Parvovirus B19 and Human Parvovirus 4 Encode Similar Proteins in a Reading Frame Overlapping the VP1 Capsid Gene.

Viruses frequently contain overlapping genes, which encode functionally unrelated proteins from the same DNA or RNA region but in different reading frames. Yet, overlapping genes are often overlooked during genome annotation, in particular in DNA viruses. Here we looked for the presence of overlapping genes likely to encode a functional protein in human parvovirus B19 (genus Erythroparvovirus), using an experimentally validated software, Synplot2. Synplot2 detected an open reading frame, X, conserved in all erythroparvoviruses, which overlaps the VP1 capsid gene and is under highly significant selection pressure. In a related virus, human parvovirus 4 (genus Tetraparvovirus), Synplot2 also detected an open reading frame under highly significant selection pressure, ARF1, which overlaps the VP1 gene and is conserved in all tetraparvoviruses. These findings provide compelling evidence that the X and ARF1 proteins must be expressed and functional. X and ARF1 have the exact same location (they overlap the region of the VP1 gene encoding the phospholipase A2 domain), are both in the same frame (+1) with respect to the VP1 frame, and encode proteins with similar predicted properties, including a central transmembrane region. Further studies will be needed to determine whether they have a common origin and similar function. X and ARF1 are probably translated either from a polycistronic mRNA by a non-canonical mechanism, or from an unmapped monocistronic mRNA. Finally, we also discovered proteins predicted to be expressed from a frame overlapping VP1 in other species related to parvovirus B19: porcine parvovirus 2 (Z protein) and bovine parvovirus 3 (X-like protein).

WIV, a protein domain found in a wide number of arthropod viruses, which probably facilitates infection.

The most powerful approach to detect distant homologues of a protein is based on structure prediction and comparison. Yet this approach is still inapplicable to many viral proteins. Therefore, we applied a powerful sequence-based procedure to identify distant homologues of viral proteins. It relies on three principles: (1) traces of sequence similarity can persist beyond the significance cutoff of homology detection programmes; (2) candidate homologues can be identified among proteins with weak sequence similarity to the query by using 'contextual' information, e.g. taxonomy or type of host infected; (3) these candidate homologues can be validated using highly sensitive profile-profile comparison. As a test case, this approach was applied to a protein without known homologues, encoded by ORF4 of Lake Sinai viruses (which infect bees). We discovered that the ORF4 protein contains a domain that has homologues in proteins from >20 taxa of viruses infecting arthropods. We called this domain 'widespread, intriguing, versatile' (WIV), because it is found in proteins with a wide variety of functions and within varied domain contexts. For example, WIV is found in the NSs protein of tospoviruses, a global threat to food security, which infect plants as well as their arthropod vectors; in the RNA2 ORF1-encoded protein of chronic bee paralysis virus, a widespread virus of bees; and in various proteins of cypoviruses, which infect the silkworm Bombyx mori. Structural modelling with AlphaFold indicated that the WIV domain has a previously unknown fold, and bibliographical evidence suggests that it facilitates infection of arthropods.

Orthoparamyxovirinae C Proteins Have a Common Origin and a Common Structural Organization.

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the subfamily Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication, and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports the assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a mini-replicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as the NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins form two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representatives of these two clusters, our discovery that they have a similar structure indicates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strikingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C), depending on which cluster they belong to.

A novel emaravirus comprising five RNA segments is associated with ringspot disease in oak.

We report the complete nucleotide sequence of the genome of a novel virus in ringspot-diseased common oak (Quercus robur L.). The newly identified pathogen is associated with leaf symptoms such as mottle, chlorotic spots and ringspots on diseased trees. High-throughput sequencing (HTS, Illumina RNASeq) was used to explore the virome of a ringspot-diseased oak that had chlorotic ringspots of suspected viral origin on leaves for several years. Bioinformatic analysis of the HTS dataset followed by RT-PCR enabled us to determine complete sequences of four RNA genome segments of a novel virus. These sequences showed high similarity to members of the genus Emaravirus, which includes segmented negative-stranded RNA viruses of economic importance. To verify the ends of each RNA, we conducted rapid amplification of cDNA ends (RACE). We identified an additional genome segment (RNA 5) by RT-PCR using a genus-specific primer (PDAP213) to the conserved 3´ and 5´termini in order to amplify full-length genome segments. RNA 5 encodes a 21-kDa protein that is homologous to the silencing suppressor P8 of High Plains wheat mosaic virus. The five viral RNAs were consistently detected by RT-PCR in ringspot-diseased oaks in Germany, Sweden, and Norway. We conclude that the virus represents a new member of the genus Emaravirus affecting oaks in Germany and in Scandinavia, and we propose the name "common oak ringspot-associated emaravirus" (CORaV).

