A Prolonged Outbreak of Human Adenovirus A31 (HAdV-A31) Infection on a Pediatric Hematopoietic Stem Cell Transplantation Ward with Whole Genome Sequencing Evidence of International Linkages.

A surge in hematopoietic stem cell transplantation (HSCT) human adenovirus A31 (HAdV-A31) infections was initially observed in late 2014/2015 at SickKids (SK) Hospital, Toronto, Canada. In response, enhanced laboratory monitoring for all adenovirus infections was conducted. Positive samples underwent genotyping, viral culture, and, in selected cases, whole-genome sequencing (WGS). HAdV-A31 specimens/DNA obtained from four international pediatric HSCT centers also underwent WGS. During the SK outbreak period (27 October 2014 to 31 October 2018), 17/20 HAdV-A31 isolates formed a distinct clade with 0 to 8 mutations between the closest neighbors. Surveillance before and after the outbreak detected six additional HAdV-A31 HSCT cases; three of the four sequenced cases clustered within the outbreak clade. Two SK outbreak isolates were identical to sequences from two patients in an outbreak in England. Three SK non-outbreak sequences also had high sequence similarity to strains from three international centers. Environmental PCR testing of the HSCT ward showed significant adenovirus contamination. Despite intense infection control efforts, we observed re-occurrence of infection with the outbreak strain. Severe but nonfatal infection was observed more commonly with HAdV-A31 compared to other genotypes, except HAdV-C1. Our findings strongly implicate nosocomial spread of HAdV-A31 over 10 years on a HSCT unit and demonstrate the value of WGS in defining and mapping the outbreak. Close linkages among strains in different countries suggest international dissemination, though the mechanism is undetermined. This large, extended outbreak emphasizes the pre-eminent role of HAdV-A31 in causing intractable pediatric HSCT outbreaks of severe illness worldwide.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE: Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Half the story: Thermal effects on within-host infectious disease progression in a warming climate.

Immune defense is temperature dependent in cold-blooded vertebrates (CBVs) and thus directly impacted by global warming. We examined whether immunity and within-host infectious disease progression are altered in CBVs under realistic climate warming in a seasonal mid-latitude setting. Going further, we also examined how large thermal effects are in relation to the effects of other environmental variation in such a setting (critical to our ability to project infectious disease dynamics from thermal relationships alone). We employed the three-spined stickleback and three ecologically relevant parasite infections as a "wild" model. To generate a realistic climatic warming scenario we used naturalistic outdoors mesocosms with precise temperature control. We also conducted laboratory experiments to estimate thermal effects on immunity and within-host infectious disease progression under controlled conditions. As experimental readouts we measured disease progression for the parasites and expression in 14 immune-associated genes (providing insight into immunophenotypic responses). Our mesocosm experiment demonstrated significant perturbation due to modest warming (+2°C), altering the magnitude and phenology of disease. Our laboratory experiments demonstrated substantial thermal effects. Prevailing thermal effects were more important than lagged thermal effects and disease progression increased or decreased in severity with increasing temperature in an infection-specific way. Combining laboratory-determined thermal effects with our mesocosm data, we used inverse modeling to partition seasonal variation in Saprolegnia disease progression into a thermal effect and a latent immunocompetence effect (driven by nonthermal environmental variation and correlating with immune gene expression). The immunocompetence effect was large, accounting for at least as much variation in Saprolegnia disease as the thermal effect. This suggests that managers of CBV populations in variable environments may not be able to reliably project infectious disease risk from thermal data alone. Nevertheless, such projections would be improved by primarily considering prevailing thermal effects in the case of within-host disease and by incorporating validated measures of immunocompetence.

An impedance-based approach using human iPSC-derived cardiomyocytes significantly improves in vitro prediction of in vivo cardiotox liabilities.

