Corrigendum: The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

[This corrects the article DOI: 10.3389/fimmu.2022.1093242.].

The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial. Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths. Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Are You Ready to Negotiate Your First Employment Contract? Experience of More Than 700 Plastic Surgeons.

BACKGROUND: Plastic surgeons have been shown to be unprepared to negotiate their first employment contracts. Previous survey studies have attempted to assess plastic surgeons' first employment contracts to outline common pitfalls in contract negotiation. With this study, the authors aim to expand these previous studies and help plastic surgeons become prepared to negotiate their employment contracts. METHODS: A seven-question, cross-sectional survey was sent to attending-level surgeon members of the California Society of Plastic Surgeons, the American Society of Plastic Surgeons, the Texas Society of Plastic Surgeons, and the American Cleft Palate-Craniofacial Association. Questions investigated plastic surgeons' first contracts. Correlations were determined using a two-sample Wilcoxon rank sum test in an attempt to link these questions with overall satisfaction. RESULTS: From the 3908 distributed surveys, 782 (20 percent) responses were collected, and 744 were included for analysis. The majority of respondents were found to join a group-centered, private practice following residency. Surprisingly, 69 percent of surgeons did not use attorney assistance when negotiating their contract. Although greater than 70 percent of respondents reported a salary of $200,000 or less, satisfaction with one's contract was most strongly correlated with a salary of greater than $300,000 (p < 0.0001). However, only 12 percent of respondent surgeons were able to secure such a salary. CONCLUSIONS: This study examined the largest, most diverse plastic surgeon cohort to date regarding surgeons' first employment contract. Although the authors' findings indicate that certain factors should be prioritized when approaching a first employment contract, they ultimately recommend that all surgeons take into account their personal priorities and attempt to proactively define their terms of employment before signing a contract.

Enhanced Killing of Triple-Negative Breast Cancer Cells by Reassortant Reovirus and Topoisomerase Inhibitors.

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast cancer. Mammalian orthoreovirus (reovirus) selectively infects and kills transformed cells, and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent against several cancers. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and if reovirus-induced cytotoxicity of breast cancer cells can be enhanced by modulating the activity of host molecules and pathways. Here, we generated reassortant reoviruses by forward genetics with enhanced infective and cytotoxic properties in triple-negative breast cancer cells. From a high-throughput screen of small-molecule inhibitors, we identified topoisomerase inhibitors as a class of drugs that enhance reovirus infectivity and cytotoxicity of triple-negative breast cancer cells. Treatment of triple-negative breast cancer cells with topoisomerase inhibitors activates DNA damage response pathways, and reovirus infection induces robust production of type III, but not type I, interferon (IFN). Although type I and type III IFNs can activate STAT1 and STAT2, triple-negative breast cancer cellular proliferation is only negatively affected by type I IFN. Together, these data show that reassortant viruses with a novel genetic composition generated by forward genetics in combination with topoisomerase inhibitors more efficiently infect and kill triple-negative breast cancer cells.IMPORTANCE Patients afflicted by triple-negative breast cancer have decreased survival and limited therapeutic options. Reovirus infection results in cell death of a variety of cancers, but it is unknown if different reovirus types lead to triple-negative breast cancer cell death. In this study, we generated two novel reoviruses that more efficiently infect and kill triple-negative breast cancer cells. We show that infection in the presence of DNA-damaging agents enhances infection and triple-negative breast cancer cell killing by reovirus. These data suggest that a combination of a genetically engineered oncolytic reovirus and topoisomerase inhibitors may provide a potent therapeutic option for patients afflicted with triple-negative breast cancer.

Ivabradine in Congestive Heart Failure: Patient Selection and Perspectives.

Heart failure (HF) is the fourth-most frequent cause of death and remains a challenge for public health. Therapy goals for HF with reduced ejection fraction (HFrEF) are the improvement in the quality of life, prolonged survival, a reduction of signs and symptoms, and the prevention of hospitalization. Angiotensin-converting enzyme inhibitors, beta-blockers, and mineralocorticoid receptor antagonists are the treatments of choice for HFrEF. Although ivabradine is not available in all countries, it is likely a new promising approach to improve outcomes in patients with HFrEF, either alone or with beta-blockers. Here, we review the current knowledge about ivabradine in HFrEF and assess its effect on outcomes in HF.

Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

Outcomes of Desidustat Treatment in People with Anemia and Chronic Kidney Disease: A Phase 2 Study.

