A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

Transient naive reprogramming corrects hiPS cells functionally and epigenetically.

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.

Modelling human blastocysts by reprogramming fibroblasts into iBlastoids.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1-4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

Comparative utility of MRI and EEG for early detection of cortical dysmaturation after postnatal systemic inflammation in the neonatal rat.

BACKGROUND: Exposure to postnatal systemic inflammation is associated with increased risk of brain injury in preterm infants, leading to impaired maturation of the cerebral cortex and adverse neurodevelopmental outcomes. However, the optimal method for identifying cortical dysmaturation is unclear. Herein, we compared the utility of electroencephalography (EEG), diffusion tensor imaging (DTI), and neurite orientation dispersion and density imaging (NODDI) at different recovery times after systemic inflammation in newborn rats. METHODS: Sprague Dawley rat pups of both sexes received single-daily lipopolysaccharide (LPS; 0.3 mg/kg i.p.; n = 51) or saline (n = 55) injections on postnatal days (P)1, 2, and 3. A subset of these animals were implanted with EEG electrodes. Cortical EEG was recorded for 30 min from unanesthetized, unrestrained pups at P7, P14, and P21, and in separate groups, brain tissues were collected at these ages for ex-vivo MRI analysis (9.4 T) and Golgi-Cox staining (to assess neuronal morphology) in the motor cortex. RESULTS: Postnatal inflammation was associated with reduced cortical pyramidal neuron arborization from P7, P14, and P21. These changes were associated with dysmature EEG features (e.g., persistence of delta waveforms, higher EEG amplitude, reduced spectral edge frequency) at P7 and P14, and higher EEG power in the theta and alpha ranges at P21. By contrast, there were no changes in cortical DTI or NODDI in LPS rats at P7 or P14, while there was an increase in cortical fractional anisotropy (FA) and decrease in orientation dispersion index (ODI) at P21. CONCLUSIONS: EEG may be useful for identifying the early evolution of impaired cortical development after early life postnatal systemic inflammation, while DTI and NODDI seem to be more suited to assessing established cortical changes.

MicroRNA-29a: a novel target for non-operative management of symptomatic lumbar spinal stenosis.

PURPOSE: Lumbar spinal stenosis (LSS) is the most common reason for spinal surgery in patients over the age of 65, and there are few effective non-surgical treatments. Therefore, the development of novel treatment or preventative modalities to decrease overall cost and morbidity associated with LSS is an urgent matter. The cause of LSS is multifactorial; however, a significant contributor is ligamentum flavum hypertrophy (LFH) which causes mechanical compression of the cauda equina or nerve roots. We assessed the role of a novel target, microRNA-29a (miR-29a), in LFH and investigated the potential for using miR-29a as a therapeutic means to combat LSS. METHODS: Ligamentum flavum (LF) tissue was collected from patients undergoing decompressive surgery for LSS and assessed for levels of miR-29a and pro-fibrotic protein expression. LF cell cultures were then transfected with either miR-29a over-expressor (agonist) or inhibitor (antagonist). The effects of over-expression and under-expression of miR-29a on expression of pro-fibrotic proteins was assessed. RESULTS: We demonstrated that LF at stenotic levels had a loss of miR-29a expression. This was associated with greater LF tissue thickness and higher mRNA levels of collagen I and III. We also demonstrated that miR29-a plays a direct role in the regulation of collagen gene expression in ligamentum flavum. Specifically, agents that increase miR-29a may attenuate LFH, while those that decrease miR-29a promote fibrosis and LFH. CONCLUSION: This study demonstrates that miR-29a may potentially be used to treat LFH and provides groundwork to initiate the development of a therapeutic product for LSS.

Health State Utility Values Associated with Cochlear Implants in Adults: A Systematic Review and Network Meta-Analysis.

