SORCS2 activity in pancreatic α-cells safeguards insulin granule formation and release from glucose-stressed ß-cells.

Sorting receptor SORCS2 is a stress-response factor protecting neurons from acute insults, such as during epilepsy. SORCS2 is also expressed in the pancreas, yet its action in this tissue remains unknown. Combining metabolic studies in SORCS2-deficient mice with ex vivo functional analyses and single-cell transcriptomics of pancreatic tissues, we identified a role for SORCS2 in protective stress response in pancreatic islets, essential to sustain insulin release. We show that SORCS2 is predominantly expressed in islet alpha cells. Loss of expression coincides with inability of these cells to produce osteopontin, a secreted factor that facilitates insulin release from stressed beta cells. In line with diminished osteopontin levels, beta cells in SORCS2-deficient islets show gene expression patterns indicative of aggravated cell stress, and exhibit defects in insulin granule maturation and a blunted glucose response. These findings corroborate a function for SORCS2 in protective stress response that extends to metabolism.

Ventilation-perfusion scan for diagnosing pulmonary embolism: do chest x-rays matter?

BACKGROUND: Ventilation-perfusion (V/Q) scan coupled with single photon emission computed tomography (SPECT) is commonly used for the diagnosis of pulmonary embolism (PE). An abnormal chest x-ray (CXR) is deemed to hinder the interpretation of V/Q scan and therefore a normal CXR is recommended prior to V/Q scan. AIMS: To determine if an abnormal CXR impacted on V/Q scan interpretation and subsequent management. METHODS: A retrospective cohort analysis of all patients who underwent a V/Q scan for diagnosis of suspected acute PE between March 2016 and 2022 was performed. CXR reports were reviewed and classified as normal or abnormal. Low-dose computerised tomography was routinely performed in patients above the age of 70. Data regarding V/Q scan results and subsequent management including initiation of anticoagulation for PE or further diagnostic investigations were collected. RESULTS: A total of 340 cases were evaluated. Of the positive V/Q scans (92/340), 98.3% of the normal CXR were anticoagulated compared to 100% of the abnormal CXR group. Of the negative V/Q scans (239/340), no cases were started on anticoagulation and no further investigations were performed across both normal and abnormal CXR groups. Indeterminate results occurred in only 9 cases with no significant difference in management between normal and abnormal CXR groups. CONCLUSION: An abnormal CXR does not affect the reliability of V/Q scan interpretation in the diagnosis of PE when coupled with SPECT. Unless clinically indicated, the mandate by clinical society guidelines for a normal CXR prior to V/Q should be revisited.

Clofazimine pharmacokinetics in HIV-infected adults with diarrhea: Implications of diarrheal disease on absorption of orally administered therapeutics.

Oral drug absorption kinetics are usually established in populations with a properly functioning gastrointestinal tract. However, many diseases and therapeutics can alter gastrointestinal physiology and cause diarrhea. The extent of diarrhea-associated impact on drug pharmacokinetics has not been quantitatively described. To address this knowledge gap, we used a population pharmacokinetic modeling approach with data collected in a phase IIa study of matched human immunodeficiency virus (HIV)-infected adults with/without cryptosporidiosis and diarrhea to examine diarrhea-associated impact on oral clofazimine pharmacokinetics. A population pharmacokinetic model was developed with 428 plasma samples from 23 HIV-infected adults with/without Cryptosporidium infection using nonlinear mixed-effects modeling. Covariates describing cryptosporidiosis-associated diarrhea severity (e.g., number of diarrhea episodes, diarrhea grade) or HIV infection (e.g., viral load, CD4+ T cell count) were evaluated. A two-compartment model with lag time and first-order absorption and elimination best fit the data. Maximum diarrhea grade over the study duration was found to be associated with a more than sixfold reduction in clofazimine bioavailability. Apparent clofazimine clearance, intercompartmental clearance, central volume of distribution, and peripheral volume of distribution were 3.71 L/h, 18.2 L/h (interindividual variability [IIV] 45.0%), 473 L (IIV 3.46%), and 3434 L, respectively. The absorption rate constant was 0.625 h-1 (IIV 149%) and absorption lag time was 1.83 h. In conclusion, the maximum diarrhea grade observed for the duration of oral clofazimine administration was associated with a significant reduction in clofazimine bioavailability. Our results highlight the importance of studying disease impacts on oral therapeutic pharmacokinetics to inform dose optimization and maximize the chance of treatment success.

Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context.

BACKGROUND: Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits. METHODOLOGY: Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41-55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant's diarrhea. CONCLUSION: ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche.

The cellular organization of gastrointestinal crypts is orchestrated by different cells of the stromal niche but available in vitro models fail to fully recapitulate the interplay between epithelium and stroma. Here, we establish a colon assembloid system comprising the epithelium and diverse stromal cell subtypes. These assembloids recapitulate the development of mature crypts resembling in vivo cellular diversity and organization, including maintenance of a stem/progenitor cell compartment in the base and their maturation into secretory/absorptive cell types. This process is supported by self-organizing stromal cells around the crypts that resemble in vivo organization, with cell types that support stem cell turnover adjacent to the stem cell compartment. Assembloids that lack BMP receptors either in epithelial or stromal cells fail to undergo proper crypt formation. Our data highlight the crucial role of bidirectional signaling between epithelium and stroma, with BMP as a central determinant of compartmentalization along the crypt axis.

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells.

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

iPSC-derived reactive astrocytes from patients with multiple sclerosis protect cocultured neurons in inflammatory conditions.

Multiple sclerosis (MS) is the most common chronic central nervous system inflammatory disease. Individual courses are highly variable, with complete remission in some patients and relentless progression in others. We generated induced pluripotent stem cells (iPSCs) to investigate possible mechanisms in benign MS (BMS), compared with progressive MS (PMS). We differentiated neurons and astrocytes that were then stressed with inflammatory cytokines typically associated with MS phenotypes. TNF-α/IL-17A treatment increased neurite damage in MS neurons from both clinical phenotypes. In contrast, TNF-α/IL-17A-reactive BMS astrocytes cultured with healthy control neurons exhibited less axonal damage compared with PMS astrocytes. Accordingly, single-cell transcriptomic BMS astrocyte analysis of cocultured neurons revealed upregulated neuronal resilience pathways; these astrocytes showed differential growth factor expression. Furthermore, supernatants from BMS astrocyte/neuronal cocultures rescued TNF-α/IL-17-induced neurite damage. This process was associated with a unique LIF and TGF-ß1 growth factor expression, as induced by TNF-α/IL-17 and JAK-STAT activation. Our findings highlight a potential therapeutic role of modulation of astrocyte phenotypes, generating a neuroprotective milieu. Such effects could prevent permanent neuronal damage.

Single-cell clonal tracking of persistent T-cells in allogeneic hematopoietic stem cell transplantation.

The critical balance between intended and adverse effects in allogeneic hematopoietic stem cell transplantation (alloHSCT) depends on the fate of individual donor T-cells. To this end, we tracked αßT-cell clonotypes during stem cell mobilization treatment with granulocyte-colony stimulating factor (G-CSF) in healthy donors and for six months during immune reconstitution after transfer to transplant recipients. More than 250 αßT-cell clonotypes were tracked from donor to recipient. These clonotypes consisted almost exclusively of CD8+ effector memory T cells (CD8TEM), which exhibited a different transcriptional signature with enhanced effector and cytotoxic functions compared to other CD8TEM. Importantly, these distinct and persisting clonotypes could already be delineated in the donor. We confirmed these phenotypes on the protein level and their potential for selection from the graft. Thus, we identified a transcriptional signature associated with persistence and expansion of donor T-cell clonotypes after alloHSCT that may be exploited for personalized graft manipulation strategies in future studies.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

Population Pharmacokinetics of Piperacillin/Tazobactam Across the Adult Lifespan.

