Showing papers by "Vanderbilt University published in 2023"

COVID-19 Diagnostic Methods and Detection Techniques

Abstract: Coronavirus disease 2019 (COVID-19) is an emerging human-to-human infectious disease that broke out in early December 2019 and threatens global public health, causing widespread concern. This respiratory disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The development of rapid and reliable techniques for COVID-19 diagnosis is a significant step to prevent further infections. Combinations of genome sequencing, nucleic acid molecular testing, clustered regularly interspaced short palindromic repeats editing technology, antigen/antibody detection, and computed tomography imaging have been implemented to identify and screen COVID-19 infections. Moreover, other new diagnosis methods such as dried blood spots and biosensors are being developed and are summarized here. This manuscript reviews currently available methods for SARS-CoV-2 detection with the aim of helping researchers develop timely and effective technologies to detect this emerging virus and its variants.

Systematic Transmission Electron Microscopy‐Based Identification and 3D Reconstruction of Cellular Degradation Machinery

Abstract: Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer-scale micrographs of cellular degradation components in a fixed sample. Serial block face-scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

Hyperaminoacidemia induces pancreatic α cell proliferation via synergism between the mTORC1 and CaSR-Gq signaling pathways

Abstract: Glucagon has emerged as a key regulator of extracellular amino acid (AA) homeostasis. Insufficient glucagon signaling results in hyperaminoacidemia, which drives adaptive proliferation of glucagon-producing α cells. Aside from mammalian target of rapamycin complex 1 (mTORC1), the role of other AA sensors in α cell proliferation has not been described. Here, using both genders of mouse islets and glucagon receptor (gcgr)-deficient zebrafish (Danio rerio), we show α cell proliferation requires activation of the extracellular signal-regulated protein kinase (ERK1/2) by the AA-sensitive calcium sensing receptor (CaSR). Inactivation of CaSR dampened α cell proliferation, which was rescued by re-expression of CaSR or activation of Gq, but not Gi, signaling in α cells. CaSR was also unexpectedly necessary for mTORC1 activation in α cells. Furthermore, coactivation of Gq and mTORC1 induced α cell proliferation independent of hyperaminoacidemia. These results reveal another AA-sensitive mediator and identify pathways necessary and sufficient for hyperaminoacidemia-induced α cell proliferation.

Peptide Triazole Inhibitors of HIV-1: Hijackers of Env Metastability

Abstract: With 1.5 million new infections and 690,000 AIDS-related deaths globally each year, HIV- 1 remains a pathogen of significant public health concern. Although a wide array of effective antiretroviral drugs have been discovered, these largely target intracellular stages of the viral infectious cycle, and inhibitors that act at or before the point of viral entry still require further advancement. A unique class of HIV-1 entry inhibitors, called peptide triazoles (PTs), has been developed, which irreversibly inactivates Env trimers by exploiting the protein structure's innate metastable nature. PTs, and a related group of inhibitors called peptide triazole thiols (PTTs), are peptide compounds that dually engage the CD4 receptor and coreceptor binding sites of Env's gp120 subunit. This triggers dramatic conformational rearrangements of Env, including the shedding of gp120 (PTs and PTTs) and lytic transformation of the gp41 subunit to a post-fusion-like arrangement (PTTs). Due to the nature of their dual receptor site engagement, PT/PTT-induced conformational changes may elucidate mechanisms behind the native fusion program of Env trimers following receptor and coreceptor engagement, including the role of thiols in fusion. In addition to inactivating Env, PTT-induced structural transformation enhances the exposure of important and conserved neutralizable regions of gp41, such as the membrane proximal external region (MPER). PTT-transformed Env could present an intriguing potential vaccine immunogen prototype. In this review, we discuss the origins of the PT class of peptide inhibitors, our current understanding of PT/PTT-induced structural perturbations and viral inhibition, and prospects for using these antagonists for investigating Env structural mechanisms and for vaccine development.

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions

Abstract: Abstract Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA (≥50 bp) referred to as copy number variation (CNV). In humans, CNV may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive CNV, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