OBJECTIVES: The cost-effectiveness of bilateral cochlear implants in adults remains uncertain despite established clinical benefits. In cost-effectiveness studies, benefit is often measured by change in health state utility value (HSUV), a single number summary of health-related quality of life anchored at 0 (state of being dead) and 1 (perfect health). Small differences in bilateral cochlear implant HSUV change conclusions of published models, and invalid estimates can therefore mislead policy and funding decisions. As such, we aimed to review and synthesize published HSUV estimates associated with cochlear implants. DESIGN: We included observational or experimental studies reporting HSUV for adult patients (age ≥18 years) with at least moderate-profound sensorineural hearing loss in both ears who received unilateral or bilateral cochlear implants. We searched MEDLINE, EMBASE, PsycINFO, and Cochrane Library databases up to May 1, 2021. Study and participant characteristics and HSUV outcomes were extracted. Narrative synthesis is reported for all studies. A Bayesian network meta-analysis was conducted to generate pooled estimates for the mean difference in HSUV for three comparisons: (1) unilateral cochlear implant versus preimplant, (2) bilateral cochlear implants versus preimplant, (3) bilateral versus unilateral cochlear implants. Our principal measure was pooled mean difference in HSUV. RESULTS: Thirty-six studies reporting unique patient cohorts were identified. Health Utilities Index, 3 (HUI-3) was the most common HSUV elicitation method. HSUV from 19 preimplant mean estimates (1402 patients), 19 unilateral cochlear implant mean estimates (1701 patients), and 5 bilateral cochlear implants mean estimates (83 patients) were pooled to estimate mean differences in HUI-3 HSUV by network meta-analysis. Compared with preimplant, a unilateral cochlear implant was associated with a mean change in HSUV of +0.17 (95% credible interval [CrI] +0.12 to +0.23) and bilateral cochlear implants were associated with a mean change of +0.25 (95% CrI +0.12 to +0.37). No significant difference in HSUV was detected for bilateral compared with unilateral cochlear implants (+0.08 [95% CrI -0.06 to +0.21]). Overall study quality was moderate. CONCLUSIONS: The findings of this review and network meta-analysis comprise the best-available resource for parameterization of cost-utility models of cochlear implantation in adults and highlight the need to critically evaluate the validity of available HSUV instruments for bilateral cochlear implant populations.Protocol registration: PROSPERO (CRD42018091838).

Local magnetic delivery of adeno-associated virus AAV2(quad Y-F)-mediated BDNF gene therapy restores hearing after noise injury.

Moderate noise exposure may cause acute loss of cochlear synapses without affecting the cochlear hair cells and hearing threshold; thus, it remains "hidden" to standard clinical tests. This cochlear synaptopathy is one of the main pathologies of noise-induced hearing loss (NIHL). There is no effective treatment for NIHL, mainly because of the lack of a proper drug-delivery technique. We hypothesized that local magnetic delivery of gene therapy into the inner ear could be beneficial for NIHL. In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) and a recombinant adeno-associated virus (AAV) vector (AAV2(quad Y-F)) to deliver brain-derived neurotrophic factor (BDNF) gene therapy into the rat inner ear via minimally invasive magnetic targeting. We found that the magnetic targeting effectively accumulates and distributes the SPION-tagged AAV2(quad Y-F)-BDNF vector into the inner ear. We also found that AAV2(quad Y-F) efficiently transfects cochlear hair cells and enhances BDNF gene expression. Enhanced BDNF gene expression substantially recovers noise-induced BDNF gene downregulation, auditory brainstem response (ABR) wave I amplitude reduction, and synapse loss. These results suggest that magnetic targeting of AAV2(quad Y-F)-mediated BDNF gene therapy could reverse cochlear synaptopathy after NIHL.

Ice-POP: Ice Packs for Postoperative Pain: A Randomized Controlled Trial.