BACKGROUND AND OBJECTIVE: Piperacillin/tazobactam is one of the most frequently used antimicrobials in older adults. Using an opportunistic study design, we evaluated the pharmacokinetics of piperacillin/tazobactam as a probe drug to evaluate changes in antibacterial drug exposure and dosing requirements, including in older adults. METHODS: A total of 121 adult patients were included. The population pharmacokinetic models that best characterized the observed plasma concentrations of piperacillin and tazobactam were one-compartment structural models with zero-order input and linear elimination. RESULTS: Among all potential covariates, estimated creatinine clearance had the most substantial impact on the elimination clearance for both piperacillin and tazobactam. After accounting for renal function and body size, there was no remaining impact of frailty on the pharmacokinetics of piperacillin and tazobactam. Monte Carlo simulations indicated that renal function had a greater impact on the therapeutic target attainment than age, although these covariates were highly correlated. Frailty, using the Canadian Study of Health and Aging Clinical Frailty Scale, was assessed in 60 patients who were ≥ 65 years of age. CONCLUSIONS: The simulations suggested that adults ≤ 50 years of age infected with organisms with higher minimum inhibitory concentrations may benefit from continuous piperacillin/tazobactam infusions (12 g/day of piperacillin component) or extended infusions of 4 g every 8 hours. However, for a target of 50% fT + minimum inhibitory concentration, dosing based on renal function is generally preferable to dosing by age, and simulations suggested that patients with creatinine clearance ≥ 120 mL/min may benefit from infusions of 4 g every 8 hours for organisms with higher minimum inhibitory concentrations.

A single-cell RNA labeling strategy for measuring stress response upon tissue dissociation.

Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response.

[Primary total knee arthroplasty following trauma]. / Primäre Kniegelenkendoprothetik nach Trauma.

BACKGROUND: Primary total knee arthroplasty following complex knee joint trauma is only performed occasionally. In most cases a reconstruction is carried out. OBJECTIVE: Are there confirmed indications for primary total knee arthroplasty following trauma? Which special features should be paid attention to? MATERIAL AND METHODS: A selective literature search was carried out. The spectrum of indications and recommendations for action for primary total knee arthroplasty following trauma are presented, particularly against the background of demographic changes. RESULTS: The spectrum of indications for primary total knee arthroplasty following trauma is limited. This has so far been carried out only in centers with the appropriate equipment and expertise, also for the management of complications but despite good overall results is still carried out only rarely. There is a lack of studies with large patient collectives. CONCLUSION: Primary total knee arthroplasty following trauma is a safe procedure within the range of indications. The standard procedure for the vast majority of cases of complex knee trauma is a reconstruction.

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model.

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology.

Single cell- and spatial 'Omics revolutionize physiology.

Single cell multi- 'Omics and Spatial Transcriptomics are prominent technological highlights of recent years, and both fields still witness a ceaseless firework of novel approaches for high resolution profiling of additional omics layers. As all life processes in organs and organisms are based on the functions of their fundamental building blocks, the individual cells and their interactions, these methods are of utmost worth for the study of physiology in health and disease. Recent discoveries on embryonic development, tumor immunology, the detailed cellular composition and function of complex tissues like for example the kidney or the brain, different roles of the same cell type in different organs, the oncogenic program of individual tumor entities, or the architecture of immunopathology in infected tissue are based on single cell and spatial transcriptomics experiments. In this review, we will give a broad overview of technological concepts for single cell and spatial analysis, showing both advantages and limitations, and illustrate their impact with some particularly impressive case studies.

Tongue immune compartment analysis reveals spatial macrophage heterogeneity.

The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication, and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here, we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single-cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1+ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2+ macrophages were detected in deeper muscular tissue. In silico analysis indicated that the two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo. Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

Population Pharmacokinetics of Moxifloxacin in Children.