BACKGROUND: Desidustat (ZYAN1) is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) that stimulates erythropoiesis. Stabilizing HIF via PHI is developing as a new therapeutic approach to treat anemia secondary to chronic kidney disease (CKD). This trial evaluated the safety, tolerability, and efficacy of Desidustat in adult CKD patients with anemia, who were not on dialysis. METHODS: This was a Phase 2, randomized, double-blind, 6-week, placebo-controlled, dose-ranging, safety and efficacy study. A total of 117 eligible patients were randomized to 4 arms: 100, 150, 200 mg, or placebo. The investigational product was administered every alternate day for 6 weeks in fasting conditions. The primary endpoint was change in hemoglobin (Hb) from baseline to week 6. RESULTS: Baseline demographics were well balanced among all the treatment arms. In the modified intent-to-treat (mITT) population, a mean Hb increase of 1.57, 2.22, and 2.92 g/dL in Desidustat 100, 150, and 200 mg arms, respectively, was observed post 6 weeks treatment. The responder rate (≥1 g/dL increase) was 66% in 100 mg, 75% in 150 mg, and 83% in 200 mg treatment arms, in the mITT population. Eighteen patients had at least one treatment emergent adverse event (TEAE), and 5 patients reported at least one drug-related mild TEAE. No death or serious adverse event was reported during the trial. CONCLUSION: There was dose-related increase in Hb across all doses compared to placebo in mITT and per-protocol populations. Desidustat also increased pharmacokinetic parameters Cmax and AUC in dose-related manner. There was no significant change in vital signs, electrocardiographic parameters, or safety laboratory values. Clinical Trial Registration Number CTRI/2017/05/008534 (registered on May 11, 2017).

West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-ß mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.IMPORTANCE West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

Carglumic Acid Treatment of a Patient with Recurrent Valproic Acid-induced Hyperammonemia: A Rare Case Report.

Valproic acid, first manufactured as an anticonvulsant, is commonly used to treat both neurological and psychiatric conditions. A rare and deadly side effect of this medication is hyperammonemia, presenting as lethargy, confusion, seizure, and, ultimately, coma. In rare circumstances, hyperammonemia can be recurrent and devastating, especially in patients with an underlying N-acetyl glutamate synthase (NAGS) deficiency, as the valproic acid can enhance this enzyme deficiency and inhibit the conversion of ammonia into urea in the liver. For these subtypes of patients, the United States Food and Drug Administration (US FDA) has recently approved carglumic acid, a medication that can act as a scavenger by effectively increasing the levels of NAGS, ultimately enhancing the conversion of ammonia to urea. In our case report, we have mentioned a patient with treatment-resistant bipolar disorder, who presented with elevated ammonia levels secondary to valproic acid treatment. Valproic acid was the only drug that was effective in his case, so we initiated therapy to reduce his elevated ammonia levels. After a thorough evaluation, we found the patient had a genetic NAGS deficiency. Carglumic acid was initiated and proved efficacious in our patient.

Progestin-based contraception regimens modulate expression of putative HIV risk factors in the vaginal epithelium of pig-tailed Macaques.

PROBLEM: In women, the use of progestin-based contraception may increase the risk of vaginal HIV acquisition. We previously showed in macaques that there is a significantly higher simian-human immunodeficiency virus (SHIV) acquisition rate in the luteal phase of the menstrual cycle, which presents a naturally high-progesterone state, and this may be attributable to altered expression of innate immune factors. We hypothesized that progestin-based contraception, especially depot medroxyprogesterone acetate (DMPA), would, in a similar way, affect mucosal immune factors that influence HIV acquisition risk. METHOD OF STUDY: We used a pig-tailed macaque model to evaluate the effects of two progestin-based contraceptives, DMPA, and levonorgestrel (LNG)/ethinyl estradiol (EE)-based combined oral contraceptives (COCs), on innate mucosal factors. We compared the vaginal epithelial thickness data from previous studies and used cytokine profiling and microarray analysis to evaluate contraception-induced molecular changes in the vagina. RESULTS: The administration of DMPA caused a reduction in the thickness of the vaginal epithelium relative to that of the follicular or luteal phase. DMPA also induced a significant increase in vaginal levels of the anti-inflammatory cytokine IL-10. Both DMPA- and LNG-based contraception induced a signature of gene expression similar to that of the luteal phase, only more exacerbated, including widespread downregulation of antiviral genes. CONCLUSION: The use of progestin-based contraception might engender a milieu that poses an increased risk of HIV acquisition as compared to both the luteal and follicular phases of the menstrual cycle.

Pulmonary Involvement in Crohn's Disease: A Rare Case Report.

Crohn's disease (CD) is a granulomatous inflammatory disease that can involve any part of the gastrointestinal tract, from mouth to anus. In most cases, it remits and relapses in the terminal ileum, requiring treatment via steroid boluses. In rare cases, however, CD can involve the pulmonary system presenting as dyspnea on exertion and dry cough. We present a case of a 38-year-old man who developed shortness of breath, cough, and wheezing for one month after a colectomy procedure due to recurrent toxic megacolon. He recovered and tolerated extubation successfully and was prescribed mesalamine as maintenance therapy for CD. His pulmonary symptoms after the colectomy, along with his imaging and pulmonary function tests, indicated pulmonary involvement in the lungs as a progression of the primary inflammatory bowel disease. After confirming this diagnosis, he was treated with oral high-dose steroids after successful diagnosis, and the patient's symptoms improved dramatically. This case highlights often overlooked CD bronchopulmonary involvement in the postoperative period.

A Review of the Mechanism of Antagonism of N-methyl-D-aspartate Receptor by Ketamine in Treatment-resistant Depression.

