Expanded molecular detection of MPL codon p.W515 and p.S505N mutations in myeloproliferative neoplasms.

BACKGROUND: Patients negative for the JAK2 p.V617F somatic variant are frequently reflexed to testing for MPL exon 10 variants. Detection of these variants via multiplexed allele-specific PCR followed by fragment analysis has been previously published. The present study builds on this concept by improving the detection of the p.W515A variant, adding a second allele-specific primer to detect the p.W515R variant, and incorporating an improved primer for p.S505N detection. METHODS: The W515 amplification employs 5'-labeled allele-specific forward primers to detect p.W515K, p.W515L, p.W515R, and p.W515A. The p.S505N amplification includes an allele-specific reverse primer with a tail extension. Fragments were subject to capillary electrophoresis on an ABI 3500 Genetic Analyzer and analyzed using GeneMapper 6.0 (Thermo Fisher Scientific). RESULTS: Thirty MPL-negative and 13 MPL-positive samples previously tested by a reference laboratory were tested with the MPL LDT. Results were 100% concordant. The MPL LDT has a limit of detection of at least 5% VAF for the p.W515 variants and 10% VAF for the p.S505N variant. CONCLUSION: Current MPL assays are predominantly focused on p.W515L/K and p.S505N mutations. We have engineered an MPL test for detecting p.W515A/L/K/R and p.S505N variants, thereby increasing the diagnostic yield with little additional expense or technician time.

Impact of a Rapid Molecular Test for Klebsiella pneumoniae Carbapenemase and Ceftazidime-Avibactam Use on Outcomes After Bacteremia Caused by Carbapenem-Resistant Enterobacterales.

BACKGROUND: Patients with bacteremia due to carbapenem-resistant Enterobacterales (CRE) experience delays until appropriate therapy and high mortality rates. Rapid molecular diagnostics for carbapenemases and new ß-lactam/ß-lactamase inhibitors may improve outcomes. METHODS: We conducted an observational study of patients with CRE bacteremia from 2016 to 2018 at 8 New York and New Jersey medical centers and assessed center-specific clinical microbiology practices. We compared time to receipt of active antimicrobial therapy and mortality between patients whose positive blood cultures underwent rapid molecular testing for the Klebsiella pneumoniae carbapenemase (KPC) gene (blaKPC) and patients whose cultures did not undergo this test. CRE isolates underwent antimicrobial susceptibility testing by broth microdilution and carbapenemase profiling by whole-genome sequencing. We also assessed outcomes when ceftazidime-avibactam and polymyxins were used as targeted therapies. RESULTS: Of 137 patients with CRE bacteremia, 89 (65%) had a KPC-producing organism. Patients whose blood cultures underwent blaKPC PCR testing (n = 51) had shorter time until receipt of active therapy (median: 24 vs 50 hours; P = .009) compared with other patients (n = 86) and decreased 14-day (16% vs 37%; P = .007) and 30-day (24% vs 47%; P = .007) mortality. blaKPC PCR testing was associated with decreased 30-day mortality (adjusted odds ratio: .37; 95% CI: .16-.84) in an adjusted model. The 30-day mortality rate was 10% with ceftazidime-avibactam monotherapy and 31% with polymyxin monotherapy (P = .08). CONCLUSIONS: In a KPC-endemic area, blaKPC PCR testing of positive blood cultures was associated with decreased time until appropriate therapy and decreased mortality for CRE bacteremia, and ceftazidime-avibactam is a reasonable first-line therapy for these infections.

Racial/ethnic disparities on inflammation and response to methylprednisolone in severe COVID-19 pneumonia.