The Influence of Anti-LGBTQIA+ Legislation on Graduate Medical Education

Abstract: More than 400 state bills that discriminate against lesbian, gay, bisexual, transgender, queer, intersex, asexual, and other sexual and gender diverse identities (LGBTQIA+) are active or have passed in the 2023 US legislative session.1 Across the United States, 70% of state legislatures, largely concentrated in the Midwest and South, are considering at least one anti-LGBTQIA+ policy.1 These bills target the rights of LGBTQIA+ people, who represent over 7% of US adults, in essential areas of life: health care, education, public space, free speech and expression, credit, and federally funded programs.2 Examples include censoring of LGBTQIA+ content in education and media, restricting transgender students from participating in school activities, prohibiting individuals from using public bathrooms concordant with their gender identity, criminalizing gender-affirming care, allowing religious exemptions for discrimination against LGBTQIA+ persons, and restricting free speech and gender expression.1 Accordingly, patients living in affected regions face outright discrimination in everyday life and are fearful about their access to health care.3-6 Effects of anti-LGBTQIA+ legislation are relevant not only to patients but also to the health care workforce. Residents are an especially vulnerable population given their clinical and educational responsibilities and reporting roles.7 In response to the evolving sociopolitical landscape, we aim to describe the influence of anti-LGBTQIA+ legislation on graduate medical education (GME) and offer solutions for GME leaders to support their trainees and to advocate for change.Educators and academic institutions should not underestimate the consequences and insidiousness of anti-LGBTQIA+ legislation. While it may seem that efforts to oppress LGBTQIA+ people affect only a minority of the workforce, most LGBTQIA+-identifying residents are not “out” to their peers, mentors, and supervisors.8 Meanwhile, physicians who do not identify as LGBTQIA+ will have patients, children, other family members, and friends who are affected by these policies. The constitutional ambiguity of many of these laws leaves enforcement at the discretion of subjective and bias-prone policing. For example, the Tennessee Senate Bill 0003 regarding “male or female impersonators” can be interpreted to broadly outlaw gender expression that is discordant from one's sex assigned at birth.9 Therefore, a resident assigned male at birth who wears traditionally feminine clothing, such as heels or a cropped shirt, could be charged with a misdemeanor or felony—risking their future medical license and hospital credentials. Such expression is part of the rich history and culture belonging to the LGBTQIA+ community. Because free speech and expression are protected US rights, it is unacceptable for residents to sacrifice their identity for their training and careers.Bills aimed at silencing LGBTQIA+ voices and advocacy in education are also highly relevant to GME. Florida's proposed House Bill 999 law would force postsecondary institutions to restrict funding for and remove LGBTQIA+ affinity organizations, and any majors, minors, or concentrations related to diversity, equity, and inclusion.10 Furthermore, restrictions on LGBTQIA+-related health care, including bans on gender-affirming care, require programs to offer alternative training opportunities, such as simulations or clinical experiences in other states, for trainees in various medical (eg, primary care, endocrinology, infectious disease) and surgical (eg, urology, plastic surgery) specialties.Although highly rewarding, practicing medicine is a high pressure, often stressful occupation that changes continuously in response to patient and societal expectations. In recent years, attention on physician mental health has increased with efforts underway to improve well-being throughout medical training.11 Despite these efforts, mental illness is pervasive in health care—nearly one-third of physicians have clinically significant depressive symptoms, and suicide risk is at least 3 times greater than the general population.12 Amid anti-LGBTQIA+ legislation, LGBTQIA+ residents face stressors associated with not only their physician roles, but also their gender and sexual identities. As a result of mistreatment and stigmatization in society, the risk of suicide in these individuals is 4 times higher than the general population.13 For an LGBTQIA+ resident, the intersection of roles and identities amplifies the already unacceptable mental illness burden in the physician population. Studies surveying residents and medical students show that LGBTQIA+ trainees report disparate rates of discrimination, mistreatment, and burnout compared with their heteronormative peers.8 Although we prioritize physician and trainee well-being to improve patient care, attrition and burnout are also financially devastating for health care institutions. A recent analysis found the annual cost of turnover and reduced clinical hours associated with burnout was approximately $7,600 per employed physician for an estimated total of $4.6 billion nationally.14 These findings demonstrate that supporting LGBTQIA+ residents and improving advocacy for LGBTQIA+ identities in GME have benefits beyond personal and patient care-related outcomes.Lifestyle is increasingly important to senior medical students when choosing residency programs.15 Considering safety and mental health consequences, we expect many LGBTQIA+ medical students will avoid ranking programs in geographies with restrictive and discriminatory policies, such as the Midwest and the South.16 Even without legislation, there are serious threats to the safety of LGBTQIA+ individuals in many states and cities. Surveys show that up to 19% of lesbian women and gay men have been assaulted because of their sexual orientation, 40% have been threatened with physical violence, and 94% have experienced some type of victimization, including verbal abuse.17 Because no studies have examined how anti-LGBTQIA+ policy has influenced the Match process in recent years, further research should investigate whether LGBTQIA+ medical students are disproportionately more likely to avoid certain specialties or remain unmatched as a result of limited acceptable positions. This reality is harmful to students who are thus restricted in rank options, patients who deserve and benefit from a diverse health care workforce, and programs that seek to attract diverse and talented residents and fellows to advance their specialties. The consequences amplify an already limited pipeline to the diversification of faculty within academia and GME leadership positions. GME offices and program directors should consider several institutional- and systems-level actions, as the rights of their LGBTQIA+ workforce are increasingly threatened.Faculty mentoring is important for trainee wellness, especially as part of a structured program.18 By providing residents an opportunity to form longitudinal relationships with faculty, a safety net of community support within the residency program is created that can extend beyond the clinical environment.19 Some mentor-trainee relationships are substantially strengthened by concordant interests and identities; however, it may be difficult for LGBTQIA+ trainees to find a faculty mentor with similar identities and experiences. Active efforts should be made to recruit diverse and inclusive faculty who can offer this mentorship and to train allies to bolster this mentorship. Additionally, residents may feel less vulnerable and more supported if there is representation of LGBTQIA+ identities in program and institutional leadership. Faculty members are better equipped to educate peers, leaders, and trainees about the LGBTQIA+ experience in ways residents cannot in their subordinate roles. Although legislation in some states targets the formation of affinity groups for people with LGBTQIA+ identities, creating an institutionally backed organization to give a voice to LGBTQIA+ residents and faculty should be prioritized wherever possible. Finally, our institution developed a 1-year fellowship program in LGBTQIA+ health to provide focused clinical expertise, research experience, and leadership training.Unfortunately, the politicization of LGBTQIA+ rights has created several barriers to supporting LGBTQIA+ trainees.20 To remain apolitical or maintain certain partnerships, health care institutions may avoid committing to a formal stance on anti-LGBTQIA+ legislation, especially in states where these policies are strongly promoted by state leaders. However, GME offices that serve as liaisons between residents and the medical center administration have a responsibility to optimize the safety and well-being of residents and patients—all of whom are threatened by discriminatory policies. This is not a political issue but rather a public health concern.Residents should be offered a reasonable amount of protected time to participate in advocacy activities and speak out about these issues. For example, legislators and activist organizations often invite medical trainees and other physicians to serve as experts in discussions of new policy. LGBTQIA+ trainees should be encouraged to form partnerships with local community organizations, which can be mutually beneficial through the exchange of resources and support. Moreover, trainees who are targeted by extremists because of their identity or advocacy should be connected with resources, such as institutional security, to ensure that they feel safe.21When visiting rotations and in-person interviews were limited during the COVID-19 pandemic, residency and fellowship applicants relied heavily on online information about GME programs. Recent studies show that content about diversity, equity, and inclusion on program websites is often absent or provides little useful information about the program or GME office's commitment to sponsoring trainees who identify as LGBTQIA+.22,23 Updating these resources to include efforts by GME to protect and empower LGBTQIA+ residents may help attract residents and foster community. Allocating funds for visiting student rotations, research, or other experiential learning opportunities at the institution should also be considered.22To achieve its mission to accredit programs that train physicians who will provide high-quality care to patients, the Accreditation Council for Graduate Medical Education (ACGME) uses several outcome measures for program evaluation. These measures include the assessment of resident well-being, workforce diversity, engagement in quality improvement and patient safety, and recruitment and retention.24 While some metrics may be objective, most measures are subjective, with programs having the ability to self-assess their achievement in reaching their aims. Accordingly, the ACGME should encourage GME stakeholders to develop, evaluate, and implement evidence-based performance metrics to assess programs in efforts to improve the quality of life for LGBTQIA+ people and other marginalized communities. If performance measures are determined, implementation through the ACGME Common Program Requirements and/or specialty-/subspecialty-specific Program Requirements are potential next steps. Supporting programs and GME institutions in improving the quality of life for LGBTQIA+ residents and fellows is an essential goal.In addition, the ACGME should ensure that relevant training in LGBTQIA+ health is included as appropriate within program curricula (eg, sexual health, gender-affirming hormone therapy and surgery, expected radiological findings). Similar to obstetrics and gynecology programs in areas where abortion is banned that send residents to pursue rotations at other institutions, the same strategy may be necessary for gender-affirming care in many states.25There have been discussions and proposals for increased transparency in GME funding, with calls for shifts in how funds are allocated to improve workforce diversity and health equity.26 GME programs are supported using federal, state, and independent funds. However, the primary source of funding is from the Centers for Medicare & Medicaid Services, which provides direct GME payments to cover direct teaching costs (eg, resident and faculty salaries) and indirect GME adjustments, which are designed to support teaching hospitals in providing care and learning opportunities in higher cost settings and to underserved populations.26 With the implementation of performance-based metrics for GME programs, funding might be allocated, as appropriate, to support institutions and programs that are actively working to dispel misinformation about LGBTQIA+ health, promote inclusive attitudes and health equity in the community, and oppose anti-LGBTQIA+ legislation. Such activities could have powerful, lasting effects to improve public health.National mandates for employee assistance programs for LGBTQIA+ trainees are needed, particularly in states with restrictive laws. LGBTQIA+ populations experience a disproportionate burden of mental illness and minority stress, and anti-LGBTQIA+ policy and attitudes have been shown to exacerbate these disparities.27 A healthy workforce will require access to confidential counseling and resources for personal and professional challenges.The rights of LGBTQIA+ people, which include both those of our workforce and our patients, should not be a partisan issue but rather a public health concern. As state legislatures introduce laws that seek to oppress LGBTQIA+ individuals, resident and fellow safety and quality of life are threatened, which affects recruitment, retention, and patient care. In addition to advocating for federal nondiscrimination protections, GME leaders and faculty have a duty to create learning environments in which all trainees have an equal opportunity to thrive. Such efforts may need to extend beyond medical centers to community, state, and national advocacy.

