing burnout. This study represents the largest pathologist cohort survey to date about pathologists' burnout. Importantly, especially for those considering pathology as a career, these data are on the low end of the distribution of burnout among specialties for those in practice.

The lymph node ratio (LNR), which represents the proportion of metastatic lymph nodes resected, has been found to be a prognostic variable in several cancers, but data for Medullary thyroid carcinoma (MTC) are sparse. The aim of this study was to determine the value of the LNR in predicting outcome in patients with MTC.A retrospective multicenter study design of 107 patients with MTC who underwent total thyroidectomy with neck dissection between 1984 and 2016. The association of LNR with patient and tumor characteristics and prognostic factors was evaluated.Study population consisted of 53.3% female, mean age at diagnosis was 50.3 ± 18.4 years; 16.8% had inherited MTC. LNR was positively correlated with tumor size (p = 0.018) and inversely correlated with age at diagnosis (p = 0.024). A higher LNR was associated with extrathyroidal extension (p 0.001), multifocality (p = 0.001), bilateral tumor (p = 0.002), distant metastases (p 0.001), and tumor recurrence (OR = 14.7, p 0.001). LNR was also correlated to postoperative calcitonin levels (p 0.001) and carcinoembryonic antigen (p = 0.011). LNR0.1 was associated with shorter disease-specific survival in patients at risk: tumor larger than 20 mm at diagnosis (p = 0.013), sporadic MTC (p = 0.01), and age above 40 years at diagnosis (p = 0.004). Cox multivariate survival analysis revealed LNR as the only significant independent factor for disease free survival (p = 0.005).This study showed that LNR correlates well with patient and tumor characteristics and prognostic variables. We suggest that LNR should be considered an important parameter for predicting outcome in MTC.

Objective Von Hippel-Lindau (VHL) syndrome is a rare and complex disease. We described in 1996 a three generation VHL 2A kindred with 11 mutation carriers. We aim to share our experience regarding the long-term follow-up of this family and the management of all our other VHL patients focusing on frequently encountered neuroendocrine tumors: pheochromocytoma/paraganglioma and pancreatic neuroendocrine neoplasms (PNEN). Methods All VHL patients in follow-up at our tertiary center from 1980 to 2019 were identified. Clinical, laboratory, imaging and therapeutic characteristics were retrospectively analyzed. Results We identified 32 VHL patients in 16 different families, 7/16 were classified as VHL 2 subtype. In the previously described family, the 4 initially asymptomatic carriers developed a neuroendocrine tumor; 7 new children were born, 3 of them being mutation carriers; 2 patients died, one due to metastatic PNEN-related liver failure. Pheochromocytoma was frequent (22/32), bilateral (13/22;59%), often diagnosed in early childhood when active screening was timely performed, associated with paraganglioma in 5/22, rarely malignant (1/22) and recurred after surgery in some cases after more than 20 years. PNEN occurred in 8/32 patients (25%), and was metastatic in three. Surgery and palliative therapy allowed relatively satisfactory outcomes. Severe disabling morbidities due to central-nervous system and ophthalmologic hemangiomas, and other rare tumors as chondrosarcoma in 2 patients and polycythemia in 1 patient were observed. Conclusions Multidisciplinary approach and long-term follow-up is mandatory in VHL patients to manage the multiple debilitating morbidities and delay mortality in these complex patients.

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation., T cell receptors (TCR) are internalized when activated by their ligands. Here the authors show that the internalized TCRs are localized to endosomes expressing IRAP and Syntaxin 6 to maintain intracellular signalling capacity, whose importance is shown by the absence of efficient polyclonal anti-tumour response in mice with T-specific conditional deletion of IRAP.

