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Right ventricular (RV) dysfunction in pulmonary arterial hypertension (PAH) is associated with poor outcomes. Cardiac magnetic resonance imaging (cMRI) is the gold standard for volumetric assessment, and few reports have correlated 6-min walk distance (6MWD) and cMRI parameters in PAH. Cardiac Effort, (the number of heart beats used during 6-min walk test)/(6MWD), incorporates physiologic changes into walk distance and has been associated with stroke volume (SV) measured by nuclear imaging and indirect Fick. Here, we aimed to interrogate the relationship of Cardiac Effort and 6MWD with SV measured by the gold standard, cMRI. This was a single-center, observational, prospective study in Group 1 PAH patients. Subjects completed 6-min walk with heart rate monitoring (Cardiac Effort) and cMRI within 24 h. cMRI was correlated to Cardiac Effort and 6MWD using Spearman Correlation Coefficient. Twenty-five participants with a wide range of RV function completed both cMRI and Cardiac Effort. There was a strong correlation between left ventricle SV index and both Cardiac Effort ( r  = -0.70, p  = 0.0001) and 6MWD ( r  = 0.67, p  = 0.0002). Cardiac Effort and 6MWD were statistically separated in patients at prognostically significant thresholds of left ventricle SV index (>31 ml/m 2 ), RV Ejection Fraction (>35%), and SV/End Systolic Volume ( > 0.53). Cardiac Effort and 6MWD are noninvasive ways to gain insight into those with impaired SV. 6MWD may correlate better with SV than previously thought and heart rate monitoring provides physiologic context to the walk distance obtained., Competing Interests: DJL: receives consulting and speaking fees from United Therapeutics. United Therapeutics also provides University of Rochester with research funding for industry and investigator sponsored studies. ML: none, DH: non, RJW: none., (© 2024 The Authors. Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.

Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6 h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24 h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Electrochemical, aptamer-based (E-AB) sensors provide a generalizable strategy to quantitatively detect a variety of targets including small molecules and proteins. The key signaling attributes of E-AB sensors (sensitivity, selectivity, specificity, and reagentless and dynamic sensing ability) make them well suited to monitor dynamic processes in complex environments. A key bioanalytical challenge that could benefit from the detection capabilities of E-AB sensors is that of cell signaling, which involves the release of molecular messengers into the extracellular space. Here, we provide a perspective on why E-AB sensors are suited for this measurement, sensor requirements, and pioneering examples of cellular signaling measurements., (© 2023 The Author(s). Published on behalf of The Electrochemical Society by IOP Publishing Limited.)

Pulmonary arterial hypertension (PAH) patients have low activity. Activity intensity or duration could be a measure of clinical status or improvement. We aimed to determine whether standard or novel actigraphy measures could detect increases in activity after adding therapy. This was a prospective, single-center observational study evaluating activity after adding therapy in Group 1 PAH; we also report a validation cohort. For our study, two different accelerometers were used, a wrist (ActiGraph) and chest (MC10) device. Patients were analyzed in two groups, Treatment Intensification (TI, adding therapy) or Stable. Both groups had baseline monitoring periods of 7 days; the TI group had follow-up at 3 months, while Stables had follow-up within 4 weeks to assess stability. Activity time and steps were reported from both devices' proprietary algorithms. In ActiGraph only, steps in 1-min intervals throughout the day were ranked (not necessarily contiguous). Average values for each week were calculated and compared using nonparametric testing. Thirty patients had paired data (11 Stable and 19 TI). There was no between-group difference at baseline; we did not observe therapy-associated changes on average daily steps or activity time/intensity. The top 5 min of steps (capacity) increased after adding therapy; there was no difference in the stable group. This key finding was validated in a previously reported randomized trial studying a behavioral intervention to increase exercise. Total daily activity metrics are influenced by both disease and non-disease factors, making therapy-associated change difficult to detect. Peak minute steps were a treatment-responsive marker in both a pharmacologic and training intervention., Competing Interests: D. L. receives consulting and speaking fees from United Therapeutics. The University of Rochester receives research funds from United Therapeutics. None declared (A. L., M. L., R. J. W., A. H., J. A. and E. B.) related to this research., (© 2023 The Authors. Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

Abnormally elevated expression of tRNA is a common feature of breast tumours. Rather than a uniform increase in all tRNAs, some are deregulated more strongly than others. Elevation of particular tRNAs has been associated with poor prognosis for patients, and experimental models have demonstrated the ability of some tRNAs to promote proliferation or metastasis. Each tRNA isoacceptor is encoded redundantly by multiple genes, which are commonly dispersed across several chromosomes. An unanswered question is whether the consistently high expression of a tRNA in a cancer type reflects the consistent activation of the same members of a gene family, or whether different family members are activated from one patient to the next. To address this question, we interrogated ChIP-seq data to determine which tRNA genes were active in individual breast tumours. This revealed that distinct sets of tRNA genes become activated in individual cancers, whereas there is much less variation in the expression patterns of families. Several pathways have been described that are likely to contribute to increases in tRNA gene transcription in breast tumours, but none of these can adequately explain the observed variation in the choice of genes between tumours. Current models may therefore lack at least one level of regulation.

Advances in translational science require innovative solutions, and engagement of productive transdisciplinary teams play a critical role. While various forms of scientific meetings have long provided venues for sharing scientific findings and generating new collaborations, many conferences lack opportunities for active discussions. We describe the use of an Un-Meeting to foster innovative translational science teams through engaged discussions across multidisciplinary groups addressing a shared theme. The Un-Meeting was delivered by the University of Rochester Center for Leading Innovation and Collaboration, the national coordinating center for the National Institutes of Health Clinical and Translational Science Awards (CTSA) program. This pilot CTSA program Un-Meeting focused on engaging translational scientists, policy-makers, community members, advocates, and public health professionals to address the opioid crisis. The participant-driven format leveraged lightning talks, attendee-led idea generation, and extensive breakout discussions to foster multidisciplinary networking. Results indicated participation by a broad set of attendees and a high level of networking during the meeting. These results, coupled with the growth of the Un-Meeting across the CTSA Consortium, provide practices and models to potentially advance team and translational science. While future work will further assess the impact of Un-Meetings, this format presents a promising approach to enhance translational science., Competing Interests: There are no conflicts of interest related to the content of this manuscript., (© The Author(s) 2023.)