How Worried Should We Be? The Implications of Fabricated Survey Data for Political Science

Abstract: Abstract Surveys are ubiquitous in the study of politics, making enumerator fabrication a critical issue. A prevailing view is that faked interviews affect inferences drawn from compromised datasets. Researchers have generated theories about how fabrication might affect inferences. Yet, speculation has outpaced systematic testing. We leverage a rare dataset to address this gap: a national face-to-face survey in Venezuela in which a uniquely high volume of falsified interviews was detected, canceled, and replaced. Comparing the verified and fraudulent datasets, we find that descriptive inference is sometimes affected, but correlational results hold, even in a dataset with an unusually high-fabrication rate. Enumerators largely fabricate plausible data. Though still egregious, enumerator fabrication may not constitute a grave threat to political science research.

Analysis of genetically determined gene expression suggests role of inflammatory processes in exfoliation syndrome

Abstract: Abstract Background Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22–25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS. Results Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22–25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases. Conclusion Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.

Sometimes Less Is More: When Aggregating Networks Masks Effects

Abstract: A large body of research aims to detect the spread of something through a social network. This research often entails measuring multiple kinds of relationships among a group of people and then aggregating them into a single social network to use for analysis. The aggregation is typically done by taking a union of the various tie types. Although this has intuitive appeal, we show that in many realistic cases, this approach adds sufficient error to mask true network effects. We show that this can be the case, and demonstrate that the problem depends on: (1) whether the effect diffuses generically or in a tie-specific way, and (2) the extent of overlap between the measured network ties. Aggregating ties when diffusion is tie-specific and overlap is low will negatively bias and potentially mask network effects that are in fact present.

Is relativistic hydrodynamics always symmetric-hyperbolic in the linear regime?

Abstract: Close to equilibrium, the kinetic coefficients of a thermodynamic system must satisfy a set of symmetry conditions, which follow from the Onsager-Casimir principle. Here, we show that, if a system of hydrodynamic equations is analyzed from the perspective of the Onsager-Casimir principle, then it is possible to impose very strong symmetry conditions also on the principal part of such equations (the part with highest derivatives). In particular, we find that, in the absence of macroscopic magnetic fields and spins, relativistic hydrodynamics should always be symmetric-hyperbolic, when linearized about equilibrium. We use these results to prove that Carter's multifluid theory and the Israel-Stewart theory in the pressure frame are both symmetric-hyperbolic in the linear regime. Connections with the GENERIC formalism are also explored.

<scp>High‐Throughput</scp> Computational Investigation of Protein Electrostatics and Cavity for <scp>SAM‐Dependent</scp> Methyltransferases

Abstract: S-adenosyl methionine (SAM) -dependent methyl transferases (MTases) are a ubiquitous class of enzymes catalyzing dozens of essential life processes. Despite targeting a large space of substrates with diverse intrinsic reactivity, SAM MTases have similar catalytic efficiency. While understanding of MTase mechanism has grown tremendously through integration of structural characterization, kinetic assays, and multiscale simulations, it remains elusive regarding how these enzymes have evolved to fit the diverse chemical needs of their respective substrates. In this work, we performed a high-throughput computational analysis of 91 SAM MTases to better understand how the properties (i.e., electric field strength and active site volumes) of SAM MTases help achieve similar catalytic efficiency towards substrates of different reactivity. We found that electric field strengths have largely adjusted to make the target atom a better methyl acceptor. For MTases that target RNA/DNA- and histone protein, our results suggest that electric field strength accommodates formal hybridization state and variation in cavity volume trends with diversity of substrate classes. Metal ions in SAM MTases contribute negatively to electric field strength for methyl donation and enzyme scaffolds tend to offset these contributions. This article is protected by copyright. All rights reserved.

Sex modifies the effect of genetic risk scores for polycystic ovary syndrome on metabolic phenotypes

Abstract: Females with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women, have an increased risk of developing cardiometabolic disorders such as insulin resistance, obesity, and type 2 diabetes (T2D). While only diagnosable in females, males with a family history of PCOS can also exhibit a poor cardiometabolic profile. Therefore, we aimed to elucidate the role of sex in the cardiometabolic comorbidities observed in PCOS by conducting bidirectional genetic risk score analyses in both sexes. We first conducted a phenome-wide association study (PheWAS) using PCOS polygenic risk scores (PCOS PRS ) to identify potential pleiotropic effects of PCOS PRS across 1,380 medical conditions recorded in the Vanderbilt University Medical Center electronic health record (EHR) database, in females and males. After adjusting for age and genetic ancestry, we found that European (EUR)-ancestry males with higher PCOS PRS were significantly more likely to develop hypertensive diseases than females at the same level of genetic risk. We performed the same analysis in an African (AFR)-ancestry population, but observed no significant associations, likely due to poor trans-ancestry performance of the PRS. Based on observed significant associations in the EUR-ancestry population, we then tested whether the PRS for comorbid conditions (e.g., T2D, body mass index (BMI), hypertension, etc.) also increased the odds of a PCOS diagnosis. Only BMI PRS and T2D PRS were significantly associated with a PCOS diagnosis in EUR-ancestry females. We then further adjusted the T2D PRS for measured BMI and BMI residual (regressed on the BMI PRS and enriched for the environmental contribution to BMI). Results demonstrated that genetically regulated BMI primarily accounted for the relationship between T2D PRS and PCOS. Overall, our findings show that the genetic architecture of PCOS has distinct sex differences in associations with genetically correlated cardiometabolic traits. It is possible that the cardiometabolic comorbidities observed in PCOS are primarily explained by their shared genetic risk factors, which can be further influenced by biological variables including sex and BMI.

N-oxide ligands for selective separations of lanthanides: insights from computation

Abstract: N-oxide ligands, combined with pyridinic N groups, are predicted to offer attractive La( iii )/Ln( iii ) selectivities for rare-earth separations.

The yeast <scp>ALA</scp> synthase C‐terminus positively controls enzyme structure and function

Abstract: 5-Aminolevulinic acid synthase (ALAS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of heme biosynthesis in α-proteobacteria and several non-plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C-terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C-terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C-terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C-terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel β-sheet important to Fold-Type I PLP-dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C-terminus has a homolog-specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.