BACKGROUND: Racial/ethnic minorities are at higher risk for severe COVID-19. This may be related to social determinants that lead to chronic inflammatory states. The aims of the study were to determine if there are racial/ethnic disparities with inflammatory markers and association of methylprednisolone to in hospital survival. METHODS: This was a secondary analysis of a retrospective cohort study of patients ≥ 18 years of age and admitted for severe COVID-19 pneumonia between March and June 2020 in 13 Hospitals in New Jersey, United States. Patients who received other formulation of corticosteroids were not included. Area under the receiver operating characteristics curves were performed to test for discriminatory ability of each inflammatory makers. Univariate and multivariate Cox regression assessed the association of variables to in hospital survival. RESULTS: Propensity matched sample (n = 759) between no methylprednisolone (n = 380) and methylprednisolone (n = 379) had 338 Whites, 102 Blacks, 61 Asian/Indians, and 251 non-Black non-White Hispanics. Compared to CRP, area under receiving operating characteristic curve for d-dimer in Hispanics (0.742) was statistically different (DeLong Test P = 0.0041). Multivariate cox regression showed that different variables in Blacks [age ≥ 60 years (HR = 3.71, P = 0.0281), mechanical ventilation (HR = 5.07, P = 0.0281) and creatinine ≥ 1.5 mg/dL (HR = 3.61, P = 0.0007)], Whites [cancer (HR = 1.68, P = 0.0213), qSOFA score of 1 (HR = 1.81, P = 0.0213), qSOFA score of 2 (HR = 5.16, P < 0.0001), qSOFA score of 3 (HR = 11.81, P < 0.0001) and creatinine ≥ 1.5 mg/dL (HR = 2.16, P = 0.0006)], Hispanics [hypertension (HR = 2.52, P = 0.0007), cancer (HR = 2.99, P = 0.0244 and D-dimer ≥ 2 mcg/mL (HR = 2.22, P = 0.0077)], and Asian/Indians [ chronic kidney disease (HR = 6.36, P = 0.0031) and CRP > 20 mg/L (HR = 5.02, P = 0.0032)] were statistically significant for mortality. Low dose and high dose methylprednisolone were significantly associated with prolonged survival in Whites [low dose (HR = 0.37, P < 0.0001) and high dose (HR = 0.48, P < 0.0183)] and Asian/Indians [low dose (HR = 0.13, P = 0.0101) and high dose (HR = 0.15, P = 0.01)]. However, high dose was not associated with improved survival compared to low dose. Methylprednisolone was not associated with prolonged survival in Blacks and Hispanics. CONCLUSION: Racial/Ethnic disparities with inflammatory markers preclude the use of one marker as a predictor of survival. Methylprednisolone is associated with prolonged survival in Asian/Indians and Whites.

Efficacy and Safety of Sarilumab in Hospitalized Patients With Coronavirus Disease 2019: A Randomized Clinical Trial.

BACKGROUND: Open-label platform trials and a prospective meta-analysis suggest efficacy of anti-interleukin (IL)-6R therapies in hospitalized patients with coronavirus disease 2019 (COVID-19) receiving corticosteroids. This study evaluated the efficacy and safety of sarilumab, an anti-IL-6R monoclonal antibody, in the treatment of hospitalized patients with COVID-19. METHODS: In this adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial, adults hospitalized with COVID-19 received intravenous sarilumab 400 mg or placebo. The phase 3 primary analysis population included patients with critical COVID-19 receiving mechanical ventilation (MV). The primary outcome was proportion of patients with ≥1-point improvement in clinical status from baseline to day 22. RESULTS: There were 457 and 1365 patients randomized and treated in phases 2 and 3, respectively. In phase 3, patients with critical COVID-19 receiving MV (n = 298; 28.2% on corticosteroids), the proportion with ≥1-point improvement in clinical status (alive, not receiving MV) at day 22 was 43.2% for sarilumab and 35.5% for placebo (risk difference, +7.5%; 95% confidence interval [CI], -7.4 to 21.3; P =.3261), a relative risk improvement of 21.7%. In post hoc analyses pooling phase 2 and 3 critical patients receiving MV, the hazard ratio for death for sarilumab vs placebo was 0.76 (95% CI, .51 to 1.13) overall and 0.49 (95% CI, .25 to .94) in patients receiving corticosteroids at baseline. CONCLUSIONS: This study did not establish the efficacy of sarilumab in hospitalized patients with severe/critical COVID-19. Post hoc analyses were consistent with other studies that found a benefit of sarilumab in patients receiving corticosteroids. CLINICAL TRIALS REGISTRATION: NCT04315298.