Four erroneous beliefs thwarting more trustworthy research.

A range of problems currently undermines public trust in biomedical research. We discuss four erroneous beliefs that may prevent the biomedical research community from recognizing the need to focus on deserving this trust, and thus which act as powerful barriers to necessary improvements in the research process.

Phase Ib study of the combination of pexidartinib (PLX3397), a CSF-1R inhibitor, and paclitaxel in patients with advanced solid tumors.

PURPOSE: To evaluate the safety, recommended phase II dose (RP2D) and efficacy of pexidartinib, a colony stimulating factor receptor 1 (CSF-1R) inhibitor, in combination with weekly paclitaxel in patients with advanced solid tumors. PATIENTS AND METHODS: In part 1 of this phase Ib study, 24 patients with advanced solid tumors received escalating doses of pexidartinib with weekly paclitaxel (80 mg/m2). Pexidartinib was administered at 600 mg/day in cohort 1. For subsequent cohorts, the dose was increased by ⩽50% using a standard 3+3 design. In part 2, 30 patients with metastatic solid tumors were enrolled to examine safety, tolerability and efficacy of the RP2D. Pharmacokinetics and biomarkers were also assessed. RESULTS: A total of 51 patients reported ≥1 adverse event(s) (AEs) that were at least possibly related to either study drug. Grade 3-4 AEs, including anemia (26%), neutropenia (22%), lymphopenia (19%), fatigue (15%), and hypertension (11%), were recorded in 38 patients (70%). In part 1, no maximum tolerated dose was achieved and 1600 mg/day was determined to be the RP2D. Of 38 patients evaluable for efficacy, 1 (3%) had complete response, 5 (13%) partial response, 13 (34%) stable disease, and 17 (45%) progressive disease. No drug-drug interactions were found. Plasma CSF-1 levels increased 1.6- to 53-fold, and CD14dim/CD16+ monocyte levels decreased by 57-100%. CONCLUSIONS: The combination of pexidartinib and paclitaxel was generally well tolerated. RP2D for pexidartinib was 1600 mg/day. Pexidartinib blocked CSF-1R signaling, indicating potential for mitigating macrophage tumor infiltration.

Overlapping genes and the proteins they encode differ significantly in their sequence composition from non-overlapping genes.

Overlapping genes represent a fascinating evolutionary puzzle, since they encode two functionally unrelated proteins from the same DNA sequence. They originate by a mechanism of overprinting, in which point mutations in an existing frame allow the expression (the "birth") of a completely new protein from a second frame. In viruses, in which overlapping genes are abundant, these new proteins often play a critical role in infection, yet they are frequently overlooked during genome annotation. This results in erroneous interpretation of mutational studies and in a significant waste of resources. Therefore, overlapping genes need to be correctly detected, especially since they are now thought to be abundant also in eukaryotes. Developing better detection methods and conducting systematic evolutionary studies require a large, reliable benchmark dataset of known cases. We thus assembled a high-quality dataset of 80 viral overlapping genes whose expression is experimentally proven. Many of them were not present in databases. We found that overall, overlapping genes differ significantly from non-overlapping genes in their nucleotide and amino acid composition. In particular, the proteins they encode are enriched in high-degeneracy amino acids and depleted in low-degeneracy ones, which may alleviate the evolutionary constraints acting on overlapping genes. Principal component analysis revealed that the vast majority of overlapping genes follow a similar composition bias, despite their heterogeneity in length and function. Six proven mammalian overlapping genes also followed this bias. We propose that this apparently near-universal composition bias may either favour the birth of overlapping genes, or/and result from selection pressure acting on them.

Bioinformatic and mutational analysis of ophiovirus movement proteins, belonging to the 30K superfamily.

Ophioviridae is a family of segmented, negative-sense, single-stranded RNA plant viruses. We showed that their cell-to-cell movement protein (MP) is an isolated member of the 30K MP superfamily with a unique structural organization. All 30K MPs share a core domain that contains a nearly-invariant signature aspartate. We examined its role in the MP of Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV). Alanine substitution of this aspartate prevented plasmodesmata accumulation of MP(MiLBVV), while MP(CPsV) was not affected. The capacity of ophiovirus MPs to increase the plasmodesmata size exclusion limit and non-cell autonomous protein feature was abolished in both mutants. To investigate the role of the signature aspartate in cell-to-cell movement, we constructed a new movement-deficient Tobacco mosaic virus vector used for trans-complementation assays. We showed that both ophiovirus MP mutants lack the cell-to-cell movement capacity, confirming that this signature aspartate is essential for viral cell-to-cell movement.

Sequence analysis reveals a conserved extension in the capping enzyme of the alphavirus supergroup, and a homologous domain in nodaviruses.