Data from PI3K Inhibition Restores and Amplifies Response to Ruxolitinib in Patients with Myelofibrosis

Abstract: <div>AbstractPurpose:<p>Treatment options are limited beyond JAK inhibitors for patients with primary myelofibrosis (MF) or secondary MF. Preclinical studies have revealed that PI3Kδ inhibition cooperates with ruxolitinib, a JAK1/2 inhibitor, to reduce proliferation and induce apoptosis of <i>JAK2</i>^V617F-mutant cell lines.</p>Patients and Methods:<p>In a phase I dose-escalation and -expansion study, we evaluated the safety and efficacy of a selective PI3Kδ inhibitor, umbralisib, in combination with ruxolitinib in patients with MF who had a suboptimal response or lost response to ruxolitinib. Enrolled subjects were required to be on a stable dose of ruxolitinib for ≥8 weeks and continue that MTD at study enrollment. The recommended dose of umbralisib in combination with ruxolitinib was determined using a modified 3+3 dose-escalation design. Safety, pharmacokinetics, and efficacy outcomes were evaluated, and spleen size was measured with a novel automated digital atlas.</p>Results:<p>Thirty-seven patients with MF (median age, 67 years) with prior exposure to ruxolitinib were enrolled. A total of 2 patients treated with 800 mg umbralisib experienced reversible grade 3 asymptomatic pancreatic enzyme elevation, but no dose-limiting toxicities were seen at lower umbralisib doses. Two patients (5%) achieved a durable complete response, and 12 patients (32%) met the International Working Group-Myeloproliferative Neoplasms Research and Treatment response criteria of clinical improvement. With a median follow-up of 50.3 months for censored patients, overall survival was greater than 70% after 3 years of follow-up.</p>Conclusions:<p>Adding umbralisib to ruxolitinib in patients was well tolerated and may resensitize patients with MF to ruxolitinib without unacceptable rates of adverse events seen with earlier generation PI3Kδ inhibitors. Randomized trials testing umbralisib in the treatment of MF should be pursued.</p></div>

PI3K Inhibition Restores and Amplifies Response to Ruxolitinib in Patients with Myelofibrosis

Abstract: Treatment options are limited beyond JAK inhibitors for patients with primary myelofibrosis (MF) or secondary MF. Preclinical studies have revealed that PI3Kδ inhibition cooperates with ruxolitinib, a JAK1/2 inhibitor, to reduce proliferation and induce apoptosis of JAK2V617F-mutant cell lines.In a phase I dose-escalation and -expansion study, we evaluated the safety and efficacy of a selective PI3Kδ inhibitor, umbralisib, in combination with ruxolitinib in patients with MF who had a suboptimal response or lost response to ruxolitinib. Enrolled subjects were required to be on a stable dose of ruxolitinib for ≥8 weeks and continue that MTD at study enrollment. The recommended dose of umbralisib in combination with ruxolitinib was determined using a modified 3+3 dose-escalation design. Safety, pharmacokinetics, and efficacy outcomes were evaluated, and spleen size was measured with a novel automated digital atlas.Thirty-seven patients with MF (median age, 67 years) with prior exposure to ruxolitinib were enrolled. A total of 2 patients treated with 800 mg umbralisib experienced reversible grade 3 asymptomatic pancreatic enzyme elevation, but no dose-limiting toxicities were seen at lower umbralisib doses. Two patients (5%) achieved a durable complete response, and 12 patients (32%) met the International Working Group-Myeloproliferative Neoplasms Research and Treatment response criteria of clinical improvement. With a median follow-up of 50.3 months for censored patients, overall survival was greater than 70% after 3 years of follow-up.Adding umbralisib to ruxolitinib in patients was well tolerated and may resensitize patients with MF to ruxolitinib without unacceptable rates of adverse events seen with earlier generation PI3Kδ inhibitors. Randomized trials testing umbralisib in the treatment of MF should be pursued.

Cytoskeleton and Motors: The Overview

Abstract: The cytoskeleton is an essential housekeeping part of a cell, which consists of several networks of protein polymers. This overview summarizes the content of the articles discussing cytoskeletal components and their functions.

Data-Driven Identification of Dynamic Quality Models in Drinking Water Networks

Abstract: Traditional control and monitoring of water quality in drinking water distribution networks (WDNs) rely on mostly model- or toolbox-driven approaches, where the network topology and parameters are assumed to be known. In contrast, system identification (SysID) algorithms for generic dynamic system models seek to approximate such models using only input-output data without relying on network parameters. The objective of this paper is to investigate SysID algorithms for water quality model approximation. This research problem is challenging due to (1) complex water quality and reaction dynamics; and (2) the mismatch between the requirements of SysID algorithms and the properties of water quality dynamics. In this paper, we present the first attempt to identify water quality models in WDNs using only input-output experimental data and classical SysID methods without knowing any WDN parameters. Properties of water quality models are introduced, the ensuing challenges caused by these properties when identifying water quality models are discussed, and remedial solutions are given. Through case studies, we demonstrate the applicability of SysID algorithms, show the corresponding performance in terms of accuracy and computational time, and explore the possible factors impacting water quality model identification.

Linked fire activity and climate whiplash in California during the early Holocene

Abstract: Recent wildfire activity in semi-arid regions like western North America exceeds the range of historical records. High-resolution paleoclimate archives such as stalagmites could illuminate the link between hydroclimate, vegetation change, and fire activity in pre-anthropogenic climate states beyond the timescale of existing tree-ring records. Here we present an analysis of levoglucosan, a combustion-sensitive anhydrosugar, and lignin oxidation products (LOPs) in a stalagmite from White Moon Cave in the California Coast Range in order to reconstruct fire activity and vegetation composition across the 8.2 kyr event. Elevated levoglucosan concentrations suggest increased fire activity while altered LOP compositions indicate a shift toward more woody vegetation during the event, with the shift in vegetation preceding the increase in fire activity. These changes are concurrent with increased hydroclimate volatility as shown by carbon and calcium isotope proxies. Together, these records suggest that climate whiplash (oscillations between extreme wetness and aridity) and fire activity in California, both projected to increase with anthropogenic climate change, were tightly coupled during the early Holocene.