Development and Validation of Two RT-qPCR Diagnostic Assays for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 Genomic Targets across Two Specimen Types.

Following the outbreak and subsequent pandemic of coronavirus disease 2019 (COVID-19), clinical diagnostic laboratories worldwide sought accurate and reliable testing methodologies. However, many laboratories were and still are hindered by a number of factors, including an unprecedented demand for testing, reagent and laboratory supply shortages and availability of qualified staff. To respond to these concerns, two separate laboratory-developed tests were validated for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using two different specimen types. In addition, these assays target different genomic regions of SARS-CoV-2, allowing for viral detection and mitigating genetic variation. Lower limit of detection and clinical evaluation studies showed detection of SARS-CoV-2 at 500 cp/mL with nasopharyngeal and saliva samples. These multiplexed RT-qPCR assays, although based on modified CDC, New York State Department of Health, and World Health Organization Emergency Use Authorization tests, allow for higher throughput and rapid turnaround time, benefiting patients, clinicians, and communities as a whole. These cost-effective tests also use readily obtainable reagents, circumventing commercial assay supply chain issues. The laboratory-developed tests described here have improved patient care and are highly adaptable should the need arise at other clinical diagnostic laboratories. Furthermore, the foundation and design of these assays may be modified in the future for detection of COVID-19 variants or other RNA-based viral detection tests.

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders.

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.

Methylprednisolone and 60 Days in Hospital Survival in Coronavirus Disease 2019 Pneumonia.

OBJECTIVES: To determine methylprednisolone's dose, duration, and administration from onset of symptoms and association with 60 days in hospital survival of coronavirus disease 2019 pneumonia. DESIGN: Cohort study. SETTING: Thirteen hospitals in New Jersey, United States during March to June 2020. PATIENTS: Seven-hundred fifty-nine hospitalized coronavirus disease 2019 patients. INTERVENTIONS: We performed a propensity matched cohort study between patients who received methylprednisolone and no methylprednisolone. Patients in the methylprednisolone group were further differentiated into dose (high dose and low dose), duration, and administration from onset of symptoms. MEASUREMENTS AND MAIN RESULTS: In the propensity matched sample, 99 out of 380 (26%) in no methylprednisolone, 69 out of 215 (31.9%) in low-dose methylprednisolone, and 74 out of 164 (55.2%) high-dose methylprednisolone expired. Overall median survival for no methylprednisolone (25.0 d), low-dose methylprednisolone (39.0 d), high-dose methylprednisolone (20.0 d), less than or equal to 7 days duration (19.0 d), 7-14 days duration (30.0 d), greater than 14 days duration (44.0 d), onset of symptoms less than or equal to 7 days (20.0 d), and onset of symptoms 7-14 days (27.0 d) were statistically significant (log-rank p ≤ 0.001). Multivariate Cox regression showed nursing home residents, coronary artery disease, and invasive mechanical ventilation were independently associated with mortality. Methylprednisolone was associated with reduced mortality compared with no methylprednisolone (hazard ratio, 0.40; 95% CI, 0.27-0.59; p < 0.001) but no added benefit with high dose. Low-dose methylprednisolone for 7-14 days was associated with reduced mortality compared with less than or equal to 7 days (hazard ratio, 0.45; 95% CI, 0.22-0.91; p = 0.0273), and no additional benefit if greater than 14 days (hazard ratio, 1.27; 95% CI, 0.60-2.69; p = 0.5434). Combination therapy with tocilizumab was associated with reduced mortality over monotherapy (p < 0.0116). CONCLUSIONS: Low-dose methylprednisolone was associated with reduced mortality if given greater than 7 days from onset of symptoms, and no additional benefit greater than 14 days. High dose was associated with higher mortality.