The biochemical processes involved in depression go beyond serotonin, norepinephrine, and dopamine. The N-methyl-D-aspartate (NMDA) receptor has a major role in the neurophysiology of depression. Ketamine, one of the prototypical NMDA antagonists, works rapidly in controlling depressive symptoms, including acutely suicidal behavior, by just a single injection. Ketamine may rapidly increase the glutamate levels and lead to structural neuronal changes. Increased neuronal dendritic growth may contribute to synaptogenesis and an increase in brain-derived neurotrophic factor (BDNF). Activation of the mechanistic target of rapamycin (mTOR), as well as increased levels of BDNF, may increase long-term potentiation and result in an improvement in the symptoms of depression. The mechanisms of ketamine's proposed effect as an off-label treatment for resistant depression are outlined in this paper.

BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data.

B cells play a critical role in the immune response by producing antibodies, which display remarkable diversity. Here we describe a bioinformatic pipeline, BALDR (BCR Assignment of Lineage using De novo Reconstruction) that accurately reconstructs the paired heavy and light chain immunoglobulin gene sequences from Illumina single-cell RNA-seq data. BALDR was accurate for clonotype identification in human and rhesus macaque influenza vaccine and simian immunodeficiency virus vaccine induced vaccine-induced plasmablasts and naïve and antigen-specific memory B cells. BALDR enables matching of clonotype identity with single-cell transcriptional information in B cell lineages and will have broad application in the fields of vaccines, human immunodeficiency virus broadly neutralizing antibody development, and cancer.BALDR is available at https://github.com/BosingerLab/BALDR .

Pressure Sores and Systemic Inflammatory Response Syndrome: UC Davis Quality Improvement Initiative.

BACKGROUND: The National Pressure Ulcer Advisory Panel estimates pressure sore care to approach $11 billion annually. It is not uncommon for these patients to present to the emergency department (ED) with a chief concern of a pressure sore, while concurrently carrying an undiagnosed infectious process that is the culprit for the acute presentation, rather than the chronic pressure injury. We aim to identify patients who met systemic inflammatory response syndrome (SIRS) criteria at ED presentation who were referred to plastic and reconstructive surgery for pressure sore debridement prior to a complete medical workup. We hypothesize that a restructuring of the ED triaging system would help conserve hospital resources, reduce costs of pressure sore management, and improve patient care and outcomes by first treating primary, underlying pathologies. METHODS: This is a retrospective chart review of 36 patients who presented to the University of California, Davis Medical Center Emergency Department with a pressure sore and met SIRS criteria, but obtained a plastic surgery consult prior to a full medical workup. We defined SIRS based on standardized criteria: temperature greater than 100.4°F or less than 96.8°F, pulse rate greater than 90 beats/min, respiratory rate greater than 20 breaths/min or PaCO2 less than 32 mm Hg, white blood cell count greater than 12,000, less than 4000, or greater than 10% bands. RESULTS: Fifty percent of patients (18/36) met SIRS criteria at ED presentation for their pressure sores. Of these SIRS patients, 9 (50%) had a diagnosis of urinary tract infection or urosepsis, 6 (33.3%) had sepsis of undefined origin, and 3 (16.7%) had other diagnoses such as osteomyelitis or acute respiratory distress syndrome. CONCLUSIONS: Half of patients consulted while in the University of California, Davis Medical Center Emergency Department with pressure sores met SIRS criteria and received a plastic and reconstructive surgery consult prior to a full medical workup. We propose a new algorithm for triaging pressure sore patients be established in our institution that emphasizes a medical and surgical collaborative approach in order to reduce cost, conserve resources, and improve patient care.

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

Diagnosis and Management of a Cardiac Amyloidosis Case Mimicking Hypertrophic Cardiomyopathy.

Cardiac amyloidosis is an acquired heart disease secondary to the deposition of ß-pleated amyloid proteins in heart tissue. Amyloid light chain (AL) amyloidosis is usually secondary to multiple myeloma and can rapidly deteriorate cardiac function, with high mortality. Up to 50% of AL patients have cardiac involvement presenting as heart failure, conduction abnormalities, and cardiomyopathies. One of the rare presentations is the likely simulation of disease with hypertrophic cardiomyopathies like left ventricular outflow tract (LVOT) obstruction due to the systolic anterior motion of the mitral valve and irregular septal hypertrophy secondary to amyloid deposits. We present a case of cardiac amyloidosis secondary to multiple myeloma who presented with dynamic LVOT obstruction resembling hypertrophic obstructive cardiomyopathy and complicated by acute pulmonary edema. These complicated cases can be initially treated for pulmonary edema with an elevation of the head of the bed, furosemide, and nitroglycerin intravenously. For multiple myeloma, chemotherapy was continued. Beta-blockers, calcium channel blockers and angiotensin-converting enzyme inhibitors, and aldosterone receptor blocker were avoided due to poor tolerability. After symptomatic control, the patient can likely be scheduled for septal myotomy and the placement of a pacemaker or implantable cardiac defibrillator to prevent any arrhythmias causing sudden cardiac death in these subsets of patients.