Data from An Active Learning Framework Improves Tumor Variant Interpretation

Abstract: <div>Abstract<p>For precision medicine to reach its full potential for treatment of cancer and other diseases, protein variant effect prediction tools are needed to characterize variants of unknown significance (VUS) in a patient's genome with respect to their likelihood to influence treatment response and outcomes. However, the performance of most variant prediction tools is limited by the difficulty of acquiring sufficient training and validation data. To overcome these limitations, we applied an iterative active learning approach starting from available biochemical, evolutionary, and functional annotations. With active learning, VUS that are most challenging to classify by an initial machine learning model are functionally evaluated and then reincorporated with the phenotype information in subsequent iterations of algorithm training. The potential of active learning to improve variant interpretation was first demonstrated by applying it to synthetic and deep mutational scanning datasets for four cancer-relevant proteins. The utility of the approach to guide interpretation and functional validation of tumor VUS was then probed on the nucleotide excision repair (NER) protein xeroderma pigmentosum complementation group A (XPA), a potential biomarker for cancer therapy sensitivity. A quantitative high-throughput cell-based NER activity assay was used to validate XPA VUS selected by the active learning strategy. In all cases, active learning yielded a significant improvement in variant effect predictions over traditional learning. These analyses suggest that active learning is well suited to significantly improve interpretation of VUS and cancer patient genomes.</p>Significance:<p>A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.</p></div>

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth

Abstract: Abstract Background Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. Methods To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven Braf V600E /Pten -/- /Cxcr2 -/- and NRas Q61R /INK4a -/- /Cxcr2 -/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in Braf V600E /Pten -/- and NRas Q61R /INK4a -/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). Results Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1 , a key tumor suppressive transcription factor, was the only gene significantly induced with a log 2 fold-change greater than 2 in these three different melanoma models. Conclusions Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Editor's evaluation: Mutation of vsx genes in zebrafish highlights the robustness of the retinal specification network

