Multidimensional biomarker predicts disease control in response to immunotherapy in recurrent or metastatic head and neck squamous-cell carcinoma.

PURPOSE: Anti-PD-1 therapy provides clinical benefit in 40-50% of patients with relapsed and/or metastatic head and neck squamous cell carcinoma (RM-HNSCC). Selection of anti- PD-1 therapy is typically based on patient PD-L1 immunohistochemistry (IHC) which has low specificity for predicting disease control. Therefore, there is a critical need for a clinical biomarker that will predict clinical benefit to anti-PD-1 treatment with high specificity. METHODS: Clinical treatment and outcomes data for 103 RM-HNSCC patients were paired with RNA-sequencing data from formalin-fixed patient samples. Using logistic regression methods, we developed a novel biomarker classifier based on expression patterns in the tumor immune microenvironment to predict disease control with monotherapy PD-1 inhibitors (pembrolizumab and nivolumab). The performance of the biomarker was internally validated using out-of-bag methods. RESULTS: The biomarker significantly predicted disease control (65% in predicted non-progressors vs. 17% in predicted progressors, p < 0.001) and was significantly correlated with overall survival (OS; p = 0.004). In addition, the biomarker outperformed PD-L1 IHC across numerous metrics including sensitivity (0.79 vs 0.64, respectively; p = 0.005) and specificity (0.70 vs 0.61, respectively; p = 0.009). CONCLUSION: This novel assay uses tumor immune microenvironment expression data to predict disease control and OS with high sensitivity and specificity in patients with RM-HNSCC treated with anti-PD-1 monotherapy.

Hepatitis B Virus Reactivation Following Treatment of HNSCC With Cisplatin.

Hepatitis B Virus (HBV) reactivation is a known complication of intense immunosuppression with B-cell depleting monoclonal antibody therapy and transplantation immunosuppression. HBV reactivation has occurred following treatment with chemotherapy regimens for hematologic malignancies and solid tumors. There are 2 prior case reports of HBV reactivation following cisplatin monotherapy for head and neck squamous cell carcinoma (HNSCC). Here, we present a case of a 49-year-old Caucasian male with a past medical history of laryngeal squamous cell carcinoma (SCC). There are no consensus guidelines on how to define hepatitis B reactivation. There are guidelines on when to initiate prophylaxis with Entecavir while on immunosuppressive therapy with risk according to medication category and hepatitis B surface antigen/hepatitis B core antibody IgG serology. CDC recommends screening everyone. American Society of Clinical Oncology (ASCO) now with a recent update in 2020 recommends screening everyone. There is a definite role of immunosuppression in HBV reactivation, however, there is also direct enhancement by cisplatin of viral replication by creating endoplasmic reticulum stress which increases HBV DNA indirectly. Finally, cytotoxicity enhances HBV reactivation and immune reconstitution post withdrawing immunosuppressive treatment. Because of the effects of chemotherapy, aka cisplatin goes beyond immunosuppression-related reactivation of HBV, our recommendations are in line with CDC and ASCO to screen all patients for HBV before onset of chemotherapy and start Entecavir/Tenofovir Disoproxil Fumarate before the onset of chemotherapy for HBV-positive patients.

MelRisk: Using neutrophil-to-lymphocyte ratio to improve risk prediction models for metastatic cutaneous melanoma in the sentinel lymph node.

BACKGROUND: Identifying metastatic melanoma in the sentinel lymph node (SLN) is important because 80% of SLN biopsies are negative and 11% of patients develop complications. The neutrophil-to-lymphocyte ratio (NLR), a biomarker of micrometastatic disease, could improve prediction models for SLN status. We externally validated existing models and developed 'MelRisk' prognostic score to better predict SLN metastasis. METHODS: The models were externally validated using data from a multicenter cohort study of 1,251 adults. Additionally, we developed and internally validated a new prognostic score `MelRisk', using candidate predictors derived from the extant literature. RESULTS: The Karakousis model had a C-statistic of 0.58 (95% CI, 0.54-0.62). The Sondak model had a C-statistic of 0.57 (95% CI 0.53-0.61). The MIA model had a C-statistic of 0.60 (95% CI. 0.56-0.64). Our 'MelRisk' model (which used Breslow thickness, ulceration, age, anatomical site, and the NLR) showed an adjusted C-statistic of 0.63 (95% CI, 0.56-0.64). CONCLUSION: Our prediction tool is freely available in the Google Play Store and Apple App Store, and we invite colleagues to externally validate its performance .