Soil organisms are a crucial part of the terrestrial biosphere and are essential for ecosystem functioning. A major part of soil and sediment ecosystems are nematodes worms, which can be used as a bioindicator of soil status. These worms represent one of the most numerous animal phyla on earth, filling all trophic levels in the soil food web. Overall nematode abundance is related to net ecosystem productivity, and regional variations in abundance provides insight into local patterns of soil fertility and functioning. Methods for extracting nematodes from soils have been established, but these can be cumbersome, or require specialist equipment or consumables, meaning they are not always suitable for the field or remote areas. We have built on previous methods to develop a simple, more compact, and zero-waste method of extracting nematodes, using basic equipment. We demonstrate this in a small collection of soils from deforested, native forest, and reforested sites. On a larger scale, this method can be used to reflect overall ecosystem function, indicating current soil status, and future success and proliferation of reforested sites., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

Electrochemical aptamer-based sensors (EABs) using self-assembled monolayers on gold working-electrodes have now been in-vivo demonstrated for multiple-analytes, demonstrating their sensitivity and specificity even in a continuous sensing format. However, longevity has been demonstrated for only 24 hours and sensitivity has been challenging for highly dilute analytes (nM regime). A novel approach is reported here using electrochemical aptamer-based sensing that is not covalently-bound to a gold-working electrode but where aptamers are freely mobile in solution. This alternative approach has the potential to improve longevity by reducing electrode surface degradation and improving sensitivity using aptamer binding constructs that are not available for aptamers when covalently bound to the electrode. Specifically, a molecular-beacon (fluorescent) cortisol aptamer was adapted into an amperometry solution-phase cortisol EAB sensor, demonstrating ∼5% signal gain starting at only 10 nM and a saturated signal gain of ∼70% at several μM. A robust signal was achieved due to use of methylene-blue redox-tagged aptamer that was measured through amperometry with interdigitated electrodes. While this result demonstrates the basic feasibility of solution-phase EAB sensors, the result also required a self-assembled monolayer alkylthiolate blocking-layer on the gold working electrode which restricts potential device longevity. These results cumulatively suggest that initial significance of solution-phase EAB sensors may be strongest for point-of-care type testing applications and further development would be required for long-lasting continuous sensing applications.

Background: There is a paucity of literature describing the recovery trajectory after surgery for upper extremity ischemia. Using Patient-Reported Outcomes Measurement Information System (PROMIS) Physical Function (PF), Upper Extremity (UE), Pain Interference (PI), and Depression domains, we aimed to describe the postoperative recovery of such patients., Methods: We queried our PROMIS database for patients undergoing surgery for vasospastic or occlusive disease over a 4.5-year period. Inclusion criteria were preoperative, early (average 3 weeks) and late (average 6 months) postoperative PROMIS PF and/or UE, PI, and Depression scores. The change in PROMIS scores was calculated for each time point. Changes in PROMIS scores were compared with minimal clinically important difference estimates., Results: We identified 13 patients undergoing 13 surgical interventions that met inclusion criteria. More than one-half of our patients were men (n = 7 [54%]), and more than one-half of the surgeries (n = 7 [54%]) were for isolated occlusive diagnoses, with the remainder for vasospastic disease. At short-term postoperative follow-up, the change in PROMIS PF, UE, PI, and Depression scores was -6.34 (SD: 9.13), -6.81 (SD: 9.61), 3.16 (SD: 5.78), and -3.05 (SD: 8.37), respectively. At mid-term postoperative follow-up, the change in PROMIS PF, UE, PI, and Depression scores was 4.45 (SD: 10.33), 8.04 (SD: 13.84), -7.03 (SD: 7.06), and -12.27 (SD: 10.85), respectively., Conclusions: Our findings suggest patients undergoing surgical treatment for upper extremity ischemia experience a worsening of functional symptoms initially, as expected, followed by notable improvement.

Ion channel proteins showed great promise in the field of nanopore sensing and molecular flux imaging applications due to the atomic-level precision of the pore size and a high signal-to-noise ratio. More specifically, ion channel probes, where the protein channels are integrated at the end of a solid probe, can achieve highly localized detection. Metal probe materials such as gold and silver have been developed to support lipid bilayers and enable the use of smaller probes, or nanoneedles, compared to more traditional glass micropipette ion channel probes. Silver probes are preferable because they support sustained DC stable channel current due to the AgCl layer formed around the tip during the fabrication process. However, one of the current challenges in ion channel measurements is maintaining a single-channel recording. Multiple protein insertions complicate data analysis and destabilize the bilayer. Herein, we combine the promising probe material (Ag/AgCl) with an approach based on current feedback-controlled tip positioning to maintain long-term single-channel recordings for up to 3 h. We develop a hybrid positioning control system, where the channel current is used as feedback to control the vertical movement of the silver tip and, subsequently, control the number of protein channels inserted in the lipid membrane. Our findings reveal that the area of the lipid bilayer decreases with moving the silver tip up (i.e., decreasing the displacement in the z -direction). By reducing the bilayer area around the fine silver tip, we minimize the probability of multiple insertions and remove unwanted proteins. In addition, we characterize the effect of lipid properties such as fluidity on the lipid membrane area. We believe that the use of silver nanoneedles, which enables DC stable channel current, coupled with the developed tip displacement mechanism will offer more opportunities to employ these probes for chemical imaging and mapping different surfaces.

Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, many of which are produced by integrating transgenes into the genomes of mammalian cells. However, expression is highly variable and can be unstable during prolonged culture. This is often due to epigenetic mechanisms silencing the transgenes. To combat this problem, vectors have been engineered to include ubiquitous chromatin opening elements (UCOEs) that protect against silencing. Here, we recount the evidence that UCOEs can modify chromatin environments and benefit biomanufacturing., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Background: Team development and idea generation are key intertwined steps in translational science that need a framework to accommodate unstructured, participatory interactions. To this end, we introduced Un-Meetings to the Clinical and Translational Science Awards (CTSA) Program, innovative events that facilitate cross-disciplinary idea generation and informal discussions between translational scientists, policy makers, community members, advocates, and public health professionals. Here we describe a mixed methods study to characterize the conceptual diversity and clusterization of ideas generated through an Opioid Crisis Un-Meeting., Methods: An Un-Meeting targeting translation science approaches to the opioid crisis were hosted at the University of Rochester Center for Leading Innovation and Collaboration (CLIC). We used semantic analysis and conceptual mapping of keywords to analyze how attendee-led idea generation sessions identified topics for breakout discussions., Results: One hundred and two individuals from 40 institutions proposed 150 unique ideas that were grouped into 23 breakout sessions. Network analysis showed that diverse pools of experts were bridged by topics addressing the complexities of the opioid crisis. Two clusters emerged: (1) systems, contexts, and community engagement, and (2) technologies, innovations, and treatment advancements., Conclusions: The cross-disciplinary nature of topic areas that bridge across thematic communities provide opportunities for CTSA programs to engage and support development of diverse translational teams. Potential opportunities for team building include technological advancements of opioid prevention, treatment, surveillance, systems approaches, and studies focusing on special populations and health disparities. The analysis method here may be useful in identifying naturally emerging teams of experts and community gaps when addressing large problems., (© The Author(s) 2022.)