Abstract: Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors’ specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR/Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted toward photoreceptor or Müller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species. Editor's evaluation This study provides important insights into how tissue specification networks, while often employing conserved genes across species, can differ in their network architecture, resulting in differences in how they buffer perturbations. This is shown for the Visual System Homeobox genes (VSX) in the zebrafish retinal specification pathway, where yet-to-be-defined compensatory mechanisms prevent microphthalmia in the absence of VSX function, something not observed in humans or mice. The evidence supporting the conclusions of the study is solid and provides a foundation for further molecular and genetic analysis of retinal specification. This work is relevant to developmental biologists with interests in tissue specification and gene regulatory networks. https://doi.org/10.7554/eLife.85594.sa0 Decision letter eLife's review process Introduction The organogenesis of the vertebrate eye is a complex multistep process entailing the sequential activation of genetic programs responsible for the initial specification of the eye field, the patterning of the eye primordium into sub-domains, and the determination of the different neuronal types. Although we are far from understanding the precise architecture of the gene regulatory networks (GRNs) controlling eye formation, many of their central nodes have been already identified (Buono and Martinez-Morales, 2020; Fuhrmann, 2010; Heavner and Pevny, 2012; Martinez-Morales, 2016). They comprise transcriptional regulators recruited repeatedly for key developmental decisions at different stages of eye formation, and which mutation in humans is often associated to severe ocular malformations: that is, microphthalmia, anophthalmia, and coloboma. This is the case for SIX3, PAX6, RAX, SOX2, VSX2, or OTX2 (Gregory-Evans et al., 2004; Gregory-Evans et al., 2013). Among the main regulators, the visual system homeobox transcription factors, Vsx1 and Vsx2, have been shown to control the development of visual circuits in vertebrate and invertebrate species (Burmeister et al., 1996; Erclik et al., 2008; Focareta et al., 2014). Vsx2, initially termed as Chx10, was the first gene of the family characterized in vertebrates (Liu et al., 1994). Vsx2/Chx10 shows a conserved expression pattern across vertebrate species, both in the retina (i.e. early in all optic cup precursors, and later in retinal bipolar cells), as well as in hindbrain and spinal cord interneurons (Ferda Percin et al., 2000; Kimura et al., 2013; Liu et al., 1994; Passini et al., 1997). A nonsense mutation in Vsx2 (Y176stop) turned to be the molecular cause of the phenotype exhibited by the classical mutant mice ocular retardation (or), which displays microphthalmia and optic nerve aplasia (Burmeister et al., 1996; Truslove, 1962). The phenotypic analysis of or mutants, as well as the examination of human patients with hereditary microphthalmia, revealed an essential role for Vsx2 in neuro-epithelial proliferation and bipolar cells differentiation (BarYosef et al., 2004; Burmeister et al., 1996; Ferda Percin et al., 2000). Subsequent studies indicated that, during optic cup formation, Vsx2 is a key factor in the binary decision between neural retina and retinal-pigmented epithelium (RPE) lineages. Genetic studies in mice and chick revealed that Vsx2 acts, downstream of the neural retina inducing ligands (i.e. FGFs), as a repressor of Mitf and Tfec genes and hence of the RPE identity (Horsford et al., 2005; Nguyen and Arnheiter, 2000; Rowan et al., 2004). A few years after Vsx2 identification, a closely related paralog, Vsx1, was reported in several vertebrate species (Chen and Cepko, 2000; Chow et al., 2001; Levine et al., 1994; Passini et al., 1997). The proteins encoded by these paralogous genes have similar domains’ architecture, including well-conserved paired-like homeodomain and CVC (Chx10/Vsx-1 and ceh-10) regulatory modules, and share biochemical properties, binding with high affinity to the same DNA sequence motif ‘TAATTAGC’ (Capowski et al., 2016; Dorval et al., 2005; Ferda Percin et al., 2000; Heon, 2002). Although both genes display partially overlapping expression patterns in the retina, Vsx2 precedes Vsx1 expression in undifferentiated progenitors in all vertebrate models analysed. Furthermore, once retinal precursors exit the cell cycle, they are expressed in complementary sets of differentiated bipolar cells. Thus, Vsx1 is restricted to different types of ON and OFF cone bipolar cells in mice, and Vsx2 to S4 bipolar and Müller cells in zebrafish (Ohtoshi et al., 2004; Shi et al., 2011; Vitorino et al., 2009). In contrast to Vsx2, Vsx1 seems to have a minor contribution to retinal specification in mammals. A single case of sporadic microphthalmia has been associated to Vsx1 mutation in humans (Matías-Pérez et al., 2018), and its mutation in mice does not affect early retinal development even in a Vsx2 mutant background (Chow et al., 2004; Clark et al., 2008). However, Vsx1 mutation has been linked to inherited corneal dystrophies in humans, and is associated to abnormal electroretinogram (ERGs) recordings either in mice or in patients (Chow et al., 2004; Heon, 2002; Mintz-Hittner et al., 2004). Despite all these advances on the developmental role of Vsx genes, many questions remain open. A fundamental issue is to understand to which extent Vsx gene functions are conserved across vertebrates. Previous antisense oligonucleotides or morpholino studies in zebrafish have shown that vsx2 knockdown results in microphthalmia and optic cup folding defects (Barabino et al., 1997; Clark et al., 2008; Gago-Rodrigues et al., 2015; Vitorino et al., 2009). However, these findings have not been validated using knockout lines, neither the role of vsx1 and vsx2 in fate determination and bipolar cells differentiation has been sufficiently explored in teleost fish. To gain insight into the universality and diversity of Vsx functions, we have generated zebrafish mutants for vsx1 and vsx2 harboring deletions within the homeodomain-encoding exons. Surprisingly, eye morphology and size appear normal either in the individual or in the double vsx1/vsx2 mutants, thus indicating that vsx genes are not essential to initiate retinal development in zebrafish. The absence of early retinal malformations facilitates the phenotypic analysis of the mutants at later embryonic and larval stages. Defects in the visual background adaptation (VBA) reflex are observed in vsx1 mutant, and appear enhanced in double mutant larvae, suggesting partial or complete blindness. Analysis of ERG responses confirms vision loss, showing that the amplitude of the b-wave recordings is reduced in vsx1 mutants, and absent in double mutants. Interestingly, a single wild type copy of vsx1 is sufficient to prevent VBA and ERG defects, indicating that vsx2 loss of function can be compensated by vsx1. The analysis of neuronal-specific markers confirmed that retinal progenitors fail to differentiate into bipolar cells in double mutant embryos. Instead, we show that precursors at the inner nuclear layer (INL) can remain proliferative, undergo apoptosis, or be rerouted toward other retinal lineages, particularly differentiating as Müller glial cells. Finally, we investigate whether transcriptional adaptation (El-Brolosy et al., 2019) may compensate for vsx1/vsx2 loss-of-function during retinal specification. The transcriptomic analysis of core components of the retinal specification GRN do not support a transcriptional adaptation mechanism in vsx1/vsx2 double mutants, rather suggesting that the network robustness is by itself sufficient to sustain early eye development even in the absence of vsx1 and vsx2 function. In summary, whereas our work shows a conserved role for Vsx genes during bipolar cell differentiation, also indicates that their hierarchic weight within the eye GRNs varies considerably across vertebrate species. Results Zebrafish vsx double mutants show normal eye size but affected lamination of the retina Despite the additional round of genome duplication occurring in the teleost lineage after the split with sarcopterygians (Meyer and Schartl, 1999), a single copy of both vsx1 and vsx2 was retained in zebrafish. In order to investigate the role of Vsx transcription factors during visual system formation in zebrafish, we generated mutants for both paralogs using CRISPR/Cas9. To optimize the generation of null animals, we targeted conserved regions encoding for the DNA binding domain of the proteins in their corresponding loci at chromosome 17 (Figure 1a). We generated a 245 bp deletion in vsx1 encompassing exon3, intron3, and exon4 of the gene (vsx1∆245). This mutation results in an in-frame deletion of 53 amino acids by the removal of 159 bp from exon3 (54 bp) and exon4 (105 bp; Figure 1—figure supplement 1a). In the case of vsx2, a 73 bp deletion was generated in exon 3 (vsx2∆73). This mutation deletes 24 amino acids of the core DBD of the protein and generates a premature stop codon in that domain (Figure 1—figure supplement 1b). Both deletions can be easily screened by PCR with primers flanking the mutation sites. Using Vsx1- and Vsx2-specific antibodies, we found that no Vsx2 or Vsx1 proteins could be detected by western blot in 24hpf vsxKO samples (Figure 1—figure supplement 1c, d). In addition, no maternal Vsx1 protein was detected in early 1.5hpf wildtype embryos (Figure 1—figure supplement 1c). Figure 1 with 3 supplements see all Download asset Open asset DNA-binding domain deletion of vsx genes affect neural retina formation and disrupt VBA reflex. (a) CRISPR/Cas9 DNA editing tool was used to generate deletions (green box) in the highly conserved DBD from vsx1 (top) and vsx2 (bottom) TFs. Blue boxes represent gene exons, black boxes the location of sgRNAs used to guide Cas9 endonuclease and primers for screening are depicted as opposing arrowheads. b-d and f-h. Histological sections stained with nuclear marker DAPI and phalloidin-Alexa488 for actin filaments from WT (b-d, n≥8) and vsxKO central retinas (f-h, n≥10) at 48hpf (b, f), 72hpf (c, g) and 6dpf (d, h). (e, i). Head dorsal view from 6dpf WT (e) and vsxKO (i) larvae with insets showing their pigmentation pattern (white arrowhead). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar in (b-d) and (f-h): 50 µm, scale bar in e and i: 500 µm. At 2-week post fertilization, no obvious macroscopic defects were observed in the visual system of either homozygous single mutants (i.e. vsx1∆245 or vsx2∆73) or homozygous double mutants vsx1∆245; vsx2∆73 (here termed vsxKO), which appeared normal in shape and size (Figure 1—figure supplement 2; Figure 1—figure supplement 3a–d). Homozygous single mutants, and even animals harboring a single wild type copy either of vsx1 (vsx1∆245+/-, vsx2∆73-/-) or vsx2 (vsx1∆245-/-; vsx2∆73+/-) reached adulthood and were fertile. However, double mutant larvae (vsx1∆245 -/-; vsx2∆73 -/-) died at around 3-week post fertilization, with the exception of a single unfertile escaper reaching adulthood (1 out of 152 larvae raised). For further analyses, double mutant embryos and larvae were obtained each generation by in-crossing of vsx1∆245+/-; vsx2∆73-/- or vsx1∆245-/-; vsx2∆73+/-animals. Once the proper recombinants were obtained, heterozygous lines maintenance was facilitated by the linkage between vsx1 and vsx2 mutant alleles, which tend to segregate together due to their proximity (10.6 Mb) in chromosome 17. Histological sectioning of mutant retinas at 48hpf showed a small delay in the formation of the inner plexiform layer (IPL), but no obvious macroscopic optic cup malformations when compared to WT (Figure 1b and f). At 72hpf, both the outer plexiform layer (OPL) and the IPL appeared less organized in the double mutant retinas, which showed discontinuities/fenestrae (Figure 1c and g). At 6dpf, double mutant larvae showed all the layers of a normal retina, but the thickness of the outer (ONL) and inner (INL) nuclear layers was significantly increased and reduced respectively, when compared to siblings (Figure 1d and h; Figure 1—figure supplement 3e, h, i). In addition to retinal layer formation defects, vsxKO fish presented expanded pigmentation in skin melanocytes even when exposed to bright light for 20 min (Figure 1e and i; Figure 1—figure supplement 3a, d). This phenomenon is indicative of an impaired visual background adaptation (VBA) reflex, and is often associated with blindness in zebrafish (Fleisch and Neuhauss, 2006). Visual function is impaired in single vsx1and vsxKO double mutants To test the visual performance of the vsx mutants; ERG recordings were obtained from WT and mutants at 5 dpf (Figure 2). Zebrafish retina becomes fully functional at 5 dpf with the exception of late maturing rods (Bilotta et al., 2001) and thus, the recorded field potentials were mainly contributed by cones. Wild type larvae show a standard ERG response to light flash, characterized by a large positive b-wave representing the depolarization of ON bipolar cells (Figure 2a), which also masks the initial a-wave generated by photoreceptor (PR) hyperpolarization. Representative recordings from larvae harboring different vsx genotypes are shown in Figure 2a. We found that vsx2∆73 ERG response (green curve) was similar to the WT recording (blue curve). However, recordings in vsx1∆245 larvae showed a reduced b-wave compared to WT or vsx2∆73 larvae. From the 36 double mutant larvae recorded in total, 10 of them still showed a b-wave, though reduced in comparison to vsx1∆245 mutants, and much smaller than WT recordings. Moreover, in the remaining 26 double mutants recorded only the negative a-wave but not the b-wave (gray curve) was detected, suggesting that bipolar cells differentiation and/or function might be compromised. Statistical analysis of the average amplitude showed that the b-wave is significantly decreased in both vsx1∆245 single and vsxKO double mutants in comparison to WT at all tested light intensities (Figure 2b). In addition, the b-wave response amplitude in the double mutant was significantly reduced compared to vsx1 single mutants (Figure 2b). These measurements are in line with our previous observation indicating that double mutant retinas are more affected at the cellular level than single vsx1∆245 animals (Figure 1—figure supplement 3). Figure 2 with 1 supplement see all Download asset Open asset ERG response is reduced in vsxKO larvae. (a) Representative ERG tracks at maximum light intensity from WT (blue), vsx1∆245 (red), vsx2∆73 (green) and vsxKO double mutants (grey and yellow) at 5dpf. For vsxKO larvae, two typical recordings are shown (grey and yellow tracks). (b). Averaged ERG b-wave amplitudes from WT (blue), vsx1∆245 (red), vsx2∆73 (green) and vsxKO (yellow) larvae. No significant differences were observed between WT and vsx2∆73 samples. vsx1∆245 and vsxKO mutants produce a significant reduction of the ERG b-wave amplitude compared with both WT and vsx2∆73 larvae throughout all light intensities tested (***p<0.0001, ****p<0.00001). Data are shown as mean ± SEM. In (a) and (b), vsx1∆245 (red tracks) represents both vsx1∆245-/- and vsx1∆245-/-; vsx2∆73+/-genotypes, while vsx2∆73 (green tracks) represents both vsx2∆73-/- and vsx1∆245+/-; vsx2∆73-/- genotypes. Data were collected from five independent experiments. For statistical comparison, one way ANOVA test was used. ms: milliseconds, mV: millivolts. To quantitatively characterize eye performance, optokinetic response (OKR) recordings (Rinner et al., 2005) were obtained for WT and vsx mutant fish (Figure 2—figure supplement 1). To investigate the role of Vsx transcription factors at the behavioral level, eye movement velocity was recorded at 5dpf in WT and vsxKO mutant fish. We measured eye velocity varying different parameters of the moving stimuli, such as contrast (contrast sensitivity; Figure 2—figure supplement 1a), frequency (spatial resolution; Figure 2—figure supplement 1b) and angular velocity (temporal resolution; Figure 2—figure supplement 1c). In all conditions tested, we observed a significant reduction in eye velocity for vsx1 single and vsxKO double mutants when compared with vsx2∆73 larvae and WT controls (repeated measurement, ANOVA p<0.001). Taken together these physiological recordings confirmed significant sight impairment in vsx1 mutants, a phenotype that is further aggravated by vsx2 loss in vsxKO double mutants. Extended proliferation wave and INL cell death in vsxKO double mutant retinas As vsxKO double mutants showed stronger retinal architecture and visual defects than other vsx mutant combinations, we decided to focus further phenotypic analyses on them. To assess whether our observations on the increased thickness of the ONL and the decreased width of the INL (Figure 1—figure supplement 3) correlate with a proliferation and/or cell death unbalance, we examined both parameters in vsxKO fish. To investigate proliferation defects, we quantified the number of phosphohistone H3 positive (PH3+) cells in the retina of wild type and vsxKO animals throughout the lamination process: that is, at 24, 48, 60, and 72hpf (Figure 3a–f and m; Figure 3—figure supplement 1). At 24 and 48hpf, no difference in the number of PH3 + cells were observed between WT and vsxKO retinas (Figure 3a, d and m; Figure 3—figure supplement 1). However, at 60hpf, when the proliferation wave has largely finished in WT eyes, double mutant retinas continued to divide and showed a significant increase in PH3 + cells, particularly in the outer and peripheral regions (Figure 3b, e and m). Later on, at 72hpf, PH3 + cells were only detected in the CMZ and no significant difference in the number of proliferative cells was detected between WT and vsxKO retinas (Figure 3c, f and m). To test if cell death may account for the reduced INL width observed in double mutants (Figure 1—figure supplement 3h, i), we stained retinal cryosections at different time points with anti-cleaved caspase3 (C3) antibodies to detect cells that undergo apoptosis (Figure 3g–l and n). At 60hpf, C3-positive cells (C3+) could be observed rarely in WT or vsxKO retinas (Figure 3g, j and n). However, at both 72 and 96hpf, a significant increase in the number of apoptotic C3 + cells were detected in double mutants compared to WT (Figure 3h, i, k and l). Apoptotic cells concentrated mainly in the INL layer of the retina (Figure 3k, l and n), suggesting that cell death within this layer may contribute to the decreased thickness observed in vsxKO retinas. We also observed a few apoptotic C3 + cells in the ganglion cell layer (GCL) in vsxKO embryos (Figure 3k and l) suggesting than the survival of these cells may be compromised. To investigate this point, we decided to analyze the integrity of the retinal ganglion cells’ (RGCs) projections to the optic tectum by injecting DiI and DiO tracers in WT and vsxKO double mutant eyes at 6dpf (Video 1). No obvious differences in retino-tectal projections were detected between WT and