Context.— Disagreement exists within the pathology community about the status of the job market for pathologists. Although many agree that jobs in pathology were harder to come by earlier this decade, recent evidence suggests improvement is occurring. Objective.— To assess the state of the job market for pathologists. Design.— We analyzed data from the 2018 College of American Pathologists Practice Leader Survey. This survey contains data from 253 practice leaders on practices' hiring (and retrenchments) in 2017, the skills and level of experience being sought, success in filling those positions, and expectations for hiring in the next 3 years. Results.— Among the surveyed practice leaders, 115 (45.5%) sought to hire at least 1 pathologist in 2017, and together tried to fill 246 full-time equivalent positions that year, of which 93.5 full-time equivalents (38%) were newly created. This hiring was not limited to larger, academic-based practices, but also occurred among smaller practices and practices based in nonacademic hospitals, independent laboratories, and other settings. Although some practices retrenched (60 full-time equivalents in 2017), the net increase was a healthy 187 full-time equivalents. Practices most frequently sought pathologists who had at least 2 years of experience, but the level of experience identified with the “optimal” candidate varied by desired areas of subspecialty expertise. Practice leaders also reported expected growth in hiring, with the number of positions they hope to fill in the next 3 years exceeding those vacated by retirement. Conclusions.— Our findings support the proposition that the demand for pathologists is strong, at least at the current time.

Chromosome Conformation Capture (3C) has emerged as a powerful approach for revealing the conformation and features of three-dimensional (3D) genomic organization. Yet attainment of higher resolution in organisms with compact genomes presents a challenge. Here, we describe modifications in the 3C technique that substantially enhance its resolution and sensitivity when applied to the 3D genome of budding yeast. Keys to our approach include use of a 4 bp cutter, Taq I, for cleaving the genome and quantitative PCR for measuring the frequency of ligation. Most importantly, we normalize the percent digestion at each restriction site to account for variation in accessibility of local chromatin structure under a given physiological condition. This strategy has led to the detection of physical interactions between regulatory elements and gene coding regions as well as intricate, stimulus-specific interchromosomal interactions between activated genes. We provide an algorithm that incorporates these and other modifications and allows quantitative determination of chromatin interaction frequencies in yeast under any physiological condition.

Context Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis for both locally advanced and metastatic disease. Standard treatment with combination etoposide–doxorubicin–cisplatin–mitotane (EDP-M) is highly toxic and some patients benefit from mitotane monotherapy. However, identification of these patients remains challenging. Objective We present a summary of the Israeli national referral center’s 20 years of experience in treating advanced ACC, with the aim of identifying prognostic factors and assisting in treatment decision making. Methods We conducted a retrospective multivariate analysis of patients treated for metastatic or locally advanced ACC at Hadassah Medical Center between 2000 and 2020 to determine clinical, pathological, and treatment factors correlated with overall survival (OS). Results In our cohort of 37 patients, a combination of modified European Network for the study of Adrenal Tumors (mENSAT) staging with either grade and R status, or age and symptoms was validated to stratify prognosis (P = .01 and P = .03, respectively). Patients who underwent R0 resection followed by radiotherapy or metastasectomy for oligometastatic disease had longer OS than patients with residual disease: median OS of 55 months vs 14 months, respectively, hazard ratio 3.1 (CI 1.4-6.7, P = .005). Patients treated with mitotane monotherapy had a significantly better prognosis, yet this result was attenuated in a multivariate analysis controlling for mENSAT and R status. Of patients treated with EDP-M, 41.4% experienced grade 3 or higher adverse events. Conclusion Patients with advanced ACC achieving R0 status have a better prognosis and might benefit from mitotane monotherapy.

[Introduction] Telotristat ethyl is indicated for use in combination with somatostatin analogs (SSAs) to treat carcinoid syndrome (CS) diarrhea uncontrolled by SSAs alone in adults, but long-term safety and efficacy data beyond 48 weeks are needed., [Objectives] The aims of the study were to evaluate the long-term safety and tolerability of telotristat ethyl and its effect on quality of life (QOL) in patients with CS., [Methods] In this phase 3, nonrandomized, multicenter, open-label, long-term extension study (TELEPATH), patients who participated in phase 2 or 3 trials of telotristat ethyl continued treatment at their present dose level (250 or 500 mg thrice daily) for 84 weeks. Safety and tolerability, the primary endpoint, were assessed by monitoring adverse events (AEs), serious AEs, AEs of special interest (AESIs; including liver-related AEs, depression, and gastrointestinal AEs), and deaths. The secondary objective was to evaluate changes in patients’ QOL using validated cancer questionnaires and a subjective global assessment of CS symptoms, [Results] In 124 patients exposed to telotristat ethyl for a mean of 102.6 ± 53.2 weeks, the type and frequency of AEs were consistent with those reported in previous trials. The occurrence of AESIs was not related to dosage or duration of therapy. Most AEs were mild to moderate in severity, and no deaths were related to telotristat ethyl. QOL scores remained stable, and the majority of patients reported adequate symptom relief throughout the study., [Conclusions] Safety results of TELEPATH support the long-term use of telotristat ethyl in patients with CS diarrhea. Telotristat ethyl was well-tolerated and associated with sustained improvement in QOL scores (NCT02026063)., This study was supported and conducted by Lexicon Pharmaceuticals, Inc.