Clinical and laboratory evaluation of patients with SARS-CoV-2 pneumonia treated with high-titer convalescent plasma.

Here, we report on a phase IIa study to determine the intubation rate, survival, viral clearance, and development of endogenous Abs in patients with COVID-19 pneumonia treated with convalescent plasma (CCP) containing high levels of neutralizing anti-SARS-CoV-2 Abs. Radiographic and laboratory evaluation confirmed all 51 treated patients had COVID-19 pneumonia. Fresh or frozen CCP from donors with high titers of neutralizing Abs was administered. The nonmechanically ventilated patients (n = 36) had an intubation rate of 13.9% and a 30-day survival rate of 88.9%, and the overall survival rate for a comparative group based on network data was 72.5% (1625/2241). Patients had negative nasopharyngeal swab rates of 43.8% and 73.0% on days 10 and 30, respectively. Patients mechanically ventilated had a day-30 mortality rate of 46.7%; the mortality rate for a comparative group based on network data was 71.0% (369/520). All evaluable patients were found to have neutralizing Abs on day 3 (n = 47), and all but 1 patient had Abs on days 30 and 60. The only adverse event was a mild rash. In this study on patients with COVID-19 disease, we show therapeutic use of CCP was safe and conferred transfer of Abs, while preserving endogenous immune response.

Hydroxychloroquine and tocilizumab therapy in COVID-19 patients-An observational study.

Hydroxychloroquine has been touted as a potential COVID-19 treatment. Tocilizumab, an inhibitor of IL-6, has also been proposed as a treatment of critically ill patients. In this retrospective observational cohort study drawn from electronic health records we sought to describe the association between mortality and hydroxychloroquine or tocilizumab therapy among hospitalized COVID-19 patients. Patients were hospitalized at a 13-hospital network spanning New Jersey USA between March 1, 2020 and April 22, 2020 with positive polymerase chain reaction results for SARS-CoV-2. Follow up was through May 5, 2020. Among 2512 hospitalized patients with COVID-19 there have been 547 deaths (22%), 1539 (61%) discharges and 426 (17%) remain hospitalized. 1914 (76%) received at least one dose of hydroxychloroquine and 1473 (59%) received hydroxychloroquine with azithromycin. After adjusting for imbalances via propensity modeling, compared to receiving neither drug, there were no significant differences in associated mortality for patients receiving any hydroxychloroquine during the hospitalization (HR, 0.99 [95% CI, 0.80-1.22]), hydroxychloroquine alone (HR, 1.02 [95% CI, 0.83-1.27]), or hydroxychloroquine with azithromycin (HR, 0.98 [95% CI, 0.75-1.28]). The 30-day unadjusted mortality for patients receiving hydroxychloroquine alone, azithromycin alone, the combination or neither drug was 25%, 20%, 18%, and 20%, respectively. Among 547 evaluable ICU patients, including 134 receiving tocilizumab in the ICU, an exploratory analysis found a trend towards an improved survival association with tocilizumab treatment (adjusted HR, 0.76 [95% CI, 0.57-1.00]), with 30 day unadjusted mortality with and without tocilizumab of 46% versus 56%. This observational cohort study suggests hydroxychloroquine, either alone or in combination with azithromycin, was not associated with a survival benefit among hospitalized COVID-19 patients. Tocilizumab demonstrated a trend association towards reduced mortality among ICU patients. Our findings are limited to hospitalized patients and must be interpreted with caution while awaiting results of randomized trials. Trial Registration: Clinicaltrials.gov Identifier: NCT04347993.

Hypergeometric decomposition of symmetric K3 quartic pencils.