STUDY OBJECTIVE: To evaluate the benefit of ice packs as a supplement to standard pain management following laparoscopic hysterectomy (LH). DESIGN: This Institutional Review Board-approved randomized controlled trial involved patients undergoing LH for benign conditions. Subjects were randomized to receive standard enhanced recovery after surgery pain management or standard enhanced recovery after surgery plus ice packs. SETTING: Two academic tertiary care centers PATIENTS: Patients undergoing planned outpatient LH with the minimally invasive gynecologic surgery team between February 2019 and November 2020 were considered. Patients with chronic pain, current opioid use ≥1 week, or planned overnight hospitalizations were excluded. Primary outcome data were available for 51 subjects (24 control, 27 intervention). INTERVENTIONS: Ice packs were placed on the abdomen in the operating room. MEASUREMENTS AND MAIN RESULTS: Pain was assessed at multiple time points throughout the study using a visual analogue scale (VAS). Opioid requirement was assessed using morphine milligram equivalent. There was no difference between the groups on any demographic variables. Morphine milligram equivalent requirements were also not different between the groups (p = .63). Postoperative day 1 (POD#1) VAS scores were not different (p = .89). Eighty-five percent of subjects reported feeling that their pain was controlled. Subjects who reported that they did not feel their pain was controlled did not use more opioids on POD#1 (p = .37), nor did they have higher POD#1 VAS scores (p = .55). Eighty-seven percent of the intervention subjects said they would use ice again, and 82.6% of them said they would recommend ice to others. There were no adverse events related to ice. All subjects were prescribed 20 tablets oxycodone and averaged 2.9 (SD 3.4) tablets used after discharge. CONCLUSION: Ice packs are an acceptable supplement for postoperative pain control, but they do not reduce postoperative pain or opioid usage compared to standard pain management without ice packs.

Metabolomic differentiation of tumor core versus edge in glioma.

OBJECTIVE: Gliomas exhibit high intratumor and interpatient heterogeneity. Recently, it has been shown that the microenvironment and phenotype differ significantly between the glioma core (inner) and edge (infiltrating) regions. This proof-of-concept study differentiates metabolic signatures associated with these regions, with the potential for prognosis and targeted therapy that could improve surgical outcomes. METHODS: Paired glioma core and infiltrating edge samples were obtained from 27 patients after craniotomy. Liquid-liquid metabolite extraction was performed on the samples and metabolomic data were obtained via 2D liquid chromatography-mass spectrometry/mass spectrometry. To gauge the potential of metabolomics to identify clinically relevant predictors of survival from tumor core versus edge tissues, a boosted generalized linear machine learning model was used to predict metabolomic profiles associated with O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. RESULTS: A panel of 66 (of 168) metabolites was found to significantly differ between glioma core and edge regions (p ≤ 0.05). Top metabolites with significantly different relative abundances included DL-alanine, creatine, cystathionine, nicotinamide, and D-pantothenic acid. Significant metabolic pathways identified by quantitative enrichment analysis included glycerophospholipid metabolism; butanoate metabolism; cysteine and methionine metabolism; glycine, serine, alanine, and threonine metabolism; purine metabolism; nicotinate and nicotinamide metabolism; and pantothenate and coenzyme A biosynthesis. The machine learning model using 4 key metabolites each within core and edge tissue specimens predicted MGMT promoter methylation status, with AUROCEdge = 0.960 and AUROCCore = 0.941. Top metabolites associated with MGMT status in the core samples included hydroxyhexanoycarnitine, spermine, succinic anhydride, and pantothenic acid, and in the edge samples metabolites included 5-cytidine monophosphate, pantothenic acid, itaconic acid, and uridine. CONCLUSIONS: Key metabolic differences are identified between core and edge tissue in glioma and, furthermore, demonstrate the potential for machine learning to provide insight into potential prognostic and therapeutic targets.

Chronic arsenic increases cell migration in BEAS-2B cells by increasing cell speed, cell persistence, and cell protrusion length.