Antiphospholipid syndrome (APS) is an uncommon autoantibody-mediated condition characterised by acquired thrombophilia resulting in recurrent arterial and venous thrombosis. An inciting factor allows for the exposure of endothelial phospholipids, causing antigen formation and subsequent creation of antibodies. A woman in her 70s presented after vehicular trauma, suffering broken ribs, pneumothorax and incidentally discovered left adrenal haemorrhage. Two weeks later she presented with acute-onset abdominal pain and was found to have a right adrenal gland haemorrhage on CT imaging without interval trauma occurring. The patient had antiphospholipid antibody laboratory studies drawn and was given intravenous heparin with a bridge to warfarin at discharge. Laboratory results returned positive for lupus anticoagulant, beta-2 glycoprotein and anticardiolipin antibodies indicating triple positivity, with repeated laboratory tests positive in 12 weeks' time, confirming the diagnosis. Bilateral adrenal haemorrhage, rather than traditional venous thromboembolism, was the presenting pathology in this patient's diagnosis of APS., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Square wave voltammetry (SWV) is a voltammetric technique for measuring Faradaic current while minimizing contributions from non-Faradaic processes. In square wave voltammetry, the potential waveform applied to a working electrode and the current sampling protocols followed are designed to minimize contributions from non-Faradaic processes (i.e., double layer charging) to improve voltammetric sensitivity. To achieve this, the current is measured at the end of each forward and reverse potential pulse after allowing time for non-Faradaic currents to decay exponentially. A consequence of sampling current at the end of a potential pulse is that the current data from the preceding time of the potential pulse are discarded. These discarded data can provide information about the non-Faradaic contributions as well as information about the redox system including charge transfer rates. In this paper, we introduce continuous square wave voltammetry (cSWV), which utilizes the continuous collection of current to maximize the information content obtainable from a single voltammetry sweep eliminating the need for multiple scans. cSWV enables acquiring a multitude of voltammograms corresponding to various frequencies and, thus, different scan rates from a single sweep. An application that benefits significantly from cSWV is conformation switching, functional nucleic acid sensors. We demonstrate the utility of cSWV on two representative small molecules targeting electrochemical, aptamer-based sensors. Moreover, we show that cSWV provides comparable results to those obtained from traditional square wave voltammetry, but with cSWV, we are able to acquire dynamic information about the sensor surfaces enabling rapid calibration and optimization of sensing performance. We also demonstrate cSWV on soluble redox markers. cSWV can potentially become a mainstay technique in the field of conformation switching sensors., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution., (© 2022 Geng et al.; Published by Cold Spring Harbor Laboratory Press.)

Eukaryotic chromosomes are divided into domains with distinct structural and functional properties, such as differing levels of chromatin compaction and gene transcription. Domains of relatively compact chromatin and minimal transcription are termed heterochromatic, whereas euchromatin is more open and actively transcribed. Insulators separate these domains and maintain their distinct features. Disruption of insulators can cause diseases such as cancer. Many insulators contain tRNA genes (tDNAs), examples of which have been shown to block the spread of activating or silencing activities. This characteristic of specific tDNAs is conserved through evolution, such that human tDNAs can serve as barriers to the spread of silencing in fission yeast. Here we demonstrate that tDNAs from the methylotrophic fungus Pichia pastoris can function effectively as insulators in distantly-related budding yeast. Key to the function of tDNAs as insulators is TFIIIC, a transcription factor that is also required for their expression. TFIIIC binds additional loci besides tDNAs, some of which have insulator activity. Although the mechanistic basis of TFIIIC-based insulation has been studied extensively in yeast, it is largely uncharacterized in metazoa. Utilising publicly-available genome-wide ChIP-seq data, we consider the extent to which mechanisms conserved from yeast to man may suffice to allow efficient insulation by TFIIIC in the more challenging chromatin environments of metazoa and suggest features that may have been acquired during evolution to cope with new challenges. We demonstrate the widespread presence at human tDNAs of USF1, a transcription factor with well-established barrier activity in vertebrates. We predict that tDNA-based insulators in higher organisms have evolved through incorporation of modules, such as binding sites for factors like USF1 and CTCF that are absent from yeasts, thereby strengthening function and providing opportunities for regulation between cell types., (Copyright © 2022. Published by Elsevier B.V.)

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

tRNA gene transcription by RNA polymerase III (Pol III) is a tightly regulated process, but dysregulated Pol III transcription is widely observed in cancers. Approximately 75% of all breast cancers are positive for expression of Estrogen Receptor alpha (ERα), which acts as a key driver of disease. MCF-7 cells rapidly upregulate tRNA gene transcription in response to estrogen and ChIP-PCR demonstrated ERα enrichment at tRNA Leu and 5S rRNA genes in this breast cancer cell line. While these data implicate the ERα as a Pol III transcriptional regulator, how widespread this regulation is across the 631 tRNA genes has yet to be revealed. Through analyses of ERα ChIP-seq datasets, we show that ERα interacts with hundreds of tRNA genes, not only in MCF-7 cells, but also in primary human breast tumours and distant metastases. The extent of ERα association with tRNA genes varies between breast cancer cell lines and does not correlate with levels of ERα binding to its canonical target gene GREB1. Amongst other Pol III-transcribed genes, ERα is consistently enriched at the long non-coding RNA gene RMRP, a positive regulator of cell cycle progression that is subject to focal amplification in tumours. Another Pol III template targeted by ERα is the RN7SL1 gene, which is strongly implicated in breast cancer pathology by inducing inflammatory responses in tumours. Our data indicate that Pol III-transcribed non-coding genes should be added to the list of ERα targets in breast cancer., (Copyright © 2022. Published by Elsevier B.V.)