We study the hypergeometric functions associated to five one-parameter deformations of Delsarte K3 quartic hypersurfaces in projective space. We compute all of their Picard-Fuchs differential equations; we count points using Gauss sums and rewrite this in terms of finite-field hypergeometric sums; then we match up each differential equation to a factor of the zeta function, and we write this in terms of global L-functions. This computation gives a complete, explicit description of the motives for these pencils in terms of hypergeometric motives.

Multicenter Clinical and Molecular Epidemiological Analysis of Bacteremia Due to Carbapenem-Resistant Enterobacteriaceae (CRE) in the CRE Epicenter of the United States.

Although the New York/New Jersey (NY/NJ) area is an epicenter for carbapenem-resistant Enterobacteriaceae (CRE), there are few multicenter studies of CRE from this region. We characterized patients with CRE bacteremia in 2013 at eight NY/NJ medical centers and determined the prevalence of carbapenem resistance among Enterobacteriaceae bloodstream isolates and CRE resistance mechanisms, genetic backgrounds, capsular types (cps), and antimicrobial susceptibilities. Of 121 patients with CRE bacteremia, 50% had cancer or had undergone transplantation. The prevalences of carbapenem resistance among Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli bacteremias were 9.7%, 2.2%, and 0.1%, respectively. Ninety percent of CRE were K. pneumoniae and 92% produced K. pneumoniae carbapenemase (KPC-3, 48%; KPC-2, 44%). Two CRE produced NDM-1 and OXA-48 carbapenemases. Sequence type 258 (ST258) predominated among KPC-producing K. pneumoniae (KPC-Kp). The wzi154 allele, corresponding to cps-2, was present in 93% of KPC-3-Kp, whereas KPC-2-Kp had greater cps diversity. Ninety-nine percent of CRE were ceftazidime-avibactam (CAZ-AVI)-susceptible, although 42% of KPC-3-Kp had an CAZ-AVI MIC of ≥4/4 µg/ml. There was a median of 47 h from bacteremia onset until active antimicrobial therapy, 38% of patients had septic shock, and 49% died within 30 days. KPC-3-Kp bacteremia (adjusted odds ratio [aOR], 2.58; P = 0.045), cancer (aOR, 3.61, P = 0.01), and bacteremia onset in the intensive care unit (aOR, 3.79; P = 0.03) were independently associated with mortality. Active empirical therapy and combination therapy were not associated with survival. Despite a decade of experience with CRE, patients with CRE bacteremia have protracted delays in appropriate therapies and high mortality rates, highlighting the need for rapid diagnostics and evaluation of new therapeutics.

Heat shock 70-kDa protein 5 (Hspa5) is essential for pronephros formation by mediating retinoic acid signaling.

Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and ß1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development.

barx1 is necessary for ectomesenchyme proliferation and osteochondroprogenitor condensation in the zebrafish pharyngeal arches.

Barx1 modulates cellular adhesion molecule expression and participates in specification of tooth-types, but little is understood of its role in patterning the pharyngeal arches. We examined barx1 expression during zebrafish craniofacial development and performed a functional analysis using antisense morpholino oligonucleotides. Barx1 is expressed in the rhombencephalic neural crest, the pharyngeal arches, the pectoral fin buds and the gut in contrast to its paralogue barx2, which is most prominently expressed in the arch epithelium. Additionally, barx1 transient expression was observed in the posterior lateral line ganglia and developing trunk/tail. We show that Barx1 is necessary for proliferation of the arch osteochondrogenic progenitors, and that morphants exhibit diminished and dysmorphic arch cartilage elements due to reductions in chondrocyte differentiation and condensation. Attenuation of Barx1 results in lost arch expression of osteochondrogenic markers col2a1, runx2a and chondromodulin, as well as odontogenic marker dlx2b. Further, loss of barx1 positively influenced gdf5 and chordin, markers of jaw joint patterning. FGF signaling is required for maintaining barx1 expression, and that ectopic BMP4 induces expression of barx1 in the intermediate region of the second pharyngeal arch. Together, these results indicate an essential role for barx1 at early stages of chondrogenesis within the developing zebrafish viscerocranium.