There is a strong association between arsenic exposure and lung cancer development, however, the mechanism by which arsenic exposure leads to carcinogenesis is not clear. In our previous study, we observed that when BEAS-2B cells are chronically exposed to arsenic, there is an increase in secreted TGFα, as well as an increase in EGFR expression and activity. Further, these changes were broadly accompanied with an increase in cell migration. The overarching goal of this study was to acquire finer resolution of the arsenic-dependent changes in cell migration, as well as to understand the role of increased EGFR expression and activity levels in the underlying mechanisms of cell migration. To do this, we used a combination of biochemical and single cell assays, and observed chronic arsenic treatment enhancing cell migration by increasing cell speed, cell persistence and cell protrusion length. All three parameters were further increased by the addition of TGFα, indicating EGFR activity is sufficient to enhance those aspects of cell migration. In contrast, EGFR activity was necessary for the increase in cell speed, as it was reversed with an EGFR inhibitor, AG1478, but was not necessary to enhance persistence and protrusion length. From these data, we were able to isolate both EGFR-dependent and -independent features of cell migration that were enhanced by chronic arsenic exposure.

Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways.

Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10-15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-ßgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health.

Intracerebroventricular enzyme replacement therapy with ß-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.

Autosomal recessive mutations in the galactosidase ß1 (GLB1) gene cause lysosomal ß-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human ß-gal (rhß-gal) produced in Chinese hamster ovary cells enabled direct and precise rhß-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rhß-gal was sufficient for normalizing ß-gal activity and mediating substrate clearance for several weeks. We found that rhß-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor-mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhß-gal (100 µg) resulted in broad bilateral biodistribution of rhß-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhß-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of ß-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhß-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches.

Palliative care education and research at US cancer centers: A national survey.

BACKGROUND: Palliative care (PC) education and research are essential to developing a skilled workforce and evidence base to support the delivery of quality cancer care. The current state of PC education and research at US cancer centers is unclear. In this national survey, the education and research programs of the National Cancer Institute (NCI)-designated and nondesignated cancer centers and the changes between 2009 and 2018 are compared. METHODS: Between April and August 2018, PC program leaders at all NCI-designated cancer centers and a random sample of nondesignated centers were sent a survey to examine the structure, processes, and outcomes of their programs on the basis of questions from a 2009 national survey. This preplanned analysis focused on education and research. RESULTS: There were 52 of 61 (85%) NCI-designated and 27 of 38 (71%) nondesignated cancer centers that responded. NCI-designated centers were more likely than nondesignated centers to have a PC fellowship program (87% vs 30%; P < .001), training for advanced practice providers (71% vs 44%; P = .03), PC research program (58% vs 15%; P < .001), peer-reviewed funding (43% vs 11%; P = .005), and philanthropic grants (41% vs 7%; P = .002). There were few significant improvements in PC education or research between 2009 and 2018 for both groups, notable exceptions include an increase in PC fellowships (38% vs 87%; P < .001) and mandatory PC rotations for medical oncology fellows (29% vs 55%; P = .02) at NCI-designated cancer centers. CONCLUSIONS: PC education and research are more developed at NCI-designated cancer centers. Despite some progress over the past decade, it is relatively slow and suboptimal.

Neural correlates of visual stimulus encoding and verbal working memory differ between cochlear implant users and normal-hearing controls.

A common concern for individuals with severe-to-profound hearing loss fitted with cochlear implants (CIs) is difficulty following conversations in noisy environments. Recent work has suggested that these difficulties are related to individual differences in brain function, including verbal working memory and the degree of cross-modal reorganization of auditory areas for visual processing. However, the neural basis for these relationships is not fully understood. Here, we investigated neural correlates of visual verbal working memory and sensory plasticity in 14 CI users and age-matched normal-hearing (NH) controls. While we recorded the high-density electroencephalogram (EEG), participants completed a modified Sternberg visual working memory task where sets of letters and numbers were presented visually and then recalled at a later time. Results suggested that CI users had comparable behavioural working memory performance compared with NH. However, CI users had more pronounced neural activity during visual stimulus encoding, including stronger visual-evoked activity in auditory and visual cortices, larger modulations of neural oscillations and increased frontotemporal connectivity. In contrast, during memory retention of the characters, CI users had descriptively weaker neural oscillations and significantly lower frontotemporal connectivity. We interpret the differences in neural correlates of visual stimulus processing in CI users through the lens of cross-modal and intramodal plasticity.