BACKGROUND: Members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis E virus and rubella virus. They encode a capping enzyme with methyltransferase-guanylyltransferase (MTase-GTase) activity, which is an attractive drug target owing to its unique mechanism. However, its experimental study has proven very difficult. RESULTS: We examined over 50 genera of viruses by sequence analyses. Earlier studies showed that the MTase-GTase contains a "Core" region conserved in sequence. We show that it is followed by a long extension, which we termed "Iceberg" region, whose secondary structure, but not sequence, is strikingly conserved throughout the alphavirus supergroup. Sequence analyses strongly suggest that the minimal capping domain corresponds to the Core and Iceberg regions combined, which is supported by earlier experimental data. The Iceberg region contains all known membrane association sites that contribute to the assembly of viral replication factories. We predict that it may also contain an overlooked, widely conserved membrane-binding amphipathic helix. Unexpectedly, we detected a sequence homolog of the alphavirus MTase-GTase in taxa related to nodaviruses and to chronic bee paralysis virus. The presence of a capping enzyme in nodaviruses is biologically consistent, since they have capped genomes but replicate in the cytoplasm, where no cellular capping enzyme is present. The putative MTase-GTase domain of nodaviruses also contains membrane-binding sites that may drive the assembly of viral replication factories, revealing an unsuspected parallel with the alphavirus supergroup. CONCLUSIONS: Our work will guide the functional analysis of the alphaviral MTase-GTase and the production of domains for structure determination. The identification of a homologous domain in a simple model system, nodaviruses, which replicate in numerous eukaryotic cell systems (yeast, flies, worms, mammals, and plants), can further help crack the function and structure of the enzyme.

An innovative lab-based training program to help patient groups understand their disease and the research process.

Genuine partnership between patient groups and medical experts is important but challenging. Our training program meets this challenge by organizing hands-on, lab-based training sessions for members of patient groups. These sessions allow "trainees" to better understand their disease and the biomedical research process, and strengthen links between patients and local researchers. Over the past decade, we and our partner institutes have received more than 900 French patients, with the participation of over 60 researchers and clinicians.

Directed growth of electroactive metal-organic framework thin films using electrophoretic deposition.

Electrophoretic deposition (EPD) is used to assemble metal-organic framework (MOF) materials in nano- and micro-particulate, thin-film form. The flexibility of the method is demonstrated by the successful deposition of 4 types of MOFs: NU-1000, UiO-66, HKUST-1, and Al-MIL-53. Additionally, EPD is used to pattern the growth of NU-1000 thin films that exhibit full electrochemical activity.

Evolution and structural organization of the C proteins of paramyxovirinae.

The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus. We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, α-helical C-terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations.

Powerful sequence similarity search methods and in-depth manual analyses can identify remote homologs in many apparently "orphan" viral proteins.

The genome sequences of new viruses often contain many "orphan" or "taxon-specific" proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as "genus specific" by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virions.

Viral proteins originated de novo by overprinting can be identified by codon usage: application to the "gene nursery" of Deltaretroviruses.

A well-known mechanism through which new protein-coding genes originate is by modification of pre-existing genes, e.g. by duplication or horizontal transfer. In contrast, many viruses generate protein-coding genes de novo, via the overprinting of a new reading frame onto an existing ("ancestral") frame. This mechanism is thought to play an important role in viral pathogenicity, but has been poorly explored, perhaps because identifying the de novo frames is very challenging. Therefore, a new approach to detect them was needed. We assembled a reference set of overlapping genes for which we could reliably determine the ancestral frames, and found that their codon usage was significantly closer to that of the rest of the viral genome than the codon usage of de novo frames. Based on this observation, we designed a method that allowed the identification of de novo frames based on their codon usage with a very good specificity, but intermediate sensitivity. Using our method, we predicted that the Rex gene of deltaretroviruses has originated de novo by overprinting the Tax gene. Intriguingly, several genes in the same genomic region have also originated de novo and encode proteins that regulate the functions of Tax. Such "gene nurseries" may be common in viral genomes. Finally, our results confirm that the genomic GC content is not the only determinant of codon usage in viruses and suggest that a constraint linked to translation must influence codon usage.

Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form ß-strands, namely ß1 and ß2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the ß1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within ß1 without preventing the membrane association or movement function of P4.

Evolution of viral proteins originated de novo by overprinting.