BACKGROUND/OBJECTIVE: Moxifloxacin is a fluoroquinolone that is commonly used in adults, but not children. Certain clinical situations compel pediatric clinicians to use moxifloxacin, despite its potential for toxicity and limited pharmacokinetics (PK) data. Our objective was to further characterize the pharmacokinetics of moxifloxacin in children. METHODS: We performed an opportunistic, open-label population PK study of moxifloxacin in children < 18 years of age who received moxifloxacin as part of standard care. A set of structural PK models and residual error models were explored using nonlinear mixed-effects modeling. Covariates with known biological relationships were investigated for their influence on PK parameters. RESULTS: We obtained 43 moxifloxacin concentrations from 14 participants who received moxifloxacin intravenously (n = 8) or orally (n = 6). The dose of moxifloxacin was 10 mg/kg daily in participants ≤ 40 kg and 400 mg daily in participants > 40 kg. The population mean clearance and mean volume of distribution were 18.2 L/h and 167 L, respectively. The oral absorption was described by a first-order process. The estimated extent of oral bioavailability was highly variable (range 20-91%). Total body weight was identified as a covariate on clearance and volume of distribution, and substantially reduced the random unexplained inter-individual variability for both parameters. No participants experienced suspected serious adverse reactions related to moxifloxacin. CONCLUSION: These data add to the existing literature to support use of moxifloxacin in children in certain situations; however, further prospective studies on the safety and efficacy of moxifloxacin are needed.

Adverse Reactions in a Phase 1 Trial of the Anti-Malarial DM1157: An Example of Pharmacokinetic Modeling and Simulation Guiding Clinical Trial Decisions.

INTRODUCTION: There is an urgent need to develop new drugs to treat malaria due to increasing resistance to first-line therapeutics targeting the causative organism, Plasmodium falciparum (P. falciparum). One drug candidate is DM1157, a small molecule that inhibits the formation of hemozoin, which protects P. falciparum from heme toxicity. We describe a first-in-human, phase 1 trial of DM1157 in healthy adult volunteers that was halted early because of significant toxicity. METHODS: Adverse events were summarized using descriptive statistics. We used pharmacokinetic modeling to quantitatively assess whether the DM1157 exposure needed for P. falciparum inhibition was achievable at safe doses. RESULTS: We found that there was no dose where both the safety and efficacy target were simultaneously achieved; conversely, the model predicted that 27 mg was the highest dosage at which patients would consistently maintain safe exposure with multiple dosing. By pre-defining dose escalation stopping rules and conducting an interim pharmacokinetic/pharmacodynamic analysis, we determined that the study would be unable to safely achieve a dosage needed to observe an anti-malarial effect, thereby providing strong rationale to halt the study. CONCLUSION: This study provides an important example of the risks and challenges of conducting early phase research as well as the role of modeling and simulation to optimize participant safety (ClinicalTrials.gov, NCT03490162).

Short- vs Standard-Course Outpatient Antibiotic Therapy for Community-Acquired Pneumonia in Children: The SCOUT-CAP Randomized Clinical Trial.