State of palliative care services at US cancer centers: An updated national survey.

BACKGROUND: This study examined the changes in outpatient palliative care services at US cancer centers over the past decade. METHODS: Between April and August 2018, all National Cancer Institute (NCI)-designated cancer centers and a random sample of 1252 non-NCI-designated cancer centers were surveyed. Two surveys used previously in a 2009 national study were sent to each institution: a 22-question cancer center executive survey regarding palliative care infrastructure and attitudes toward palliative care and an 82-question palliative care program leader survey regarding detailed palliative care structures and processes. Survey findings from 2018 were compared with 2009 data from 101 cancer center executives and 96 palliative care program leaders. RESULTS: The overall response rate was 69% (140 of 203) for the cancer center executive survey and 75% (123 of 164) for the palliative care program leader survey. Among NCI-designated cancer centers, a significant increase in outpatient palliative care clinics was observed between 2009 and 2018 (59% vs 95%; odds ratio, 12.3; 95% confidence interval, 3.2-48.2; P < .001) with no significant changes in inpatient consultation teams (92% vs 90%; P = .71), palliative care units (PCUs; 26% vs 40%; P = .17), or institution-operated hospices (31% vs 18%; P = .14). Among non-NCI-designated cancer centers, there was no significant increase in outpatient palliative care clinics (22% vs 40%; P = .07), inpatient consultation teams (56% vs 68%; P = .27), PCUs (20% vs 18%; P = .76), or institution-operated hospices (42% vs 23%; P = .05). The median interval from outpatient palliative care referral to death increased significantly, particularly for NCI-designated cancer centers (90 vs 180 days; P = 0.01). CONCLUSIONS: Despite significant growth in outpatient palliative care clinics, there remain opportunities for improvement in the structures and processes of palliative care programs.

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming.

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

The effect of itraconazole on the pharmacokinetics of lorlatinib: results of a phase I, open-label, crossover study in healthy participants.

Background The third-generation tyrosine kinase inhibitor lorlatinib is approved for the treatment of ALK-positive metastatic NSCLC. CYP3A plays a major role in lorlatinib metabolism; therefore, a drug-drug interaction study was warranted to evaluate the impact of the strong CYP3A inhibitor, itraconazole, on lorlatinib plasma exposure. Methods This phase 1, open-label, 2-period, crossover study estimated the effects of itraconazole on the plasma pharmacokinetics and safety of lorlatinib in healthy participants (NCT02838264). Single-dose lorlatinib 50 mg (n = 2), 75 mg (n = 2) and 100 mg (n = 12) was administered in Period 1. In Period 2, itraconazole oral solution 200 mg/day was administered on Days 1-11, and single-dose lorlatinib on Day 5. Blood samples were collected up to 168 h after lorlatinib dosing. Results During daily dosing with itraconazole (Period 2), the ratios of the adjusted geometric means for area under the plasma concentration-time profile extrapolated to infinity (AUCinf) and maximum plasma concentration (Cmax) of single-dose lorlatinib 100 mg were 141.79% (90% confidence interval, 128.71%, 156.21%) and 124.39% (110.20%, 140.41%), respectively, compared with Period 1 (lorlatinib alone). Lorlatinib was well tolerated alone and with itraconazole. No serious adverse events or withdrawals were reported. Conclusions Co-administration of itraconazole and lorlatinib increased the plasma exposure of lorlatinib relative to lorlatinib alone in healthy participants. Therefore, concomitant use of lorlatinib with strong CYP3A inhibitors should be avoided. If this combination is unavoidable, the starting dose of lorlatinib should be reduced from 100 mg to 75 mg.

Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.

STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. PARTICIPANTS/MATERIALS, SETTING, METHODS: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. MAIN RESULTS AND THE ROLE OF CHANCE: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.