Multi-level analysis of reproduction in an Antarctic midge identifies female and male accessory gland products that are altered by larval stress and impact progeny viability.

The Antarctic midge, Belgica antarctica, is a wingless, non-biting midge endemic to Antarctica. Larval development requires at least 2 years, but adults live only 2 weeks. The nonfeeding adults mate in swarms and females die shortly after oviposition. Eggs are suspended in a gel of unknown composition that is expressed from the female accessory gland. This project characterizes molecular mechanisms underlying reproduction in this midge by examining differential gene expression in whole males, females, and larvae, as well as in male and female accessory glands. Functional studies were used to assess the role of the gel encasing the eggs, as well as the impact of stress on reproductive biology. RNA-seq analyses revealed sex- and development-specific gene sets along with those associated with the accessory glands. Proteomic analyses were used to define the composition of the egg-containing gel, which is generated during multiple developmental stages and derived from both the accessory gland and other female organs. Functional studies indicate the gel provides a larval food source as well as a buffer for thermal and dehydration stress. All of these function are critical to juvenile survival. Larval dehydration stress directly reduces production of storage proteins and key accessory gland components, a feature that impacts adult reproductive success. Modeling reveals that bouts of dehydration may have a significant impact on population growth. This work lays a foundation for further examination of reproduction in midges and provides new information related to general reproduction in dipterans. A key aspect of this work is that reproduction and stress dynamics, currently understudied in polar organisms, are likely to prove critical in determining how climate change will alter their survivability.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Do Mosquitoes Sleep?

Sleep is a phenomenon conserved across the animal kingdom, where studies on Drosophila melanogaster have revealed that sleep phenotypes and molecular underpinnings are similar to those in mammals. However, little is known about sleep in blood-feeding arthropods, which have a critical role in public health as disease vectors. Specifically, sleep studies in mosquitoes are lacking despite considerable focus on how circadian processes, which have a central role in regulating sleep/wake cycles, impact activity, feeding, and immunity. Here, we review observations which suggest that sleep-like states likely occur in mosquitoes and discuss the potential role of sleep in relation to mosquito biology and their ability to function as disease vectors.

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs.

The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

Interactions with ectoparasitic mites induce host metabolic and immune responses in flies at the expense of reproduction-associated factors.

Parasites cause harm to their hosts and represent pervasive causal agents of natural selection. Understanding host proximate responses during interactions with parasites can help predict which genes and molecular pathways are targets of this selection. In the current study, we examined transcriptional changes arising from interactions between Drosophila melanogaster and their naturally occurring ectoparasitic mite, Gamasodes queenslandicus. Shifts in host transcript levels associated with behavioural avoidance revealed the involvement of genes underlying nutrient metabolism. These genetic responses were reflected in altered body lipid and glycogen levels in the flies. Mite infestation triggered a striking immune response, while male accessory gland protein transcript levels were simultaneously reduced, suggesting a trade-off between host immune responses to parasite challenge and reproduction. Comparison of transcriptional analyses during mite infestation to those during nematode and parasitoid attack identified host genes similarly expressed in flies during these interactions. Validation of the involvement of specific genes with RNA interference lines revealed candidates that may directly mediate fly-ectoparasite interactions. Our physiological and molecular characterization of the Drosophila-Gamasodes interface reveals new proximate mechanisms underlying host-parasite interactions, specifically host transcriptional shifts associated with behavioural avoidance and infestation. The results identify potential general mechanisms underlying host resistance and evolutionarily relevant trade-offs.

Molecular mechanisms underlying milk production and viviparity in the cockroach, Diploptera punctata.

Viviparous reproduction is characterized by maternal retention of developing offspring within the reproductive tract during gestation, culminating in live birth. In some cases, a mother will provide nutrition beyond that present in the yolk; this is known as matrotrophic viviparity. While this phenomenon is best associated with mammals, it is observed in insects such as the viviparous cockroach, Diploptera punctata. Female D. punctata carry developing embryos in the brood sac, a reproductive organ that acts as both a uterus and a placenta by protecting and providing a nutritive secretion to the intrauterine developing progeny. While the basic physiology of D. punctata pregnancy has been characterized, little is known about the molecular mechanisms underlying this phenomenon. This study combined RNA-seq analysis, RNA interference, and other assays to characterize molecular and physiological changes associated with D. punctata reproduction. A comparison of four stages of the female reproductive cycle and males revealed unique gene expression profiles corresponding to each stage and between sexes. Differentially regulated transcripts of interest include the previously identified family of milk proteins and transcripts associated with juvenile hormone metabolism. RNA interference and methoprene application experiments established the potential impacts of bothbreakdown and synthesis reduction of juvenile hormone in maintaining pregnancy in D. punctata. These studies provide the comprehensive molecular mechanisms associated with cockroach viviparity, which will be a critical resource for comparative purposes among viviparity in insect systems.