Zebrafish dlx2a contributes to hindbrain neural crest survival, is necessary for differentiation of sensory ganglia and functions with dlx1a in maturation of the arch cartilage elements.

The Dlx genes are expressed in a coordinate manner, establishing proximal-distal polarity within the pharyngeal arches. In zebrafish, dlx2a is expressed in the migrating cranial neural crest that contributes to the pharyngeal arches. Expression of dlx2a in the arches is subsequently followed by overlapping expression of the physically linked dlx1a gene, and of other paralogues that include dlx5a/dlx6a and dlx3b/dlx4b. To investigate the patterning and establishment of arch proximodistal polarity in zebrafish, we characterized the function of dlx2a and dlx1a, using antisense morpholino oligonucleotides (MOs). We show that embryos injected with dlx1a and dlx2a MOs exhibit reduced and dysmorphic arch cartilage elements. The combined loss of dlx1a and dlx2a causes severe arch cartilage dysmorphology, revealing a role for these genes in maturation and patterning of arch chondrogenesis. Knockdown of dlx2a affects migrating neural crest cells as evidenced by reduced expression of crestin, and sox9a transcripts, in addition to increased levels of apoptosis. During pharyngogenesis, loss of dlx2a results in aberrant barx1 expression and the absence of goosecoid transcripts in the dorsal region of the ceratohyal arch. Defects in the differentiation of ectomesenchymal derivatives, including sensory ganglia and cartilage elements, indicate a role for dlx2a in specification and maintenance of cranial neural crest.

Isolation and expression of zebrafish zinc-finger transcription factor gene tsh1.

We report the expression patterns of tsh1, a zebrafish homologue of the Drosophila homeotic gene teashirt. Expression of tsh1 is first detected at the 2-somite stage (10h post-fertilization, hpf) at the anterior end of the spinal cord. Expression expands toward the posterior spinal cord, and by the prim-5 stage (24 hpf) tsh1 transcripts are detected throughout spinal cord. Between the 14- and 25-somite stage (16-24 hpf), spinal cord expression shows a clear anterior boundary at the rostral margin of rhombomere 7. Around the prim-25 stage (36 hpf), while the spinal expression of tsh1 decreases, new expression is detected in the pectoral fin buds and dorsal forebrain. By the long-pec stage (48 hpf), spinal cord expression is undetectable, but strong expression is observed in the rhombencephalon, telencephalon, tectum opticum, midbrain-hindbrain boundary, in the first pharyngeal arch and in the eyes. This expression persists at least until the larval stages. Retinoic acid signaling influences tsh1 expression. Zebrafish tsh1 expression was induced in the anterior neural tube in embryos treated briefly with exogenous retinoic acid. Furthermore, tsh1 expression was down-regulated in the spinal cord in the zebrafish neckless mutant in which RA signaling is disrupted due to a missense mutation in the gene encoding retinaldehyde dehydrogenase type 2.

Echinacea purpurea for prevention of experimental rhinovirus colds.

A randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the ability of Echinacea purpurea to prevent infection with rhinovirus type 39 (RV-39). Forty-eight previously healthy adults received echinacea or placebo, 2.5 mL 3 times per day, for 7 days before and 7 days after intranasal inoculation with RV-39. Symptoms were assessed to evaluate clinical illness. Viral culture and serologic studies were performed to evaluate the presence of rhinovirus infection. A total of 92% of echinacea recipients and 95% of placebo recipients were infected. Colds developed in 58% of echinacea recipients and 82% of placebo recipients (P=.114, by Fisher's exact test). Administration of echinacea before and after exposure to rhinovirus did not decrease the rate of infection; however, because of the small sample size, statistical hypothesis testing had relatively poor power to detect statistically significant differences in the frequency and severity of illness.

Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches.

The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.