Dual combination therapy with a phosphodiesterase-5 inhibitor (PDE5i) and endothelin receptor antagonist is recommended for most patients with intermediate-risk pulmonary arterial hypertension (PAH). The RESPITE and REPLACE studies suggest that switching from a PDE5i to a soluble guanylate cyclase (sGC) activator may provide clinical improvement in this situation. The optimal approach to escalation or transition of therapy in this or other scenarios is not well defined. We developed an expert consensus statement on the transition to sGC and other treatment escalations and transitions in PAH using a modified Delphi process. The Delphi process used a panel of 20 physicians with expertise in PAH. Panelists answered three questionnaires on the management of treatment escalations and transitions in PAH. The initial questionnaire included open-ended questions. Later questionnaires consolidated the responses into statements that panelists rated on a Likert scale from -5 ( strongly disagree ) to +5 ( strongly agree ) to determine consensus. The Delphi process produced several consensus recommendations. Escalation should be considered for patients who are at high risk or not achieving treatment goals, by adding an agent from a new class, switching from oral to parenteral prostacyclins, or increasing the dose. Switching to a new class or within a class should be considered if tolerability or other considerations unrelated to efficacy are affecting adherence. Switching from a PDE5i to an SGC activator may benefit patients with intermediate risk who are not improving on their present therapy. These consensus-based recommendations may be helpful to clinicians and beneficial for patients when evidence-based guidance is unavailable., Competing Interests: Franck F. Rahaghi reports consultation, research, and speakership honoraria from Bayer and Janssen, consultation and speakership from United Therapeutics, and consultation fees from Acceleron. Vijay P. Balasubramanian reports a research grant from United Therapeutics and serves on a speakers bureau for Bayer. Robert C. Bourge reports research grant support to my institution from United Therapeutics and Bayer, and service on a Scientific Advisory Board at United Therapeutics. Murali M. Chakinala reports grants or contracts from Actelion/Janssen, Bayer, Medtronic, NIH, Reata, Liquidia, Phase Bio, Complexa, United Therapeutics, Altavant, Trio Health Analytics, Reata, Acceleron, Arena, and Gossamer; consulting fees from Altavant, Vaderis Therapeutics, Aerovate, Reata, VWave, and Arena; honoraria from Bayer, Gilead, Simply Speaking, WebMD, and United Therapeutics; support for attending meetings and/or travel from Actelion/Janssen, United Therapeutics, Bayer, Acceleron, Reata, and Gilead; participation on a Data Safety Monitoring Board or Advisory Board for Actelion/Janssen, Express Scripts, Phase Bio, Altavant, Gossamer, United Therapeutics, Bayer, Acceleron, and Liquidia; and leadership or a fiduciary role in the Pulmonary Hypertension Association and the Cure HHT Global Research and Medical Advisory Board. Michael S. Eggert reports research contracts with United Therapeutics (BREEZE and ADVANCE Outcomes), Acceleron, and Actelion. Jean M. Elwing reports grants from Actelion, Acceleron, Reata, United Therapeutics, Liquidia, Phase Bio, Complexa, Gossamer Bio, Bayer, Arena, Eiger, Akros, Bellerophon, and Lung LLC and consulting fees from United Therapeutics, Acceleron, Liquidia, Altavant, Bayer, Gossamer Bio, Actelion, Bayer. Jeremy Feldman reports consulting and giving talks for Bayer, United Therapeutics, and Jansen. Christopher King reports personal fees from Actelion, personal fees from Genentech, United Therapeutics, and Boehringer Ingelheim; has served on advisory boards for and is on the Speakers’ Bureau of Actelion, Boehringer‐Ingelheim Pharmaceuticals, and United Therapeutics. James R. Klinger reports that his institution receives research funding from United Technologies, service on a Steering Committee and a Clinical Outcomes Committee for Bayer, and a leadership role in the Pulmonary Hypertension Association. Stephen C. Mathai reports personal fees from United Therapeutics; participation on a data safety monitoring board or advisory board from United Therapeutics, Actelion, and Bayer, and leadership or a fiduciary role in the PCORI Rare Disease Advisory Panel and the World Symposium on Pulmonary Hypertension. John Wesley McConnell reports consulting fees from Actelion, Bayer, Gossamer, Altavant, and Liquidia; honoraria from Actelion, Bayer, Simply Speaking, Impact PH, and Reata, and participation on a data safety monitoring board or advisory board for Actelion, Liquidia, Gossamer, and Altavant. Harold I. Palevsky reports honoraria for serving on scientific advisory boards for Acceleron, Actelion/Janssen, PhaseBio and United Therapeutics, and serving on a DSMB for United Therapeutics. Ricardo Restrepo‐Jaramillo reports serving on speaker's bureau for United Therapeutics, Bayer, and Actelion. Zeenat Safdar reports serving on a speakers bureau, consultation and advisory boards for Actelion, United Therapeutics, Boehringer Ingelheim, Bayer, and Roche. Jeffrey S. Sager reports personal fees from Bayer pharmaceuticals, grants and personal fees from United Therapeutics, grants and personal fees from Janssen (J and J), and grants from Reata outside the submitted work. Namita Sood reports a speaking fee from Bayer. Roxana Sulica reports research grants from Bayer, United Therapeutics, Complexa, and Reata, and serves on advisory boards for Actelion, Bayer, United Therapeutics, and Reata. R. James White reports research grants from United Therapeutics, Reata, Bayer, Merck, and Janssen and consulting fees from Merck and Bayer. Nicholas S. Hill reports honoraria from Axon Research for this study; grants to his institution from Actelion, Bayer, Gilead, and United Therapeutics; consulting fees from United Therapeutics; and participation in Data Safety Monitoring Boards for United Therapeutics and Pfizer. All authors had access to the Delphi questionnaire analysis and data and participated in the review, revision, and approval of the content of the manuscript for submission. Charles D. Burger reports no disclosures or conflicts of interest., (© 2022 The Authors. Pulmonary Circulation published by John Wiley & Sons Ltd on behalf of Pulmonary Vascular Research Institute.)