Enzymatic control of cycloadduct conformation ensures reversible 1,3-dipolar cycloaddition in a prFMN-dependent decarboxylase.

The UbiD enzyme plays an important role in bacterial ubiquinone (coenzyme Q) biosynthesis. It belongs to a family of reversible decarboxylases that interconvert propenoic or aromatic acids with the corresponding alkenes or aromatic compounds using a prenylated flavin mononucleotide cofactor. This cofactor is suggested to support (de)carboxylation through a reversible 1,3-dipolar cycloaddition process. Here, we report an atomic-level description of the reaction of the UbiD-related ferulic acid decarboxylase with substituted propenoic and propiolic acids (data ranging from 1.01-1.39 Å). The enzyme is only able to couple (de)carboxylation of cinnamic acid-type compounds to reversible 1,3-dipolar cycloaddition, while the formation of dead-end prenylated flavin mononucleotide cycloadducts occurs with distinct propenoic and propiolic acids. The active site imposes considerable strain on covalent intermediates formed with cinnamic and phenylpropiolic acids. Strain reduction through mutagenesis negatively affects catalytic rates with cinnamic acid, indicating a direct link between enzyme-induced strain and catalysis that is supported by computational studies.

Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes.

The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMNreduced species to the active prFMNiminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.

Terminal Alkenes from Acrylic Acid Derivatives via Non-Oxidative Enzymatic Decarboxylation by Ferulic Acid Decarboxylases.

Fungal ferulic acid decarboxylases (FDCs) belong to the UbiD-family of enzymes and catalyse the reversible (de)carboxylation of cinnamic acid derivatives through the use of a prenylated flavin cofactor. The latter is synthesised by the flavin prenyltransferase UbiX. Herein, we demonstrate the applicability of FDC/UbiX expressing cells for both isolated enzyme and whole-cell biocatalysis. FDCs exhibit high activity with total turnover numbers (TTN) of up to 55000 and turnover frequency (TOF) of up to 370 min-1. Co-solvent compatibility studies revealed FDC's tolerance to some organic solvents up 20 % v/v. Using the in-vitro (de)carboxylase activity of holo-FDC as well as whole-cell biocatalysts, we performed a substrate profiling study of three FDCs, providing insights into structural determinants of activity. FDCs display broad substrate tolerance towards a wide range of acrylic acid derivatives bearing (hetero)cyclic or olefinic substituents at C3 affording conversions of up to >99 %. The synthetic utility of FDCs was demonstrated by a preparative-scale decarboxylation.

The role of conserved residues in Fdc decarboxylase in prenylated flavin mononucleotide oxidative maturation, cofactor isomerization, and catalysis.

The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu-Arg-Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277-Arg173-Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu-Arg-Glu network, as only Glu → Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu-Arg-Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu-Arg-Glu network in both catalysis and oxidative maturation.

The challenge of comprehensive and consistent sequence variant interpretation between clinical laboratories.

PURPOSE: Genetic testing has shifted from academic laboratories with expertise in specific genes to commercial laboratories that offer tests of a diverse array of genes. The purpose of this comparative study was to determine whether one academic laboratory's model of variant interpretation is similar to that of several commercial laboratories. METHODS: The Collagen Diagnostic Laboratory (CDL) received, over a 14-month period, 38 requests to interpret variants originally identified by an outside laboratory (OL). The interpretations by the OL and CDL were compared and discrepancies were assessed. RESULTS: Interpretations from the OL and CDL were concordant in 11 inquiries (29%); discrepancies were moderate in 11 instances (29%) and significant in 16 (42%). Factors that caused discrepancies included the following: (i) private data were not shared in a public database (n = 9); (ii) publicly available allele frequency data were not referenced and used as evidence (n = 5); and (iii) important aspects of protein structure and function were not taken into account (n = 13). CONCLUSION: Comprehensive interpretation of sequence variants depends on good functional tests and well-curated variant databases. Provision of clinical information to the clinical laboratory, mandatory submission of identified variants with phenotype data to common resources, and collaboration between clinical laboratories and recognized experts is likely to improve consistency in variant interpretation among clinical laboratories.Genet Med 18 1, 20-24.