IMPORTANCE: Childhood community-acquired pneumonia (CAP) is usually treated with 10 days of antibiotics. Shorter courses may be effective with fewer adverse effects and decreased potential for antibiotic resistance. OBJECTIVE: To compare a short (5-day) vs standard (10-day) antibiotic treatment strategy for CAP in young children. DESIGN, SETTING, AND PARTICIPANTS: Randomized double-blind placebo-controlled clinical trial in outpatient clinic, urgent care, or emergency settings in 8 US cities. A total of 380 healthy children aged 6 to 71 months with nonsevere CAP demonstrating early clinical improvement were enrolled from December 2, 2016, to December 16, 2019. Data were analyzed from January to September 2020. INTERVENTION: On day 6 of their originally prescribed therapy, participants were randomized 1:1 to receive 5 days of matching placebo or 5 additional days of the same antibiotic. MAIN OUTCOMES AND MEASURES: The primary end point was the end-of-treatment response adjusted for duration of antibiotic risk (RADAR), a composite end point that ranks each child's clinical response, resolution of symptoms, and antibiotic-associated adverse effects in an ordinal desirability of outcome ranking (DOOR). Within each DOOR rank, participants were further ranked by the number of antibiotic days, assuming that shorter antibiotic durations were more desirable. Using RADAR, the probability of a more desirable outcome was estimated for the short- vs standard-course strategy. In a subset of children, throat swabs were collected between study days 19 and 25 to quantify antibiotic resistance genes in oropharyngeal flora. RESULTS: A total of 380 children (189 randomized to short course and 191 randomized to standard course) made up the study population. The mean (SD) age was 35.7 (17.2) months, and 194 participants (51%) were male. Of the included children, 8 were Asian, 99 were Black or African American, 234 were White, 32 were multiracial, and 7 were of unknown or unreported race; 33 were Hispanic or Latino, 344 were not Hispanic or Latino, and 3 were of unknown or unreported ethnicity. There were no differences between strategies in the DOOR or its individual components. Fewer than 10% of children in either strategy had an inadequate clinical response. The short-course strategy had a 69% (95% CI, 63-75) probability of a more desirable RADAR outcome compared with the standard-course strategy. A total of 171 children were included in the resistome analysis. The median (range) number of antibiotic resistance genes per prokaryotic cell (RGPC) was significantly lower in the short-course strategy compared with the standard-course strategy for total RGPC (1.17 [0.35-2.43] vs 1.33 [0.46-11.08]; P = .01) and ß-lactamase RGPC (0.55 [0.18-1.24] vs 0.60 [0.21-2.45]; P = .03). CONCLUSIONS AND RELEVANCE: In this study, among children responding to initial treatment for outpatient CAP, a 5-day antibiotic strategy was superior to a 10-day strategy. The shortened approach resulted in similar clinical response and antibiotic-associated adverse effects, while reducing antibiotic exposure and resistance. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02891915.

Human Monoclonal Antibody Cocktail for the Treatment or Prophylaxis of Middle East Respiratory Syndrome Coronavirus.

BACKGROUND: REGN3048 and REGN3051 are human monoclonal antibodies (mAb) targeting the spike glycoprotein on the Middle East respiratory syndrome coronavirus (MERS-CoV), which binds to the receptor dipeptidyl peptidase-4 (DPP4) and is necessary for infection of susceptible cells. METHODS: Preclinical study: REGN3048, REGN3051 and isotype immunoglobulin G (IgG) were administered to humanized DPP4 (huDPP4) mice 1 day prior to and 1 day after infection with MERS-CoV (Jordan strain). Virus titers and lung pathology were assessed. Phase 1 study: healthy adults received the combined mAb (n = 36) or placebo (n = 12) and followed for 121 days. Six dose levels were studied. Strict safety criteria were met prior to dose escalation. RESULTS: Preclinical study: REGN3048 plus REGN3051, prophylactically or therapeutically, was substantially more effective for reducing viral titer, lung inflammation, and pathology in huDPP4 mice compared with control antibodies and to each antibody monotherapy. Phase 1 study: REGN3048 plus REGN3051 was well tolerated with no dose-limiting adverse events, deaths, serious adverse events, or infusion reactions. Each mAb displayed pharmacokinetics expected of human IgG1 antibodies; it was not immunogenic. CONCLUSIONS: REGN3048 and REGN3051 in combination were well tolerated. The clinical and preclinical data support further development for the treatment or prophylaxis of MERS-CoV infection.

Maximizing transcription of nucleic acids with efficient T7 promoters.

In vitro transcription using T7 bacteriophage polymerase is widely used in molecular biology. Here, we use 5'RACE-Seq to screen a randomized initially transcribed region of the T7 promoter for cross-talk with transcriptional activity. We reveal that sequences from position +4 to +8 downstream of the transcription start site affect T7 promoter activity over a 5-fold range, and identify promoter variants with significantly enhanced transcriptional output that increase the yield of in vitro transcription reactions across a wide range of template concentrations. We furthermore introduce CEL-Seq+ , which uses an optimized T7 promoter to amplify cDNA for single-cell RNA-Sequencing. CEL-Seq+ facilitates scRNA-Seq library preparation, and substantially increases library complexity and the number of expressed genes detected per cell, highlighting a particular value of optimized T7 promoters in bioanalytical applications.