Maf1 is a transcription factor that is conserved in sequence and structure between yeasts, animals and plants. Its principal molecular function is also well conserved, being to bind and repress RNA polymerase (pol) III, thereby inhibiting synthesis of tRNAs and other noncoding RNAs. Restrictions on tRNA production and hence protein synthesis can provide a mechanism to preserve resources under conditions that are suboptimal for growth. Accordingly, Maf1 is found in some organisms to influence growth and/or stress survival. Because of their sessile nature, plants are especially vulnerable to environmental changes and molecular adaptations that enhance growth under benign circumstances can increase sensitivity to external challenges. We tested if Maf1 depletion in the model plant Arabidopsis affects growth, pathogen resistance and tolerance of drought or soil salinity, a common physiological challenge that imposes both osmotic and ionic stress. We find that disruption of the Maf1 gene or RNAi-mediated depletion of its transcript is well-tolerated and confers a modest growth advantage without compromising resistance to common biotic and abiotic challenges., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Oral treprostinil has been shown to improve exercise capacity and delay disease progression in patients with pulmonary arterial hypertension (PAH), but its effects on hemodynamics are not well-characterized. The FREEDOM-EV trial was a Phase III, international, placebo-controlled, double-blind, event-driven study in 690 participants with PAH who were taking a single oral PAH therapy. FREEDOM-EV demonstrated a significantly reduced risk for clinical worsening with oral treprostinil taken three times daily and did not uncover new safety signals in PAH patients. Sixty-one participants in the FREEDOM-EV trial volunteered for a hemodynamics sub-study. Pulmonary artery compliance (PAC), a ratio of stroke volume to pulmonary pulse pressure, significantly increased from Baseline to Week 24 in the oral treprostinil group compared with the placebo group (geometric mean 26.4% active vs. -6.0% placebo; ANCOVA p=0.007). There was a significant increase in cardiac output in the oral treprostinil group compared to the placebo group (geometric mean 11.3% active vs. -6.4% placebo; ANCOVA p=0.005) and a corresponding significant reduction in pulmonary vascular resistance (PVR) (geometric mean -21.5 active vs. -1.8% placebo; ANCOVA p=0.02) from Baseline to Week 24. These data suggest that increased compliance contributes to the physiological mechanism by which oral treprostinil improves exercise capacity and delays clinical worsening for patients with PAH., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In model organisms, RNA-sequencing (RNA-seq) is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often over-represented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that the differential expression of genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms., Competing Interests: RW, EM, SW, EB No competing interests declared, (© 2022, White et al.)

Developmental consequences of prenatal drug exposure have been reported in many human cohorts and animal studies. The long-lasting impact on the offspring-including motor and cognitive impairments, cranial and cardiac anomalies and increased prevalence of ADHD-is a socioeconomic burden worldwide. Identifying the molecular changes leading to developmental consequences could help ameliorate the deficits and limit the impact. In this study, we have used zebrafish, a well-established behavioral and genetic model with conserved drug response and reward pathways, to identify changes in behavior and cellular pathways in response to developmental exposure to amphetamine, nicotine or oxycodone. In the presence of the drug, exposed animals showed altered behavior, consistent with effects seen in mammalian systems, including impaired locomotion and altered habituation to acoustic startle. Differences in responses seen following acute and chronic exposure suggest adaptation to the presence of the drug. Transcriptomic analysis of exposed larvae revealed differential expression of numerous genes and alterations in many pathways, including those related to cell death, immunity and circadian rhythm regulation. Differential expression of circadian rhythm genes did not correlate with behavioral changes in the larvae, however, two of the circadian genes, arntl2 and per2 , were also differentially expressed at later stages of development, suggesting a long-lasting impact of developmental exposures on circadian gene expression. The immediate-early genes, egr1, egr4, fosab , and junbb , which are associated with synaptic plasticity, were downregulated by all three drugs and in situ hybridization showed that the expression for all four genes was reduced across all neuroanatomical regions, including brain regions implicated in reward processing, addiction and other psychiatric conditions. We anticipate that these early changes in gene expression in response to drug exposure are likely to contribute to the consequences of prenatal exposure and their discovery might pave the way to therapeutic intervention to ameliorate the long-lasting deficits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mech, Merteroglu, Sealy, Teh, White, Havelange, Brennan and Busch-Nentwich.)

RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations ( e.g ., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a protocol to extract total nucleic acid from individual zebrafish embryos. Individual embryos are lysed in 96-well plates and nucleic acid is extracted using SPRI beads. The total nucleic acid can be genotyped and then DNase I treated to produce RNA samples for sequencing. This protocol allows for processing large numbers of individual samples, with the ability to genotype each sample, which makes it possible to undertake transcriptomic analysis on mutants at timepoints before the phenotype is visible. Graphic abstract: Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

Clinical trials of Dll4 (Delta-like 4) neutralizing antibodies (Dll4nAbs) in cancer patients are ongoing. Surprisingly, pulmonary hypertension (PH) occurs in 14% to 18% of patients treated with Dll4nAbs, but the mechanisms have not been studied. Here, PH progression was measured in mice treated with Dll4nAbs. We detected Notch signaling in lung tissues and analyzed pulmonary vascular permeability and inflammation. Notch target gene array was performed on adult human pulmonary microvascular endothelial cells (ECs) after inhibiting Notch cleavage. Similar mechanisms were studied in PH mouse models and pulmonary arterial hypertension patients. The rescue effects of constitutively activated Notch1 in vivo were also measured. We observed that Dll4nAbs induced PH in mice as indicated by significantly increased right ventricular systolic pressure, as well as pulmonary vascular and right ventricular remodeling. Mechanistically, Dll4nAbs inhibited Notch1 cleavage and subsequently impaired lung endothelial barrier function and increased immune cell infiltration in vessel walls. In vitro, Notch targeted genes' expression related to cell growth and inflammation was decreased in human pulmonary microvascular ECs after the Notch1 inactivation. In lungs of PH mouse models and pulmonary arterial hypertension patients, Notch1 cleavage was inhibited. Consistently, EC cell-cell junction was leaky, and immune cell infiltration increased in PH mouse models. Overexpression activated Notch1-attenuated progression of PH in mice. In conclusion, Dll4nAbs led to PH development in mice by impaired EC barrier function and increased immune cell infiltration through inhibition of Notch1 cleavage in lung ECs. Reduced Notch1 cleavage in lung ECs could be an underlying mechanism of PH pathogenesis.