Current in vitro approaches to cardiac safety testing typically focus on mechanistic ion channel testing to predict in vivo proarrhythmic potential. Outside of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, structural and functional cardiotoxicity related to chronic dosing effects are of great concern as these effects can impact compound attrition. Development and implementation of an in vitro cardiotoxicity screening platform that effectively identifies these liabilities early in the discovery process should reduce costly attrition and decrease preclinical development time. Impedence platforms have the potential to accurately identify structural and functional cardiotoxicity and have sufficient throughput to be included in a multi-parametric optimization approach. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) have demonstrated utility in cardiac safety and toxicity screening. The work described here leverages these advantages to assess the predictive value of data generated by two impedance platforms. The response of hIPSC-CMs to compounds with known or predicted cardiac functional or structural toxicity was determined. The compounds elicited cardiac activities and/or effects on "macro" impedance often associated with overt structural or cellular toxicity, detachment, or hypertrophy. These assays correctly predicted in vivo cardiotox findings for 81% of the compounds tested and did not identify false positives. In addition, internal or literature Cmax values from in vivo studies correlated within 4 fold of the in vitro observations. The work presented here demonstrates the predictive power of impedance platforms with hIPSC-CMs and provides a means toward accelerating lead candidate selection by assessing preclinical cardiac safety earlier in the drug discovery process.

Changing expression of vertebrate immunity genes in an anthropogenic environment: a controlled experiment.

BACKGROUND: The effect of anthropogenic environments on the function of the vertebrate immune system is a problem of general importance. For example, it relates to the increasing rates of immunologically-based disease in modern human populations and to the desirability of identifying optimal immune function in domesticated animals. Despite this importance, our present understanding is compromised by a deficit of experimental studies that make adequately matched comparisons between wild and captive vertebrates. RESULTS: We transferred post-larval fishes (three-spined sticklebacks), collected in the wild, to an anthropogenic (captive) environment. We then monitored, over 11 months, how the systemic expression of immunity genes changed in comparison to cohort-matched wild individuals in the originator population (total n = 299). We found that a range of innate (lyz, defbl2, il1r-like, tbk1) and adaptive (cd8a, igmh) immunity genes were up-regulated in captivity, accompanied by an increase in expression of the antioxidant enzyme, gpx4a. For some genes previously known to show seasonality in the wild, this appeared to be reduced in captive fishes. Captive fishes tended to express immunity genes, including igzh, foxp3b, lyz, defbl2, and il1r-like, more variably. Furthermore, although gene co-expression patterns (analyzed through gene-by-gene correlations and mutual information theory based networks) shared common structure in wild and captive fishes, there was also significant divergence. For one gene in particular, defbl2, high expression was associated with adverse health outcomes in captive fishes. CONCLUSION: Taken together, these results demonstrate widespread regulatory changes in the immune system in captive populations, and that the expression of immunity genes is more constrained in the wild. An increase in constitutive systemic immune activity, such as we observed here, may alter the risk of immunopathology and contribute to variance in health in vertebrate populations exposed to anthropogenic environments.

Seasonal immunoregulation in a naturally-occurring vertebrate.

BACKGROUND: Fishes show seasonal patterns of immunity, but such phenomena are imperfectly understood in vertebrates generally, even in humans and mice. As these seasonal patterns may link to infectious disease risk and individual condition, the nature of their control has real practical implications. Here we characterize seasonal dynamics in the expression of conserved vertebrate immunity genes in a naturally-occurring piscine model, the three-spined stickleback. RESULTS: We made genome-wide measurements (RNAseq) of whole-fish mRNA pools (n = 36) at the end of summer and winter in contrasting habitats (riverine and lacustrine) and focussed on common trends to filter habitat-specific from overarching temporal responses. We corroborated this analysis with targeted year-round whole-fish gene expression (Q-PCR) studies in a different year (n = 478). We also considered seasonal tissue-specific expression (6 tissues) (n = 15) at a third contrasting (euryhaline) locality by Q-PCR, further validating the generality of the patterns seen in whole fish analyses. Extremes of season were the dominant predictor of immune expression (compared to sex, ontogeny or habitat). Signatures of adaptive immunity were elevated in late summer. In contrast, late winter was accompanied by signatures of innate immunity (including IL-1 signalling and non-classical complement activity) and modulated toll-like receptor signalling. Negative regulators of T-cell activity were prominent amongst winter-biased genes, suggesting that adaptive immunity is actively down-regulated during winter rather than passively tracking ambient temperature. Network analyses identified a small set of immune genes that might lie close to a regulatory axis. These genes acted as hubs linking summer-biased adaptive pathways, winter-biased innate pathways and other organismal processes, including growth, metabolic dynamics and responses to stress and temperature. Seasonal change was most pronounced in the gill, which contains a considerable concentration of T-cell activity in the stickleback. CONCLUSIONS: Our results suggest major and predictable seasonal re-adjustments of immunity. Further consideration should be given to the effects of such responses in seasonally-occurring disease.

Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.

Humoral, mucosal, and cell-mediated immunity against vaccine and nonvaccine genotypes after administration of quadrivalent human papillomavirus vaccine to HIV-infected children.

OBJECTIVES: To characterize the immunogenicity of a quadrivalent human papillomavirus vaccine (QHPV) in human immunodeficiency virus (HIV)-infected children, we studied their immune responses to 3 or 4 doses. METHODS: HIV-infected children aged 7-12 years with a CD4 cell percentage of ≥15% of lymphocytes, received 3 doses of QHPV with or without a fourth dose after 72 weeks. Type-specific and cross-reactive antibodies and cell-mediated immunity were measured. RESULTS: Type-specific antibodies to HPV6, 11, and 16 were detected in 100% and ≥94% of children at 4 and 72 weeks, respectively, after the third QHPV dose. Corresponding numbers for HPV18 were 97% and 76%, respectively. A fourth QHPV dose increased seropositivity to ≥96% for all vaccine genotypes. Four weeks after the third QHPV dose, 67% of vaccinees seroconverted to HPV31, an HPV16-related genotype not in the vaccine; 69% and 39% of vaccinees developed mucosal HPV16 and 18 immunoglobulin G antibodies, respectively; and 60% and 52% of vaccinees developed cytotoxic T lymphocytes (CTLs) for HPV16 and 31, respectively. CONCLUSIONS: Three QHPV doses generated robust and persistent antibodies to HPV6, 11, and 16 but comparatively weaker responses to HPV18. A fourth dose increased antibodies against all vaccine genotypes in an anamnestic fashion. CTLs and mucosal antibodies against vaccine genotypes, as well as cross-reactive antibodies and CTL against nonvaccine genotypes, were detected.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

BACKGROUND: Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs). RESULTS: VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. CONCLUSIONS: D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

Searching for answers: proper prescribing of controlled prescription drugs.

Prescription drug abuse is increasing at alarming rates in this country. Most often drugs are obtained through relatives or friends. An important step in addressing this problem is educating healthcare providers in the proper prescribing of scheduled drugs. Physicians and other healthcare workers receive little training in proper screening for substance abuse, proper prescribing of scheduled drugs, and referral for those needing treatment. Continuing medical education is one venue for addressing this problem. However, screening, brief intervention and referral for treatment (SBIRT) should be taught in medical school and residency.

Infantile Hypotonia: A Case of Spinal Muscular Atrophy With Respiratory Distress Type 1 Presenting As Infant Botulism.

Spinal muscular atrophy with respiratory distress type 1 (SMARD 1) is a rare autosomal recessive disease characterized by distal muscular atrophy and respiratory distress. It presents between six weeks and six months of age, with an eventual requirement of respiratory support. To date, no curative treatment to attenuate or stop the clinical deterioration has been found; therefore, supportive treatment is the corner stone of management. We report a 12-week-old infant with SMARD1 initially diagnosed and managed as a case of infant botulism secondary to a history of significant exposure to honey. SMARD1 and infant botulism all share characteristic clinical features, namely, respiratory distress, hypotonia, and autonomic dysfunction with typical onset of less than one year of age. This case report illustrates that SMARD1, SMA Type 1, and infant botulism share common clinical features. It is important to maintain a broad differential when evaluating an infant with hypotonia, especially when there is a lack of clinical response to conventional medical interventions directed toward the working diagnosis.

Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural variations among human adenoviruses.

Enteric adenoviruses, one of the main causes of viral gastroenteritis in the world, must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We report the 4-Å-resolution structure of a human enteric adenovirus, HAdV-F41, and compare it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base, and internal minor coat proteins IIIa and VIII are conserved, we observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, we find an organization of the external minor coat protein IX different from all previously characterized human and nonhuman mastadenoviruses. Knowledge of the structure of enteric adenoviruses provides a starting point for the design of vectors suitable for oral delivery or intestinal targeting.

PD-L1 expression on circulating tumor cells and platelets in patients with metastatic breast cancer.

BACKGROUND: Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition. METHODS: Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1. RESULTS: A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p<0.001) and a history of smoking tobacco (OR = 0.76, p<0.001). Platelet PD-L1 staining was not associated with tumor marker status, recent procedures or treatments, platelet-affecting drugs, or CTC PD-L1 expression. CONCLUSION: PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment.

Circulating tumor cell number and endocrine therapy index in ER positive metastatic breast cancer patients.

Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). The CTC-endocrine therapy index (CTC-ETI), consisting of CTC-ER (estrogen receptor), BCL2, human epidermal growth factor receptor (HER2), and Ki67 expression, might predict resistance to endocrine therapy (ET) in patients with ER-positive MBC. One hundred twenty-one patients with ER-positive/HER2-negative MBC initiating a new ET after ≥1 lines of ET were enrolled in a prospective, multi-institutional clinical trial. CTC-ETI and clinical/imaging follow-up were performed at baseline and serial time points. Progression-free survival (PFS) and rapid progression (RP; determined at the 3-month time point) were primary endpoints. Associations with clinical outcomes used logrank and Fisher's exact tests. At baseline, 36% (38/107) of patients had ≥5 CTC/7.5 ml whole blood (WB). Patients with ≥5 vs. <5 CTC/7.5 ml WB had significantly worse PFS (median 3.3 vs. 5.9 months, P = 0.03). Elevated CTC at 1 month was associated with even worse PFS (1.9 vs. 5.0 months from the 1-month sample, P < 0.001). Low, intermediate, and high CTC-ETI were observed in 71 (66%), 8 (8%), and 28 (26%) patients, with median PFS of 6.9, 8.5, and 2.8 months, respectively (P = 0.008). Patients with high vs. low CTC and CTC-ETI more frequently experienced RP (CTC: 66% vs. 41%; P = 0.03; CTC-ETI: 79% vs. 40%; P = 0.002). In conclusion, CTC enumeration and the CTC-ETI assay are prognostic at baseline and follow-up in patients with ER-positive/HER2-negative MBC starting new ET. CTC at first follow-up might identify a group of patients with ER-positive MBC that could forego ET, but CTC-ETI did not contribute further.

The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation.

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

A relatively common homozygous TRAPPC4 splicing variant is associated with an early-infantile neurodegenerative syndrome.