New protein-coding genes can originate either through modification of existing genes or de novo. Recently, the importance of de novo origination has been recognized in eukaryotes, although eukaryotic genes originated de novo are relatively rare and difficult to identify. In contrast, viruses contain many de novo genes, namely those in which an existing gene has been "overprinted" by a new open reading frame, a process that generates a new protein-coding gene overlapping the ancestral gene. We analyzed the evolution of 12 experimentally validated viral genes that originated de novo and estimated their relative ages. We found that young de novo genes have a different codon usage from the rest of the genome. They evolve rapidly and are under positive or weak purifying selection. Thus, young de novo genes might have strain-specific functions, or no function, and would be difficult to detect using current genome annotation methods that rely on the sequence signature of purifying selection. In contrast to young de novo genes, older de novo genes have a codon usage that is similar to the rest of the genome. They evolve slowly and are under stronger purifying selection. Some of the oldest de novo genes evolve under stronger selection pressure than the ancestral gene they overlap, suggesting an evolutionary tug of war between the ancestral and the de novo gene.

Detecting remote sequence homology in disordered proteins: discovery of conserved motifs in the N-termini of Mononegavirales phosphoproteins.

Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P) plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11-16aa), several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains) that could be detected simply by comparing orthologous proteins.

Randomized phase II study of bevacizumab in combination with chemotherapy in previously untreated extensive-stage small-cell lung cancer: results from the SALUTE trial.

PURPOSE: Because of promising efficacy signals in single-arm studies, a placebo-controlled, double-blind, randomized phase II trial was designed to assess the efficacy and safety of adding bevacizumab to first-line standard chemotherapy for treatment of extensive-stage small-cell lung cancer (SCLC). PATIENTS AND METHODS: Patients with SCLC were randomly assigned to receive bevacizumab or placebo, with cisplatin or carboplatin plus etoposide, for four cycles followed by single-agent bevacizumab or placebo until progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). RESULTS: Fifty-two patients were randomly assigned to the bevacizumab group and 50 to the placebo group; 69% versus 66%, respectively, completed four cycles of therapy. Median PFS was higher in the bevacizumab group (5.5 months) than in the placebo group (4.4 months; hazard ratio [HR], 0.53; 95% CI, 0.32 to 0.86). Median overall survival (OS) was similar for both groups (9.4 v 10.9 months for bevacizumab and placebo groups, respectively), with an HR of 1.16 (95% CI, 0.66 to 2.04). Overall response rates were 58% (95% CI, 43% to 71%) for the bevacizumab group and 48% (95% CI, 34% to 62%) for the placebo group. Median duration of response was 4.7 months for the bevacizumab group and 3.2 months for the placebo group. In the bevacizumab and placebo groups, 75% versus 60% of patients, respectively, experienced one or more grade 3 or higher adverse events. No new or unexpected safety signals for bevacizumab were observed. CONCLUSION: The addition of bevacizumab to cisplatin or carboplatin plus etoposide for treatment of extensive-stage SCLC improved PFS, with an acceptable toxicity profile. However, no improvement in OS was observed.

BRIDGE: an open-label phase II trial evaluating the safety of bevacizumab + carboplatin/paclitaxel as first-line treatment for patients with advanced, previously untreated, squamous non-small cell lung cancer.

BACKGROUND: Patients with predominantly squamous non-small cell lung cancer (NSCLC) have been generally excluded from studies of bevacizumab treatment, because squamous histology was identified as a possible risk factor for severe (grade ≥3) pulmonary hemorrhage (PH) in a phase II study. BRIDGE was designed to determine whether delaying initiation of bevacizumab treatment and selecting patients without baseline risk factors for PH would lower the incidence of severe PH among patients with squamous NSCLC. METHODS: Patients in this open-label, single-arm study were treated with carboplatin/paclitaxel for two cycles, followed by carboplatin/paclitaxel and bevacizumab in cycles 3 to 6, followed by bevacizumab until progression or unacceptable toxicity. Eligible patients had stage IIIb, stage IV, or recurrent squamous NSCLC. The primary end point was incidence of grade ≥3 PH. RESULTS: Grade ≥3 PH occurred in 1 of 31 patients who received ≥1 dose of bevacizumab: estimated incidence was 3.2% (90% confidence interval 0.3-13.5%). The patient experienced grade 3 PH, discontinued from the study, then experienced grade 4 PH 10 days later, and died of progressive disease. No other serious bleeding events occurred. Nine patients (29.0%) experienced grade 3 adverse events, including five with hypertension; five patients experienced grade 4 adverse events (dyspnea, PH, basal ganglia infarction, cerebral ischemia, and pain). Median progression-free survival was 6.2 months (95% confidence interval 5.32-7.62 months). CONCLUSIONS: The incidence of grade ≥3 PH was 3.2% (one patient). No new safety signals were identified. Although the rate of PH was low, the number of patients in this study was also low. Treatment of squamous NSCLC with bevacizumab should be considered experimental.

Creative research science experiences for high school students.

A French research institute raises the bar for public outreach with an educational laboratory that engages 1,000 high school students per year in mini research projects.