Aims: Activity trackers for clinical trials and remote monitoring are appealing as they provide objective data outside of the clinic setting. Algorithms determine physical activity intensity and count steps. Multiple studies show physical inactivity in pulmonary arterial hypertension (PAH). There are no studies comparing different activity trackers worn on different parts of the body in PAH. We had patients with PAH simultaneously wear two different accelerometers, compared measures between the two devices, and correlated the measures with standard clinical metrics in PAH., Methods and Results: This was a single-centre, prospective observational study. Daily physical activity and daily total steps were measured using Actigraph GT9X Link and MC10 Biostamp nPoint for 5-10 days. Actigraph was worn on the non-dominant hand and the MC10 Biostamp nPoint was worn on the chest and leg with disposable adhesives. Twenty-two participants wore both accelerometers >12 h/day for an average 7.8 days. The average activity time measured by Actigraph was significantly higher than that measured by MC10 (251 ± 25 min vs. 113 ± 18 min, P  = 0.0001). Actigraph's algorithm reported more time in light activity than moderate (190 ± 62 min vs. 60 ± 56 min, P  = 0.0001). REVEAL 2.0 scores correlated highly with activity time measured using either device. Invasively measured haemodynamics within 7 days did not correlate with activity time or daily steps., Conclusion: Different activity trackers yield discordant results in PAH patients. Further studies are needed in determining the best device, optimal wear time, and different thresholds for activities in chronic diseases., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background and Objectives: Pulmonary hypertension is commonly seen in end stage kidney disease and is most commonly due to elevated left heart pressures. There is limited data about vasodilator use during the management of Group 1 pulmonary arterial hypertension in the context of those who also have or later developed end stage kidney disease. The objective of this study was to determine safety and efficacy of vasodilator therapy in precapillary pulmonary hypertension requiring renal replacement therapy., Design: This was a single-center retrospective case series. Patients were identified from our Pulmonary Hypertension Clinic using a historical roster from 2012 to 2020. Patients were included if they >18 years of age, had Group 1 or Group 4 (precapillary) pulmonary hypertension on right heart catheterization, and also had end stage kidney disease requiring either intermittent hemodialysis or peritoneal dialysis., Results: 18 patients were identified with invasively confirmed Group 1 or Group 4 pulmonary hypertension and end stage kidney disease on renal replacement therapy. Scleroderma was the most common etiology for renal failure. 17 patients were treated with vasodilator therapy. Fifteen patients had paired right heart catheterizations that showed a significant decrease in mean pulmonary artery pressure and pulmonary vascular resistance. Therapy was relatively well tolerated but hypotension was common and midodrine was often helpful. Two patients had successful renal transplantation after starting vasodilator therapy., Conclusion: We found vasodilator therapy was reasonably well tolerated and associated with a drop in mean pressure and pulmonary vascular resistance in patients with end stage kidney disease on dialysis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this work, a novel light activatable micron-sized liposomal drug carrier that has a unique capability to release drug repetitively in proportion to the cycle number of short irradiation (5 s) of near-infrared (NIR) pulsed laser is reported. We synthesized methotrexate (MTX)-loaded liposomes based on a modified reverse-phase evaporation method. Gold nanorods (AuNR) were attached to the liposomal surfaces, enabling the liposomes to release drug under short NIR irradiation via the photothermal effect. The concentrations of methotrexate (MTX) released from the liposomes were 10.6, 29.8, 43.7 and 65.9 μg/mL after one, two, three or four NIR laser cycles (1.1 W at 1064 nm, 5 s per cycle), respectively. The current finding will provide possible solution to the previously reported inconsistency in drug release from light activatable liposomal drug carriers at each activation cycle. The repeatability of drug release described in this work is believed to be due to reversible nature of the liposomes. The liposomes release drug via lipid bilayer melting when irradiated by laser due to gold nanorods' plasmonic heat on the lipid bilayer surface and quickly regain their original structure once the laser source is removed. We provided evidence of the reversible liposomal structures by monitoring the change of number densities of liposomes using a microelectrode sensor with different laser irradiation durations and powers. We also assessed the micron-sized liposome with respect to long-term stability, drug encapsulation efficiency, and drug-releasing efficiency, demonstrating the possibility of utilizing these liposomes as long-term drug delivery vehicles for various drugs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

There is little reported on the efficacy and safety of direct oral anticoagulants (DOACs) in morbid obesity after venous thromboembolism (VTE). In this observational study, patients were followed up after intermediate- or high-risk pulmonary embolism (PE) at the University of Rochester Pulmonary Hypertension Clinic 2-4 months after the initial event. All patients had echocardiography and V/Q imaging regardless of symptoms. Outcomes of interest were the rates of recurrent VTE, thrombus resolution and development of chronic thromboembolic pulmonary hypertension (CTEPH) in patients with morbid obesity treated with a DOAC compared to treatment with vitamin K antagonists and to non-morbidly obese patients after PE. Using the electronic medical record, recurrent events were assessed up to 12 months after the event. 107 patients (body mass index (BMI)>40 kg·m -2 , n=32; BMI 30-39.9 kg·m -2 , n=39; BMI<30 kg·m -2 , n=36) attended follow-up appointments after treatment for PE. A DOAC was used in 70 patients (BMI>40 kg·m -2 , n=19; BMI 30-39.9 kg·m -2 , n=27; BMI<30 kg·m -2 , n=24). There were no recurrent events within the first 12 months of initial diagnosis based on symptoms and imaging in any patient. There was no difference in rate of residual unmatched perfusion defect with DOACs or conventional anticoagulation (49% versus 49%). This finding remained in the subset of morbidly obese patients (47% versus 50%). For the overall cohort, there was no difference in the rate of CTEPH development based on anticoagulation with a DOAC (5% versus 8% with warfarin). There were no major bleeding complications with a DOAC. DOAC therapy appears to be effective and safe in morbid obesity even after intermediate- or high-risk PE. ​., Competing Interests: Conflict of interest: D.J. Lachant has nothing to disclose. Conflict of interest: C. Bach has nothing to disclose. Conflict of interest: A. Fe has nothing to disclose. Conflict of interest: R.J. White has nothing to disclose. Conflict of interest: N.A. Lachant has nothing to disclose., (Copyright ©ERS 2021.)

Right ventricular (RV) function is a predictor of outcomes in pulmonary arterial hypertension (PAH). The 6-min walk test (6MWT) is likely an indirect measure of RV function during exercise, but changes in absolute walk distance can also be influenced by factors like effort and musculoskeletal disease. Paired 6MWT with continuous electrocardiogram monitoring was performed in stable PAH patients, patients adding PAH therapies, and healthy controls. Heart rate expenditure (HRE) was calculated (integrating pulse during 6MWT) and then divided by walk distance (HRE/d). We also evaluated changes in peak heart rate, time above age-adjusted maximum predicted heart rate, and heart rate at 6 min. HRE/d was compared to invasive hemodynamic measures in patients who had right heart catheterization performed within seven days, WHO functional class assessment, and Emphasis 10 questionnaire. We measured two 6MWT in 15 stable PAH patients, 13 treatment intensification patients, and 8 healthy controls. HRE/d was reproducible in the stable PAH group (median difference, -0.79%), while it decreased (median difference, 23%, p = 0.0001) after adding vasodilator therapy. In 11 patients with right heart catheterization, HRE/d correlated strongly with stroke volume, r  = -0.72, p  = 0.01. Peak heart rate decreased after adding vasodilator therapy. HRE/d also correlated with WHO functional class and Emphasis 10 score. Continuous heart rate monitoring during 6MWT provides valuable physiologic data accounting for effort. HRE/d appears to enhance test reproducibility in stable patients while detecting change after adding therapy as compared to walk distance alone., Competing Interests: Conflict of interest: The author(s) declare that there is no conflict of interest., (© The Author(s) 2020.)