Trafficking protein particle (TRAPP) complexes, which include the TRAPPC4 protein, regulate membrane trafficking between lipid organelles in a process termed vesicular tethering. TRAPPC4 was recently implicated in a recessive neurodevelopmental condition in four unrelated families due to a shared c.454+3A>G splice variant. Here, we report 23 patients from 17 independent families with an early-infantile-onset neurodegenerative presentation, where we also identified the homozygous variant hg38:11:119020256 A>G (NM_016146.5:c.454+3A>G) in TRAPPC4 through exome or genome sequencing. No other clinically relevant TRAPPC4 variants were identified among any of over 10,000 patients with neurodevelopmental conditions. We found the carrier frequency of TRAPPC4 c.454+3A>G was 2.4-5.4 per 10,000 healthy individuals. Affected individuals with the homozygous TRAPPC4 c.454+3A>G variant showed profound psychomotor delay, developmental regression, early-onset epilepsy, microcephaly and progressive spastic tetraplegia. Based upon RNA sequencing, the variant resulted in partial exon 3 skipping and generation of an aberrant transcript owing to use of a downstream cryptic splice donor site, predicting a premature stop codon and nonsense mediated decay. These data confirm the pathogenicity of the TRAPPC4 c.454+3A>G variant, and refine the clinical presentation of TRAPPC4-related encephalopathy.

Effect of oral arginine supplementation on exhaled nitric oxide concentration in sickle cell anemia and acute chest syndrome.

INTRODUCTION: Decreased exhaled nitric oxide levels (FE(NO)) have been described in patients with sickle cell disease (SCD) and a history of acute chest syndrome (ACS) when compared with non-ACS controls. Oral arginine supplementation has been shown to increase FE(NO) in healthy participants, but its effect in SCD patients is not known. OBJECTIVE: To determine the effect of oral arginine intake on FENO in sickle cell patients with and without history of ACS, and in healthy controls. HYPOTHESIS: No differences in the FE(NO) increase were seen in SCD patients with a history of ACS (ACS+) compared with healthy controls (HC) and SCD patients without history of ACS (ACS-). MATERIALS AND METHODS: ACS+ (n=6), ACS- (n=9), and HC (n=7) patients were studied. At baseline, and after the administration of escalating doses of oral L-arginine (0.1, 0.2, and 0.4 g/kg), serial measurements were made of the following: FE(NO), plasma concentrations of arginine, ornithine, citrulline, aspartate, glutamate, arginine/ornithine ratio, nitrite, nitrate, heart rate (HR), respiratory rate (RR), blood pressure (BP), oxygen saturation (SpO2), forced expiratory volume in 1 second (FEV1), and forced vital capacity (FVC). MAIN RESULTS: At baseline, FE(NO) did not differ among the groups. ACS- and ACS+ groups were deficient in arginine, and had decreased FEV1, FVC, and SaO2 when compared with HC patients. After arginine supplementation, FE(NO), arginine, ornithine, citrulline, nitrite, and the arginine/ornithine ratio increased similarly in all groups. Changes from baseline for HR, BP, SpO2, RR, FEV1, and FVC were minimal and similar in all groups. CONCLUSIONS: In contrast to our earlier study, ACS+ patients had similar FE(NO) values when compared with ACS- and HC patients. All SCD patients were arginine deficient at baseline and showed impairment in respiratory physiology when compared with HC patients. After arginine supplementation, FE(NO) concentration increased in all groups to a similar degree, and lung function and physiologic parameters were minimally affected. The physiologic significance of alterations in FE(NO) in SCD patients and its relationship to ACS predilection requires further delineation.

Increased Expression of Interleukin-1 Receptor Characterizes Anti-estrogen-Resistant ALDH+ Breast Cancer Stem Cells.

Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH+ cells) are enriched following AE treatment. Here, we show that the interleukin-1ß (IL-1ß) signaling pathway is activated in ALDH+ cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH+ cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH+IL1R1+ cells merits testing as a strategy to combat AE resistance in patients with residual disease.

Engineered Fibrillar Fibronectin Networks as Three-Dimensional Tissue Scaffolds.

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre-coatings in an attempt to create a cell-supporting environment in both two- and three-dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM-constituting fibrillar protein networks naturally deposited by cells. Here, the cell-free and scalable synthesis of freely suspended and mechanically robust three-dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three-phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial-to-mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre-clinical diagnostics.

Schistosoma mansoni venom allergen-like proteins: phylogenetic relationships, stage-specific transcription and tissue localization as predictors of immunological cross-reactivity.

Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A-D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity.