Circulating tumour DNA detects somatic variants contributing to spatial and temporal intra-tumoural heterogeneity in head and neck squamous cell carcinoma.

Background: As circulating tumour DNA (ctDNA) liquid biopsy analysis is increasingly incorporated into modern oncological practice, establishing the impact of genomic intra-tumoural heterogeneity (ITH) upon data output is paramount. Despite advances in other cancer types the evidence base in head and neck squamous cell carcinoma (HNSCC) remains poor. We sought to investigate the utility of ctDNA to detect ITH in HNSCC. Methods: In a pilot cohort of 9 treatment-naïve HNSCC patients, DNA from two intra-tumoural sites (core and margin) was whole-exome sequenced. A 9-gene panel was designed to perform targeted sequencing on pre-treatment plasma cell-free DNA and selected post-treatment samples. Results: Rates of genomic ITH among the 9 patients was high. COSMIC variants from 19 TCGA HNSCC genes demonstrated an 86.9% heterogeneity rate (present in one tumour sub-site only). Across all patients, cell-free DNA (ctDNA) identified 12.9% (range 7.5-19.8%) of tumour-specific variants, of which 55.6% were specific to a single tumour sub-site only. CtDNA identified 79.0% (range: 55.6-90.9%) of high-frequency variants (tumour VAF>5%). Analysis of ctDNA in serial post-treatment blood samples in patients who suffered recurrence demonstrated dynamic changes in both tumour-specific and acquired variants that predicted recurrence ahead of clinical detection. Conclusion: We demonstrate that a ctDNA liquid biopsy identified spatial genomic ITH in HNSCC and reliably detected high-frequency driver mutations. Serial sampling allowed post-treatment surveillance and early identification of treatment failure.

Integra® Dermal Regeneration Template in Complex Scalp Reconstruction.

Background/Objectives: The need for surgical reconstruction of scalp defects following the excision of cutaneous skin cancers is an increasingly common procedure. Particular challenges arise when considering options for reconstruction of large defects not amenable to local skin flap coverage. The use of skin grafts poses the risk of donor site morbidity. This paper investigates the emerging use of Integra®, a synthetic acellular dermal regeneration template, as an alternative or adjunct to skin grafting in scalp reconstruction. Methods: The study presents a retrospective analysis of 101 patients who underwent Integra®-based reconstruction of scalp defects. Demographics, procedure details, complications, need for further surgery, and time to healing were evaluated. Results: The overall success rate of the one-stage Integra®-only procedure was 95%, with a minor complication rate of 30.7%. Anticoagulation medication was identified as an independent risk factor for post-operative infection, while previous head and neck radiotherapy and increased defect depth were associated with the requirement for a second-stage skin graft. Conclusions: These findings support the consideration of Integra® as a safe and viable alternative for both partial and full thickness scalp defects in a select cohort of complex highly co-morbid patients, reducing complications and the need for additional procedures.

Prognostic biomarkers for malignant progression of oral epithelial dysplasia: an updated systematic review and meta-analysis.

Oral epithelial dysplasia (OED) is a premalignant condition that carries an appreciable risk of malignant progression. The current grading system for severity, as defined by the World Health Organization, is a valuable clinical tool, but further work is required to improve the accuracy of predicting OED malignant progression. This systematic review aimed to assess progress in prognostic biomarker discovery in OED over the past 16 years. The primary objective was to update the latest evidence on prognostic biomarkers that may predict malignant progression of OED, with strict inclusion criteria of studies with a longitudinal design and long-term follow-up data to enhance the robustness and translational clinical potential of the findings. Of 2829 studies identified through the searching of five databases, 20 met our inclusion criteria. These studies investigated a total of 32 biomarkers, 20 of which demonstrated significant potential to predict malignant progression of OED. Meta-analysis demonstrated the significant prognostic value of four biomarkers: podoplanin, EGFR expression, p16 methylation, and DNA aneuploidy. Our review has identified 20 reported biomarkers with prognostic potential to predict malignant progression in OED, but their translation into clinical practice remains elusive. Further research is required, and this should focus on validating the promising biomarkers identified in large cohort studies, with adherence to standardised reporting guidelines.

Feasibility of mass cytometry proteomic characterisation of circulating tumour cells in head and neck squamous cell carcinoma for deep phenotyping.

BACKGROUND: Circulating tumour cells (CTCs) are a potential cancer biomarker, but current methods of CTC analysis at single-cell resolution are limited. Here, we describe high-dimensional single-cell mass cytometry proteomic analysis of CTCs in HNSCC. METHODS: Parsortix microfluidic-enriched CTCs from 14 treatment-naïve HNSCC patients were analysed by mass cytometry analysis using 41 antibodies. Immune cell lineage, epithelial-mesenchymal transition (EMT), stemness, proliferation and immune checkpoint expression was assessed alongside phosphorylation status of multiple signalling proteins. Patient-matched tumour gene expression and CTC EMT profiles were compared. Standard bulk CTC RNAseq was performed as a baseline comparator to assess mass cytometry data. RESULTS: CTCs were detected in 13/14 patients with CTC counts of 2-24 CTCs/ml blood. Unsupervised clustering separated CTCs into epithelial, early EMT and advanced EMT groups that differed in signalling pathway activation state. Patient-specific CTC cluster patterns separated into immune checkpoint low and high groups. Patient tumour and CTC EMT profiles differed. Mass cytometry outperformed bulk RNAseq to detect CTCs and characterise cell phenotype. DISCUSSION: We demonstrate mass cytometry allows high-plex proteomic characterisation of CTCs at single-cell resolution and identify common CTC sub-groups with potential for novel biomarker development and immune checkpoint inhibitor treatment stratification.

Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.

Academic Training in Oral and Maxillofacial Surgery - when and how to enter the pathway.

Entering into surgical academia can seem a daunting prospect for an oral and maxillofacial surgery (OMFS) trainee. However, the streamlining of academic training by the NIHR to create the integrated academic training (IAT) pathway has simplified academic training and more clearly defined academic positions and entry points for trainees. In this article we review the current NIHR IAT pathway and the various grades and entry points available to OMF surgeons, both pre- and post-doctoral. We highlight the unique challenges facing OMF trainees and provide advice and insight from both junior and senior OMFS academics. Finally, we focus on the planning and application for a doctoral research fellowship - discussing funding streams available to OMF surgeons.

Characterizing the epithelial-mesenchymal transition status of circulating tumor cells in head and neck squamous cell carcinoma.

BACKGROUND: Circulating tumor cells (CTCs), in particular those undergoing an epithelial-mesenchymal transition (EMT), are a promising source of biomarkers in head and neck squamous cell carcinoma (HNSCC). Our aim was to validate a protocol using microfluidic enrichment (Parsortix platform) with flow-cytometry CTC characterization. METHOD: Blood samples from 20 treatment naïve HNSCC patients underwent Parsortix enrichment and flow cytometry analysis to quantify CTCs and identify epithelial or EMT subgroups-correlated to clinical outcomes and EMT gene-expression in tumor tissue. RESULTS: CTCs were detected in 65% of patients (mean count 4 CTCs/ml). CTCs correlated with advanced disease (p = 0.0121), but not T or N classification. Epithelial or EMT CTCs did not correlate with progression-free or overall survival. Tumor mesenchymal gene-expression did not correlate with CTC EMT expression (p = 0.347). DISCUSSION: Microfluidic enrichment and flow cytometry successfully characterizes EMT CTCs in HNSCC. The lack of association between tumor and CTC EMT profile suggests CTCs may undergo an adaptive EMT in response to stimuli within the circulation.

The evolving role of the superficial temporal vessels as anastomotic recipients in challenging microvascular reconstruction of the upper two-thirds of the face.

Free-tissue transfer is now the standard of care in reconstructing head and neck defects. Microvascular reconstruction of the upper two-thirds of the face, particularly following recurrent malignant disease, however, remains a challenge. Retrospective review of all patients undergoing microsurgical reconstruction for defects of the upper two-thirds of the face between 2015 and 2019 revealed 17 free-tissue transfers where the superficial temporal vessels (STVs) were used as recipient vessels. Perioperative data, including return to theatre (RTT) and flap success rates were evaluated. Three patients required reoperation for vascular compromise. One of these patients ultimately lost the flap resulting in flap survival of 94%. This flap failure was, however, secondary to external factors distant to the flap and the anastomosis. In this patient, subsequent attempt at microvascular reconstruction was carried out successfully using contralateral neck vessels and vein grafts. We demonstrate the STVs are safe and reliable in microsurgical reconstruction for a variety of defects. Close proximity to the resection, reconstructing with flaps with a short pedicle, negating the need for neck access and/or high-risk anastomosis in a previously irradiated/operated vessel depleted neck are all highlighted indications. The pre-auricular/temporal access is familiar to the reconstructive surgeon and has excellent post-operative aesthetic results. We discuss key advantages of this approach and highlight potential complications and learning points - underlining the benefit of this approach for the head and neck reconstructive surgeon.

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase.

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO2 under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.

Immediate Sample Fixation Increases Circulating Tumour Cell (CTC) Capture and Preserves Phenotype in Head and Neck Squamous Cell Carcinoma: Towards a Standardised Approach to Microfluidic CTC Biomarker Discovery.

INTRODUCTION: Research demonstrates strong evidence that circulating tumour cells (CTCs) can provide diagnostic and/or prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC) and a potential tool for therapeutic stratification. However, the question still remains as to the optimum method of CTC enrichment and how this can be translated into clinical practice. We aimed to evaluate the Parsortix microfluidic device for CTC enrichment and characterisation in HNSCC, seeking to optimise a sample collection and processing protocol that preserves CTC integrity and phenotype. METHOD: Spiking experiments of the FaDu and SCC040 HNSCC cell lines were used to determine the Parsortix capture rate of rare "CTC-like" cells. Capture rates of cancer cells spiked into EDTA blood collections tubes (BCTs) were compared to the Transfix fixative BCT and Cytodelics whole blood freezing protocol. The Lexogen Quantseq library preparation was used to profile gene expression of unfixed cells before and after microfluidic enrichment and enriched cell line spiked Transfix blood samples. An antibody panel was optimised to enable immunofluorescence microscopy CTC detection in HNSCC patient Transfix blood samples, using epithelial (EpCAM) and mesenchymal (N-cadherin) CTC markers. RESULTS: Across a spiked cell concentration range of 9-129 cells/mL, Parsortix demonstrated a mean cell capture rate of 53.5% for unfixed cells, with no significant relationship between spiked cell concentration and capture rate. Samples preserved in Transfix BCTs demonstrated significantly increased capture rates at 0 h (time to processing) compared to EDTA BCTs (65.3% vs. 51.0%). Capture rates in Transfix BCTs were maintained at 24 h and 72 h timepoints, but dropped significantly in EDTA BCTs. Gene expression profiling revealed that microfluidic enrichment of unfixed cell lines caused downregulation of RNA processing/binding gene pathways and upregulation of genes involved in cell injury, apoptosis and oxidative stress. RNA was successfully extracted and sequenced from Transfix preserved cells enriched using Parsortix, demonstrating epithelial specific transcripts from spiked cells. In a proof-of-concept cohort of four patients with advanced HNSCC, CTCs were successfully identified and visualised with epithelial and epithelial-mesenchymal phenotypes. CONCLUSION: We have optimised a protocol for detection of CTCs in HNSCC with the Parsortix microfluidic device, using Transfix BCTs. We report a significant benefit, both in terms of cell capture rates and preserving cell phenotype, for using a fixative BCT- particularly if samples are stored before processing. In the design of large cohort multi-site clinical trials, such data are of paramount importance.

UbiD domain dynamics underpins aromatic decarboxylation.

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.

Lack of predictive tools for conventional and targeted cancer therapy: Barriers to biomarker development and clinical translation.

Predictive tools, utilising biomarkers, aim to objectively assessthe potentialresponse toa particular clinical intervention in order to direct treatment.Conventional cancer therapy remains poorly served by predictive biomarkers, despite being the mainstay of treatment for most patients. In contrast, targeted therapy benefits from a clearly defined protein target for potential biomarker assessment. We discuss potential data sources of predictive biomarkers for conventional and targeted therapy, including patient clinical data andmulti-omicbiomarkers (genomic, transcriptomic and protein expression).Key examples, either clinically adopted or demonstrating promise for clinical translation, are highlighted. Following this, we provide an outline of potential barriers to predictive biomarker development; broadly discussing themes of approaches to translational research and study/trial design, and the impact of cellular and molecular tumor heterogeneity. Future avenues of research are also highlighted.

Ultra-Low DNA Input into Whole Genome Methylation Assays and Detection of Oncogenic Methylation and Copy Number Variants in Circulating Tumour DNA.

Abnormal CpG methylation in cancer is ubiquitous and generally detected in tumour specimens using a variety of techniques at a resolution encompassing single CpG loci to genome wide coverage. Analysis of samples with very low DNA inputs, such as formalin fixed (FFPE) biopsy specimens from clinical trials or circulating tumour DNA is challenging at the genome-wide level because of lack of available input. We present the results of low input experiments into the Illumina Infinium HD methylation assay on FFPE specimens and ctDNA samples. METHODS: For all experiments, the Infinium HD assay for methylation was used. In total, forty-eight FFPE specimens were used at varying concentrations (lowest input 50 ng); eighteen blood derived specimens (lowest input 10 ng) and six matched ctDNA input (lowest input 10 ng)/fresh tumour specimens (lowest input 250 ng) were processed. Downstream analysis was performed in R/Bioconductor for quality control metrics and differential methylation analysis as well as copy number calls. RESULTS: Correlation coefficients for CpG methylation were high at the probe level averaged R2 = 0.99 for blood derived samples and R2 > 0.96 for the FFPE samples. When matched ctDNA/fresh tumour samples were compared, R2 > 0.91 between the two. Results of differential methylation analysis did not vary significantly by DNA input in either the blood or FFPE groups. There were differences seen in the ctDNA group as compared to their paired tumour sample, possibly because of enrichment for tumour material without contaminating normal. Copy number variants observed in the tumour were generally also seen in the paired ctDNA sample with good concordance via DQ plot. CONCLUSIONS: The Illumina Infinium HD methylation assay can robustly detect methylation across a range of sample types, including ctDNA, down to an input of 10 ng. It can also reliably detect oncogenic methylation changes and copy number variants in ctDNA. These findings demonstrate that these samples can now be accessed by methylation array technology, allowing analysis of these important sample types.

Structure and Mechanism of Pseudomonas aeruginosa PA0254/HudA, a prFMN-Dependent Pyrrole-2-carboxylic Acid Decarboxylase Linked to Virulence.

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor. This raises the question how efficient decarboxylation of (hetero)aromatic acids is achieved by other UbiD family members. Here, we show that the Pseudomonas aeruginosa virulence attenuation factor PA0254/HudA is a pyrrole-2-carboxylic acid decarboxylase. The crystal structure of the enzyme in the presence of the reversible inhibitor imidazole reveals a covalent prFMN-imidazole adduct is formed. Substrate screening reveals HudA and selected active site variants can accept a modest range of heteroaromatic compounds, including thiophene-2-carboxylic acid. Together with computational studies, our data suggests prFMN covalent catalysis occurs via electrophilic aromatic substitution and links HudA activity with the inhibitory effects of pyrrole-2-carboxylic acid on P. aeruginosa quorum sensing.

Prognostic Significance of CD4+ and CD8+ Tumor-Infiltrating Lymphocytes in Head and Neck Squamous Cell Carcinoma: A Meta-Analysis.

OBJECTIVE: It has been suggested that the presence of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment is associated with a better prognosis in different types of cancer. In this systematic review and meta-analysis, we investigated the prognostic role of CD4+ and CD8+ TILs in head and neck squamous cell carcinoma (HNSCC). METHODS: PubMed, Cochrane, Embase, Scopus, and Web of Science were searched up to September 2020. This study was conducted following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) checklist. Risk ratios from individual studies were displayed in forest plots and the pooled hazard ratios (HR) of death and corresponding confidence intervals (CI) were calculated according to random-effects models. Risk of bias of the included studies was assessed through the Newcastle-Ottawa scale. RESULTS: 28 studies met the inclusion criteria. Studies conducted on HNSCC subsites combined reported a significant reduction in the risk of death for both high CD4+ (HR: 0.77; 95% CI: 0.65-0.93) and high CD8+ TILs (HR: 0.64; 95% CI: 0.47-0.88). High CD4+ TILs were associated with significantly better overall survival among oropharyngeal HNSCC (HR: 0.52; 95% CI: 0.31-0.89), as well as high CD8+ TILS in Human papillomavirus -ve and +ve cancers (HR: 0.39; 95% CI: 0.16-0.93 and HR: 0.40; 95% CI 0.21-0.76 respectively). CD8+ TILs were also associated with improved survival in hypopharyngeal cancers (HR = 0.43 CI: 0.30-0.63). No significant association emerged for patients with cancer of the oral cavity or larynx. CONCLUSIONS: The findings from this meta-analysis demonstrate the prognostic significance of CD8+ and CD4+ TILs in HNSCC and variation in tumor subsite warrants further focused investigation. We highlight how TILs may serve as predictive biomarkers to risk stratify patients into treatment groups, with applications in immune-checkpoint inhibitors notable areas for further research.

A Biological Route to Conjugated Alkenes: Microbial Production of Hepta-1,3,5-triene.

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.

Heterologous expression of cobalamin dependent class-III enzymes.

The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.

Influence of Climatic Factors on Human Hantavirus Infections in Latin America and the Caribbean: A Systematic Review.

BACKGROUND: With the current climate change crisis and its influence on infectious disease transmission there is an increased desire to understand its impact on infectious diseases globally. Hantaviruses are found worldwide, causing infectious diseases such as haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS)/hantavirus pulmonary syndrome (HPS) in tropical regions such as Latin America and the Caribbean (LAC). These regions are inherently vulnerable to climate change impacts, infectious disease outbreaks and natural disasters. Hantaviruses are zoonotic viruses present in multiple rodent hosts resident in Neotropical ecosystems within LAC and are involved in hantavirus transmission. METHODS: We conducted a systematic review to assess the association of climatic factors with human hantavirus infections in the LAC region. Literature searches were conducted on MEDLINE and Web of Science databases for published studies according to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) criteria. The inclusion criteria included at least eight human hantavirus cases, at least one climatic factor and study from > 1 LAC geographical location. RESULTS: In total, 383 papers were identified within the search criteria, but 13 studies met the inclusion criteria ranging from Brazil, Chile, Argentina, Bolivia and Panama in Latin America and a single study from Barbados in the Caribbean. Multiple mathematical models were utilized in the selected studies with varying power to generate robust risk and case estimates of human hantavirus infections linked to climatic factors. Strong evidence of hantavirus disease association with precipitation and habitat type factors were observed, but mixed evidence was observed for temperature and humidity. CONCLUSIONS: The interaction of climate and hantavirus diseases in LAC is likely complex due to the unknown identity of all vertebrate host reservoirs, circulation of multiple hantavirus strains, agricultural practices, climatic changes and challenged public health systems. There is an increasing need for more detailed systematic research on the influence of climate and other co-related social, abiotic, and biotic factors on infectious diseases in LAC to understand the complexity of vector-borne disease transmission in the Neotropics.

Circulating Tumour Cell Expression of Immune Markers as Prognostic and Therapeutic Biomarkers in Head and Neck Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis.

Rates of loco-regional recurrence and distant metastasis remain high among head and neck squamous cell carcinoma (HNSCC) patients, despite advancing cancer treatment modalities and therapeutic agents. One area that has generated considerable interest is the immune landscape of the tumour, heralding a wave of immune checkpoint inhibitors with notable efficacy in recurrent/metastatic HNSCC patients. However, HNSCC remains poorly served by biomarkers that can direct treatment in a personalised fashion to target the tumour heterogeneity seen between patients. Detection and analysis of circulating tumour cells (CTCs) in HNSCC has provided a previously unseen view of the metastasis forming cells that are potentially contributing to poor clinical outcomes. In particular, identifying CTC expression of phenotypic and druggable protein markers has allowed CTC sub-populations to be defined that hold prognostic value or are potential therapeutic targets themselves. The aim of this systematic review was to examine the role of CTC immune-marker expression as prognostic/therapeutic biomarkers in HNSCC by evaluating progress to date and discussing areas for future research. Our results highlight how few studies have been able to demonstrate prognostic significance of immune-marker expression in CTCs. As expected, the immune checkpoint PD-L1 was the most widely investigated marker. However, no studies evaluated CTC target immune marker expression in immunotherapy cohorts. Despite these findings, the data presented demonstrate promise that CTCs may be a source of future biomarkers for immunotherapy and will provide valuable information regarding the potential immune evasion of these metastasis forming cells.

Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope.

Reductive dehalogenases are responsible for the reductive cleavage of carbon-halogen bonds during organohalide respiration. A variety of mechanisms have been proposed for these cobalamin and [4Fe-4S] containing enzymes, including organocobalt, radical, or cobalt-halide adduct based catalysis. The latter was proposed for the oxygen-tolerant Nitratireductor pacificus pht-3B catabolic reductive dehalogenase (NpRdhA). Here, we present the first substrate bound NpRdhA crystal structures, confirming a direct cobalt-halogen interaction is established and providing a rationale for substrate preference. Product formation is observed in crystallo due to X-ray photoreduction. Protein engineering enables rational alteration of substrate preference, providing a future blue print for the application of this and related enzymes in bioremediation.