Simple Summary Neuroendocrine neoplasms of the lung (lung carcinoids) are often diagnosed when they are not surgically curable, and treatment options are limited. One of the approved options for treating inoperable tumors is everolimus, an mTOR inhibitor. Activation of mTOR inhibits autophagy, which is a cell survival mechanism; everolimus may paradoxically encourage cancer cell survival via stimulation of autophagy. Chloroquine, a known antimalarial compound, inhibits autophagy. Our research is focused on the hypothesis that autophagy plays a key role in the development of tumor resistance to everolimus, and that chloroquine addition to an mTOR inhibitor increases their inhibitory effect on tumor growth. In this study, we examined the effects of chloroquine alone or in combination with mTOR inhibitors on lung neuroendocrine tumor models (cell lines and mice). We have shown that chloroquine alone suppresses tumor cells’ viability and proliferation and increases their cytotoxicity and apoptosis; these effects are augmented when chloroquine is added to mTOR inhibitors. Apparently, chloroquine suppresses tumor cell growth in lung neuroendocrine neoplasms models, potentiating the effects of the mTOR inhibitors, and implying that more research is warranted to unravel its possible role in the clinical setting, in patients with advanced lung neuroendocrine neoplasms. Abstract (1) Background: Neuroendocrine neoplasms of the lung (LNENs, lung carcinoids) are often diagnosed at an advanced stage when they are not surgically curable, and treatment options are limited. One of the approved options for treating inoperable tumors is everolimus—an mTOR inhibitor (mTORi). Activation of mTOR, among many other effects, inhibits autophagy, which is a cell survival mechanism in general, and in tumor cells in particular. Everolimus may paradoxically encourage cancer cell survival. In practice, the drug inhibits tumor development. Chloroquine (CQ) is a known antimalarial compound that inhibits autophagy. Our research is focused on the hypothesis that autophagy plays a key role in the development of tumor resistance to mTORi, and that the addition of autophagy inhibitors to mTORi exerts a synergistic effect on suppressing tumor cell proliferation. We have recently demonstrated that the combination of CQ with different mTORi increases their potency compared with mTORi alone in both in vitro and in vivo models of pancreatic NENs. In this study, we examined the effects of CQ and mTORi on in vitro and in vivo LNEN models. Aims: Testing the effects of CQ together with mTORi on cell proliferation, apoptosis, and autophagy in in vitro and in vivo LNEN models. (2) Methods: The NCI-H727 LNEN cells were treated with CQ ± mTORi. Cells’ viability and proliferation were measured using XTT and Ki-67 FACS staining. The effects of the treatments on the mTOR pathway and autophagy were examined using Western blotting. Cytotoxicity was measured using a cytotoxicity kit; apoptosis was measured by PI FACS staining and Western blotting. We further established an LNEN subcutaneous murine xenograft model and evaluated the effects of the drugs on tumor growth. (3) Results: CQ alone suppressed LNEN cells’ viability and proliferation and increased their cytotoxicity and apoptosis; these effects were augmented when CQ was added to an mTORi. We also showed the possible mechanisms for these results: on the one hand we could see a decrease in P62 levels and the absence of LC3-II (both inversely related to autophagy) following treatment with the mTORi, and on the other hand we could demonstrate an increase in their levels when CQ was added. The effect was less apparent in the murine xenograft model. (4) Conclusions: By inhibiting autophagy and inducing apoptosis, CQ suppresses tumor cell growth in LNENs. CQ potentiates mTORi effects, implying that further studies are needed in order to elucidate its possible role in tumor inhibition in patients with LNENs.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach., Pompe disease is currently treated with enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (GAA). Here, the authors show hepatic-directed gene therapy with AAV vectors enhances GAA bioavailability compared with ERT, resulting in improved rescue of the disease phenotype in mice and broad enzyme distribution in mice and non-human primates.

SUMMARYMammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved, capable of stress-inducible gene activation or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor Heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, while an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become mammalian-like: constitutively active without intergenic coalescence. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.

Objectives This study assessed providers' present practices and perceived needs in Appalachian Kentucky to identify the standard of care and implementation of expert recommendations for managing pediatric obesity. Methods Questionnaire data were gathered from 28 providers at a pediatric obesity continuing medical education workshop in eastern Kentucky. We assessed current practices, perceived barriers to treatment, and needed resources for managing pediatric obesity. Results Respondents reported mixed adherence to expert recommendations, with providers less frequently addressing family-reported barriers to change and assessing a family's readiness to change behaviors related to pediatric obesity. Respondents also reported service barriers related to patient motivation, lack of time with patients, and a lack of referral options. Finally, providers reported needing multiple community resources to better address pediatric obesity, including improved physical education programs, access to community recreation centers, additional referral resources for multidisciplinary care, and additional training in motivational techniques. Conclusions There remains a significant need for education and guidance regarding the implementation of expert recommendations for addressing pediatric obesity in Appalachian Kentucky. Providers reported needing multiple community resources, including improved physical education programs, access to community recreation centers, additional referral resources for multidisciplinary care, and additional training in motivational techniques. We discuss the implications for disseminating and implementing expert recommendations in rural eastern Kentucky, with an emphasis on the roles of behavioral health experts.

To study the evolution and optimal management of metastatic bone disease (mBD) in patients with neuroendocrine neoplasms (NENs). Seventy-four patients were recruited from four NEN centers in this observational multicenter study. Pancreas and small bowel were the most common primaries (30 and 27%, respectively). Almost all gastrointestinal (GI)-NENs were grades 1 and 2, whereas bronchopulmonary-thymic were atypical carcinoids. Thirty-two (43%) patients had synchronous metastatic bone disease (mBD) and three patients reported bone-specific symptoms; metachronous mBD developed at a median of 35 (range: 4–395) months. Thirty-six (86%) of patients with metachronous mBD had stage IV disease at diagnosis. Somatostatin receptor functional imaging and computed tomography were the modalities mostly used for mBD identification. Fifty-two patients received assessable bone-related therapy (bisphosphonates, denosumab, local radiotherapy, and radionuclide treatment). Improvement in mBD was seen in 5, stable disease in 22, and deterioration in 25 patients. The presence of synchronous mBD and the negative outcome of bone-related therapy negatively affected overall survival (OS). In the multivariate analysis, the stronger predictor of OS was the outcome of bone-related therapy (HR: 4.753; 95% CI: 1.589–14.213). Bisphosphonates therapy was the mostly used bone-specific treatment but its monthly administration did not affect OS. At last follow-up, 39 patients were alive with OS 50 (14–463) months. Early investigation for mBD offers a prognostic marker of patients with NENs, since synchronous mBD has a negative impact on survival. The outcome of bone-related therapy affects OS but the monthly administration of bisphosphonates did not show a benefit over less intense schemes.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter’s onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

SNHG12, a long non-coding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease (PAD) has not been elucidated. Hindlimb ischemia studies using femoral artery ligation in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain and loss of function studies demonstrated that SNHG12 regulated angiogenesis - SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including insulin growth factor 2 mRNA binding protein 3 (IGF2BP3/IMP3). RNA-seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IGF2BP3/IMP3. Knockdown of SNHG12 in mice undergoing femoral artery ligation using injected gapmeRs decreased angiogenesis, an effect that was more pronounced in a model of insulin resistant db/db mice. RNA-seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12-knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.