New cofactor supports α,ß-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition.

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

Molecular Outcome, Prediction, and Clinical Consequences of Splice Variants in COL1A1, Which Encodes the proα1(I) Chains of Type I Procollagen.

Approximately 10%-20% of germline pathogenic variants alter mRNA splicing, with phenotypes often dependent on the stability of the mRNA produced by the mutant allele. To better understand the relationships between genotype, mRNA splicing, and phenotype, we examined clinical and molecular data from 243 probands with osteogenesis imperfecta (OI) representing 145 unique splicing variants within the type I procollagen gene, COL1A1. All individuals with IVSX-1G>A mutations had OI type I because the substitution shifted the splice acceptor site 1 nt downstream and destabilized the mRNA. OI phenotypes were not consistent for any other splice variant identified. We sequenced all cDNA species from cultured dermal fibroblasts from 40 individuals to identify splice outcome and compared those results to splice predictions from Human Splice Finder (HSF), Spliceport (SP), and Automatic Splice Site and Exon Definition Analyses (ASSEDA). Software-based splice predictions were correct in 42%, 55%, and 74% instances for HSF, SP, and ASSEDA, respectively. As molecular diagnostics move increasingly to DNA sequence analysis, the need to understand the effects of splice site variants will increase. These data demonstrate that caution must be exercised when using splice prediction software to predict splice outcome.

Hepatitis C polymerase chain reaction testing by GPs.

BACKGROUND: Approximately 75% of patients exposed to the hepatitis C virus will become chronically infected. Polymerase chain reaction (PCR) testing more than 6 months after exposure is necessary to identify this group. This pilot study assessed the practical ap-plication of PCR testing in the general practice context. METHODS: General practitioners of patients newly notified as positive for hepatitis C antibody between 1 August 2007 and 1 August 2012 were invited to participate. They completed a self-administered survey, recording details on the use of hepatitis C PCR testing in their patients. RESULTS: The survey found that 16 patients (46%) did not undergo any PCR testing for hepatitis C. Of those who underwent PCR testing, 11 (58%) were positive on PCR testing but only six (55%) of those with a positive PCR test were retested 6 months later. DISCUSSION: Appropriate use of PCR is necessary to identify patients with chronic hepatitis C and offer appropriate referral and treatment.

Food allergy training event for restaurant staff; a pilot evaluation.

A previous cross-sectional survey highlighted that restaurant staff in Brighton had gaps in their knowledge of food allergy, which could lead to the provision of unsafe meals to food-allergic customers. A food allergy training event was developed by a multi-disciplinary team (health service researcher, clinician, teacher and patient group representative) to equip restaurant staff with the knowledge and skills necessary to safely serve food-allergic customers. This evaluation summarises the training event's impact on participants' knowledge of food allergy and their satisfaction with the event. No attendee had previously attended any formal training on food allergy. The percentage of participants who answered all true-false questions correctly increased from 82% before the training event to 91% afterwards. The percentage of participants who were able to name at least three common allergens increased from 9% to 64%. Both quantitative and qualitative feedback was positive. Restaurant staff require a good understanding of food allergy to ensure that food-allergic customers are kept safe, and their restaurants operate within the law. This food allergy training event improved participants' absolute knowledge of food allergy, and attendees changed practice. Recommendations are made which could improve the impact and uptake of future food allergy training events.

Text 2 treat - using SMS to recall clients for treatment.

Prompt treatment of patients with genital Chlamydia shortens the period of infectivity with benefits to the individual and wider community. With large numbers of genital Chlamydia notifications, predominantly occurring in younger age groups, short message service (SMS) is a potentially useful technology for recalling this patient group quickly and efficiently. In the sexual health unit of Population Health-Midwest, Western Australia, genital Chlamydia cases were recalled for treatment with an SMS. Ninety-four per cent (n = 60) of clients responded to the SMS, with 84% (n = 54) responding on the same day they were contacted. All clients (n = 64) were treated for their infection, with 72% (n = 46) having directly-observed treatment within one day of being informed of their results via SMS. Our results suggest that SMS is a highly effective, youth-friendly communication tool.