Objective: Connective tissue disease (CTD)-associated pulmonary arterial hypertension (PAH) is the second most common etiology of PAH and carries a poor prognosis. Recently, it has been shown that female human tumor necrosis factor (TNF)-transgenic (Tg) mice die of cardiopulmonary disease by 6 months of age. This study was undertaken to characterize this pathophysiology and assess its potential as a novel model of CTD-PAH., Methods: Histologic analysis was performed on TNF-Tg and wild-type (WT) mice to characterize pulmonary vascular and right ventricular (RV) pathology (n = 40 [4-5 mice per group per time point]). Mice underwent right-sided heart catheterization (n = 29) and micro-computed tomographic angiography (n = 8) to assess vascular disease. Bone marrow chimeric mice (n = 12), and anti-TNF-treated mice versus placebo-treated mice (n = 12), were assessed. RNA sequencing was performed on mouse lung tissue (n = 6)., Results: TNF-Tg mice displayed a pulmonary vasculopathy marked by collagen deposition (P < 0.001) and vascular occlusion (P < 0.001) with associated RV hypertrophy (P < 0.001) and severely increased RV systolic pressure (mean ± SD 75.1 ± 19.3 mm Hg versus 26.7 ± 1.7 mm Hg in WT animals; P < 0.0001). TNF-Tg mice had increased α-smooth muscle actin (α-SMA) staining, which corresponded to proliferation and loss of von Willebrand factor (vWF)-positive endothelial cells (P < 0.01). There was an increase in α-SMA-positive, vWF-positive cells (P < 0.01), implicating endothelial-mesenchymal transition. Bone marrow chimera experiments revealed that mesenchymal but not bone marrow-derived cells are necessary to drive this process. Treatment with anti-TNF therapy halted the progression of disease. This pathology closely mimics human CTD-PAH, in which patient lungs demonstrate increased TNF signaling and significant similarities in genomic pathway dysregulation., Conclusion: The TNF-Tg mouse represents a novel model of CTD-PAH, recapitulates key disease features, and can serve as a valuable tool for discovery and assessment of therapeutics., (© 2020, American College of Rheumatology.)

Oxidative stress and inflammation play key roles in development of pulmonary arterial hypertension (PAH). We previously reported that an endothelial cell (EC)-specific cyclophilin A overexpression mouse developed many characteristics of PAH. In other models of cardiovascular disease, cyclophilin A stimulates smooth muscle proliferation and vascular inflammation, but mechanisms responsible for PAH have not been defined. In particular, the contribution of endothelial-to-mesenchymal transition in cyclophilin A-mediated PAH has not been studied. We identified increased levels of cyclophilin A in endothelial and neointimal cells of pulmonary arteries in patients with PAH and animal pulmonary hypertension models. In the EC-specific cyclophilin A overexpression mouse that exhibited features characteristic of PAH, lineage tracing showed high level expression of mesenchymal markers in pulmonary ECs. A significant number of mesenchymal cells in media and perivascular regions of pulmonary arterioles and alveoli were derived from ECs. Pulmonary ECs isolated from these mice showed phenotypic changes characteristic of endothelial-to-mesenchymal transition in culture. Cultured pulmonary ECs stimulated with extracellular cyclophilin A and acetylated cyclophilin A demonstrated functional changes associated with endothelial-to-mesenchymal transition such as increased cytokine release, migration, proliferation, and mitochondrial dysfunction. Acetylated cyclophilin A stimulated greater increases for most features of endothelial-to-mesenchymal transition. In conclusion, extracellular cyclophilin A (especially acetylated form) contributes to PAH by mechanisms involving increased endothelial-to-mesenchymal transition, cytokine release, EC migration, proliferation, and mitochondrial dysfunction; strengthening the basis for studying cyclophilin A inhibition as a therapy for PAH.

Background: Thrombotic disease complicates severe SARS-CoV-2 infection and is associated with increased morbidity and mortality. Various anticoagulation strategies have been evaluated in hospitalized patients to prevent complications. The impact of chronic anticoagulation before SARS-CoV-2 infection on the risk for subsequent thrombosis has not been systematically studied., Methods: This was a retrospective single-center study. All patients with positive SARS-CoV-2 PCR testing from March 13, 2020, through May 6, 2020, at the University of Rochester Medical Center were identified. We included all patients receiving therapeutic anticoagulation for at least 1 month before COVID diagnosis. We documented the rate of thrombotic complications, type of anticoagulation, bleeding complications, and mortality., Results: A total of 107 SARS-CoV2-infected patients were chronically anticoagulated before SARS-CoV-2 testing with a median age of 78. Of those, 42 required hospital admission, with 17 requiring intensive care. No patients, inpatient or outpatient, were diagnosed with a new symptomatic thrombotic complication. Three patients had minor bleeding in the hospital. Thirteen (12%) patients died (69% male)., Conclusion: Our uncontrolled findings suggest that chronic anticoagulation at the time of infection may protect against thrombotic complications and decrease disease severity., (© 2020 International Society on Thrombosis and Haemostasis.)

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology., Competing Interests: The authors have declared that no competing interests exist.

This article on clinical trial design incorporates the broad experience of members of the Pulmonary Vascular Research Institute's (PVRI) Innovative Drug Development Initiative (IDDI) as an open debate platform for academia, the pharmaceutical industry and regulatory experts surrounding the future design of clinical trials in pulmonary hypertension. It is increasingly clear that the design of phase 2 and 3 trials in pulmonary hypertension will have to diversify from the traditional randomised double-blind design, given the anticipated need to trial novel therapeutic approaches in the immediate future. This article reviews a wide range of differing approaches and places these into context within the field of pulmonary hypertension., (© The Author(s) 2020.)