double mutant larvae, indicating that the RGCs are not affected in vsxKO retinas compared to control animals. Figure 3 with 1 supplement see all Download asset Open asset Mitosis and apoptosis markers expression are increased in vsxKO retinas. (a-f). Phospho-histone H3 (PH3) antibody staining reveals cell divisions in central retina cryosections from WT (a-c) and vsxKO (d-f) samples at three different developmental stages (48, 60, and 72hpf). Increased PH3 staining was observed in vsxKO retinas at 60hpf (white arrowheads in e) compared to WT samples (white arrowheads in b). (g-l). Caspase-3 (C3) antibody staining was used to evaluate cell death in central retina cryosections from WT (g-i) and vsxKO (j-l) samples at three different developmental stages (60, 72, and 96hpf). Aberrant C3 staining was observed in vsxKO retinas at 72 and 96hpf (white arrowheads in k and l) compared to WT samples (h and i). m. Quantification of PH3 positive cells in WT and vsxKO retinas at different stages. Using an unpaired t-test, a significant increase in PH3 positive cells was observed in vsxKO samples at 60hpf compared to WT (***p<0.0001) but no significant changes were observed at other stages analysed (48 and 72hpf). n. Quantification of C3 positive cells in WT and vsxKO retinas at different stages. Significant increase in C3-positive cells was observed in vsxKO samples at 72 and 96hpf compared to WT (***p<0.0001), but no change was observed at 60hpf using an unpaired t-test. Data is shown as mean ± SD. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization. Scale bar in (a-l): 50 µm. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg vsxKO larvae show normal GCL retinotectal projections. (a, b). 3-D reconstructions of confocal stacks from zebrafish larval eyes injected with either DiO (green) or DiI (red) to label retinal ganglion cells and their projections to the optic tectum in wildtype (a, n=6) and vsxKO (b, n=8) at 6dpf. Note that vsxKO larvae show apparently normal retinotectal projections. Abnormal cell fate specification in the retina in vsxKO Our results indicated that, in contrast to Vsx2 early requirement in the mouse (Burmeister et al., 1996), vsx genes are not essential for the early specification of the neural retina in zebrafish (Figure 1; Figure 1—figure supplement 2). This fact facilitated the analysis of cell fate choices in vsxKO embryos. Although all retinal layers are present in double mutant animals (Figure 1—figure supplement 3), the identity of the cells within these layers required further investigation. To examine cell fate acquisition in the INL and ONL of mutant retinas, fluorescent antisense probes or antibodies for specific markers of PRs (prdm1a), bipolar (prox1, prkcbb), amacrine (ptf1a, pax6), and Müller glia cells (gfap) were examined at 48-72hpf (Figure 4; Figure 4—figure supplement 2). Figure 4 with 3 supplements see all Download asset Open asset Altered expression of Bipolar and Müller glia cell markers in 3dpf vsx mutant fish. (a-h). Confocal sections from in toto in situ hybridization experiments using specific fluorescent probes to label different cell types in wildtype and vsxKO retinas at 72hpf. No clear differences in the expression of the photoreceptor marker prdm1a were observed in ONL of wildtype (a) and mutant samples (e). Bipolar cell marker prkcbb expression (b, f) is considerably reduced in the INL of vsxKO mutant retinas (f, white arrowheads) compared to wildtype (b). Similar expression of the amacrine cell marker ptf1a is observed in the INL from wildtype (c) and vsxKO (g) retinas. Increased expression of the Müller glia cell marker gfap (d, h) is observed in the INL of vsxKO samples (h, white arrowheads) compared to wildtype (d) retinas. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Scale bar in (a-h): 50 µm. ONL/photoreceptors specification Prdm1a (or Blimp1) is a transcription factor that has been shown to play an early role in the specification of PR identity, mainly by the suppression of bipolar cell fate genes, including vsx2 (Brzezinski et al., 2010; Katoh et al., 2010). Conversely, vsx2 acute knockdown by electroporation in the postnatal mouse retina triggers a bipolar to rod fate shift (Goodson et al., 2020; Livne-Bar et al., 2006). In this study, the comparative analysis of the transient marker prdm1a (Wilm and Solnica-Krezel, 2005) between wild type and vsxKO embryos revealed a mild downregulation in the mutants at 72 hpf (n=6) (Figure 4a and e), which is in agreement with the delayed differentiation of the photoreceptors we observed in vsxKO animals (Figure 4—figure supplement 1). However, when we examined terminal differentiation markers for cones (Ab Zpr-1) and rods (Ab Zpr-3) at 72 and 96hpf, a delayed differentiation of both cell types was observed in double mutant embryos (Figure 4—figure supplement 1). Whereas Zpr-1 and Zpr-3 staining could be detected in the entire ONL in wild type fish from 72hpf on (Figure 4—figure supplement 1a–c, h-j), in 72 hpf vsxKO embryos the staining was restricted to a few cells in the ventral retina (Figure 4—figure supplement 1d, k) and was only extended to the entire ONL at 96 hpf (Figure 4—figure supplement 1e, l). At 6dpf, there is a significant increase of Zpr1 fluorescent intensity in vsxKO compared to WT retinas (Figure 4—figure supplement 1c, f, g), but no major differences were observed in rod stain intensity (Figure 4—figure supplement 1j, m, n). This result suggests that PRs’ differentiation program is delayed in the absence of vsx function and that cone cells are overrepresented in the ONL of the double mutants. A prolonged period of precursors’ proliferation and/or competence could account for an increased number of PRs at larval stages, and thus for an expanded thickness of the ONL layer, as observed in double mutants at 6 dpf (Figure 1; Figure 1—figure supplement 3). INL/bipolar cells specification In the zebrafish retina, vsx1 and vsx2 expression has been reported in complementary subsets of bipolar cells, with vsx1 having a broader distribution and vsx2 being restricted to a few bipolar subtypes (Vitorino et al., 2009). To analyse bipolar cell integrity in vsxKO embryos, we first performed immunohistochemistry for the general INL marker prox1 (Figure 4—figure supplement 2; Dyer, 2003) and then fluorescent in situ hybridizations for the bipolar cell marker protein kinase Cb1 (prkcbb) (Figure 4). At 48hpf, no changes in the expression of prox1 was detected between WT and vsxKO retinas (Figure 4—figure supplement 2a, e). However, at 72hpf the nuclear distribution of prox1 in the INL is affected in vsxKO samples compared to WT retinas (Figure 4—figure supplement 2b, b’, f, f’) suggesting a lack of bipolar cells in vsxKO retinas. This observation was further confirmed by the fact that at 72hpf prkcbb expression is very reduced, if not absent, in the INL of double mutant retinas compared to WT (n=5) (Figure 4b and f). These results are in agreement with our previous histological (i.e., reduced INL thickness; Figure 1—figure supplement 3) and electrophysiological (i.e. reduced b-wave, Figure 2) observations in vsxKO larvae, and confirms that vsx genes are essential for bipolar cells specification in zebrafish. Although we can also detect a mild reduction of prkcbb at the GCL, and we cannot rule out transient defects in a particular RGCs subpopulation at 72 hpf (Figure 4f), the final RGC numbers seem normal in the vsxKO retinas as determined by DiI and DiO tracers (Video 1) as well as retinal histology at 6dpf (Figure 1). INL/amacrine cells (AC) specification A detailed histological analysis of the INL architecture in wild type and vsxKO embryos suggested that ACs specification was not severely affected in the double mutant (Figure 1d and h). To confirm this point, we followed the expression of ptf1a, a transcription factor encoding gene that is expressed in horizontal and AC and has been shown to play an essential role in their specification in the mouse retina (Fujitani et al., 2006). Using a fluorescent probe against ptf1a, which is expressed transiently in all types of amacrine cells in the embryonic zebrafish retina (Jusuf and Harris, 2009), we could determine that the ACs differentiation wave progresses normally through the central retina in wild type and vsxKO embryos at 48 hpf (n=5) (Figure 4—figure supplement 2c, g). Later in development, at 72 hpf, pft1a expression was no longer detected in the central retina and appeared restricted to the most peripheral region, being expressed at similar levels in both wild type and vsxKO retinas (n=10) (Figure 4c and g). In addition, the expression of the differentiated amacrine cell marker pax6 (Hitchcock et al., 1996) is not affected in vsxKO retinas compared to WT (Figure 4—figure supplement 2d, h). These observations suggest that vsx genes in zebrafish do not play a major role for amacrine cells specification. I

Climate Change

Abstract: Climate change ubiquitously influences social determinants of health via various pathways. Disproportionately burdening communities who have contributed the least to greenhouse gas (GHG) emissions and benefitted the least from economic benefits obtained through high-emission activities that cause climate change, climate justice must be centered in any discussion of health equity. This article will explore how climate change contributes to health disparities in vulnerable populations, why this is a justice issue for primary care to address, and what we can do to promote equity, resilience, and adaption in our current economic system while mitigating GHG emissions, leveraging the health sector.