Background: Squamous cell carcinoma (SCC) of larynx is a common head and neck cancer. For cases that are node negative, the role of definitive concurrent chemoradiation is unclear and not supported by guidelines but used at provider discretion. To address this knowledge gap, we examined the oncological outcomes with additional chemotherapy and factors correlated with the chemotherapy administration., Methods: We queried the National Cancer Database for patients with early stage (T2N0M0) laryngeal SCC treated nonsurgically. Multivariable logistic regression identified predictors of chemotherapy. Multivariable Cox regression evaluated predictors of survival. Propensity matching accounted for indication biases., Results: We identified 7181 patients meeting the eligibility criteria, of which 1568 (22%) patients received chemotherapy in addition to radiation. Predictors of chemotherapy use included younger age, Caucasian race, more remote year of treatment, higher grade, sites other than glottis, treatment at a community cancer center, and use of intensity-modulated radiation therapy. Median overall survival was not significantly different in the two arms analyzed-65 months (95% confidence interval [CI] 60, 72months) with chemotherapy compared to 70 months without chemotherapy (95% CI 66, 75 months, P <.37). Predictors for survival on propensity-matched multivariable analysis were increased age, male sex, less education, lower income, higher comorbidity score, receipt of treatment at a community center, and nonglottic sites., Conclusions: This study shows no clear survival benefit with chemotherapy in early stage disease. Although this implies that chemotherapy should not be routinely delivered, individualized judgment and prospective studies are recommended as the biology behind this interesting finding is undefined., Level of Evidence: 2C (Outcomes Research)., Competing Interests: The authors declare no potential conflict of interest., (© 2019 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals, Inc. on behalf of The Triological Society.)

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling., (Copyright © 2019 Jurkiewicz et al.)

Pulmonary arterial hypertension (PAH) is a female predominant disease in which progressive vascular remodeling and vasoconstriction result in right ventricular (RV) failure and death. Most PAH patients utilize multiple therapies. In contrast, the majority of preclinical therapeutic studies are performed in male rats with a single novel drug often markedly reversing disease in the model. We sought to differentiate single drug therapy from combination therapy in female rats with severe disease. One week after left pneumonectomy, we induced PH in young female Sprague-Dawley rats with an injection of monocrotaline (45 mg/kg). Female rats were then randomized to receive combination therapy (ambrisentan plus tadalafil), ambrisentan monotherapy, tadalafil monotherapy, or vehicle. We measured RV size and function on two serial echocardiograms during the development of disease. We measured RV systolic pressure (RVSP) invasively at day 28 after monocrotaline before analyzing the vascular volume with microcomputed tomography (microCT) of the right middle lobe. RVSP was significantly lower in female rats treated with combination therapy, and combination therapy resulted in increased small vessel volume density measured by microCT compared with untreated rats. Combination-treated rats had the smallest RV end-diastolic diameter on echocardiogram as compared with the other groups. In summary, we report a female model of pulmonary hypertension that can distinguish between one and two drug therapies; this model may facilitate better preclinical drug testing for novel compounds.

Background: Initial combination therapy with ambrisentan and tadalafil reduced the risk of clinical failure events for treatment-naïve participants with pulmonary arterial hypertension (PAH) as compared to monotherapy. Previous studies in PAH have demonstrated greater treatment benefits in more symptomatic participants., Methods: AMBITION was an event-driven, double-blind study in which participants were randomized 2:1:1 to once-daily initial combination therapy with ambrisentan 10 mg plus tadalafil 40 mg, ambrisentan 10 mg plus placebo, or tadalafil 40 mg plus placebo. In this pre-specified subgroup analysis, we compared the efficacy data between those with functional class (FC) II vs. FC III symptoms at baseline., Results: This analysis included 500 participants in the previously defined primary analysis set (n = 155 FC II, n = 345 FC III). Comparing combination therapy to pooled monotherapy, the risk of clinical failure events was reduced by 79% (hazard ratio, 0.21 [95% confidence interval: 0.071, 0.63]) for FC II patients and 42% (hazard ratio, 0.58 [95% confidence interval: 0.39, 0.86]) for FC III patients. In a post-hoc analysis, the risk of first hospitalization for worsening PAH was also reduced by combination therapy, particularly for FC II patients (0 combination vs. 11 [14%] pooled monotherapy). Adverse events were frequent but comparable between the subgroups., Conclusions: Treatment benefit from initial combination therapy appeared at least as great for FC II as for FC III participants. Hospitalizations for worsening PAH were not observed in FC II participants assigned to combination. The present data support an initial combination strategy for newly diagnosed patients even when symptoms are less severe. Funded by Gilead Sciences, Inc. and GlaxoSmithKline; AMBITION ClinicalTrials.gov number, NCT01178073.

Importance: Primary Adenocarcinoma of the anus is a rare disease with a poor prognosis and thus tends to have a more aggressive treatment algorithm, typically involving a surgical approach. Prior to 2001, a few retrospective studies outlined improved outcomes with the incorporation of surgery with chemoradiation. However, since the publication of these studies, advancement in radiotherapy modalities and imaging have left the question of improved outcomes while reserving surgery for salvage., Objective: We conducted this National Cancer Database (NCDB)-driven retrospective study to analyze treatment trends and outcomes in the current time from 2004 to 2015 with respect to chemoradiation and surgery., Design: Retrospective NCDB tumor registry data review-using propensity score-adjusted multivariable analyses for survival., Setting: Database review., Participants: We selected for patients listed in the NCDB with AJCC stage 1-3 anal adenocarcinoma diagnosed between 2004 and 2015 and selected out patients with undocumented/stage 4 disease, those with radiation outside the pelvis, not treated with systemic therapy and patients lost to follow-up., Exposure(s): None., Main Outcomes and Measures: Overall survival and use of surgery in the up-front management of anal adenocarcinoma., Results: Of the 1729 patients eligible in this study, 1028 were treated with surgery as up-front management and 701 had definitive chemoradiation. Median overall survival for all patients was 55 months with a 5-year survival rate of 55%. Patients treated without surgery had worse overall survival, median survival of 45 months compared to 87 months (P < 0.0001) with 5-year survival rates of 42% and 55% in favor of incorporation of surgery. Analysis across patients treated with surgery alone, surgery followed by adjuvant chemoradiation, neoadjuvant chemoradiation followed by surgery, and chemoradiation alone had median survival rates of 78, 83, 92, and 46 months, respectively. Propensity score-adjusted multivariable analysis identified older age, grade 3, high comorbidity score, and lack of surgery as predictive of worse outcome., Conclusions and Relevance: The results of the NCDB analysis indicate improved overall survival with the incorporation of surgery into the initial management of anal adenocarcinoma when compared to chemoradiation alone, despite the omission of surgery in up to 50% of the cases logged. Our results corroborate earlier studies published prior to the year 2000 for surgery to be included in the definitive management of anal adenocarcinoma., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The one-pot conversion of glucose to ethyl levulinate over an acid-functionalised hydrothermal catalyst (derived from glucose) provides high initial yields up to 37 mol%, comparable to the homogeneous H 2 SO 4 catalyst, whilst catalyst performance is strongly influenced by green solvent choice., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)