Every refuge has its price: Ostreobium as a model for understanding how algae can live in rock and stay in business.

Ostreobium is a siphonous green alga in the Bryopsidales (Chlorophyta) that burrows into calcium carbonate (CaCO3) substrates. In this habitat, it lives under environmental conditions unusual for an alga (i.e., low light and low oxygen) and it is a major agent of carbonate reef bioerosion. In coral skeletons, Ostreobium can form conspicuous green bands recognizable by the naked eye and it is thought to contribute to the coral's nutritional needs. With coral reefs in global decline, there is a renewed focus on understanding Ostreobium biology and its roles in the coral holobiont. This review summarizes knowledge on Ostreobium's morphological structure, biodiversity and evolution, photosynthesis, mechanism of bioerosion and its role as a member of the coral holobiont. We discuss the resources available to study Ostreobium biology, lay out some of the uncharted territories in Ostreobium biology and offer perspectives for future research.

Timing of development of osteoradionecrosis post head and neck radiotherapy: does a safe time interval exist for dental extraction?

INTRODUCTION: Risk factors for developing osteoradionecrosis (ORN) are well known, but less is known about factors influencing the interval between radiotherapy and the onset of ORN. Also, it is unknown whether there is any specific period post-radiotherapy with a reduced probability of ORN when irradiated teeth require extraction. PURPOSE: The primary aim of this study was to identify factors influencing the interval in developing ORN in the following subgroups of patients: (1) patients who spontaneously developed ORN, (2) surgical-intervention-related ORN with a particular focus on patients after mandibulectomy. The secondary aim was to attempt to identify a possible time for safer dental intervention after primary treatment. MATERIALS AND METHODS: The authors retrospectively analysed 1608 head and neck cancer (HNC) patients treated in a single centre. Time intervals were measured from the end of radiotherapy to the development of ORN and further analysed in the subgroups listed above. RESULTS: In all, 141 patients (8.8%) developed intra-oral ORN. Median time from radiotherapy to ORN development in the whole cohort was 9 months. Median interval for spontaneous ORN was 8 months, 6.5 months for intervention-related ORN, and 15 months for patients post-mandibulectomy. In patients who required dental extraction preradiotherapy, median interval of ORN onset was 5 months. CONCLUSION: In our study, a slightly higher proportion of patients with intervention developed ORN earlier in comparison with spontaneous ORN. The period from 12-18 months after radiotherapy was identified as having the highest probability of developing ORN in patients after mandibulectomy. A time for safer dental intervention after primary treatment was not identified.

Shrinkage of the non-malignant prostate gland volume after receiving incidental radiotherapy for rectal cancer.

Background: The purpose of this study was to assess the impact of coincidental radiotherapy on the volume of the non-malignant prostate gland in rectal cancer patients treated with neo-adjuvant radiotherapy. Materials and methods: In this retrospective analysis, thirty male patients with rectal cancer who had neoadjuvant radiotherapy met the inclusion criteria. These patients had pre-treatment magnetic resonance imaging (MRI) and at least one post-treatment MRI of the pelvis and the whole of their prostate volume received the full prescribed radiotherapy dose; 45 Gy in 25 fractions (n = 22), 45 Gy in 20 fractions (n = 4) and 25 Gy in 5 fractions (n = 4). Results: The median age of this patient cohort was 66 years (range: 30-87). With a median interval between pre-treatment MRI and first MRI post-treatment of 2 months (range: 1-11), the mean prostate volume reduced from 36.1 cm3 [standard deviation (SD) 14.2] pre-radiotherapy to 31.3 cm3 (SD 13.0) post radiotherapy and this difference was significant (p = 0.0004). Conclusion: Radiotherapy may cause shrinkage in volume of normal (non-malignant) prostate. Further research is required in this field, since these results may be of some comfort to men contemplating the consequences of radiotherapy on their quality of life. The authors suggest recording flow-rate and international prostate symptom score (IPSS) during rectal radiotherapy as a next step.

Survival outcomes for patients with nasopharyngeal carcinoma in non-endemic region in the UK treated with intensity modulated based radiotherapy 65 Gy in 30 fractions ± weekly cisplatin chemotherapy.

Background: Nasopharyngeal carcinoma (NPC) is rare in the UK. The aim of the current study was to investigate survival outcomes for patients with NPC treated with (chemo)radiotherapy using 65 Gy in 30 fractions in a non-endemic region. Materials and methods: All consecutive 62 patients with histology proven non-metastatic nasopharyngeal carcinoma diagnosed between January 2009 to June 2019 were included in this retrospective analysis. Results: Median age was 59 years (range:19-81). The majority of patients had stage III disease (66.1%). Induction chemotherapy was given in 21% of patients and 82.3% of patients received concomitant systemic therapy. All patients were treated with 65 Gy in 30 fractions. There was disease recurrence in 17.4% patients. The 5-year disease-free, disease-specific and overall survival were 81.9%, 79.2% and 76.4%, respectively. On univariate analysis, disease recurrence was associated with N-stage (p = 0.047) and overall stage group (p = 0.023). Conclusion: To the best of authors' knowledge, this is the first report of the use of 65 Gy in 30 fractions of radiotherapy ± weekly cisplatin chemotherapy in NPC in a real-world setting. Our results are comparable to that from other non-endemic regions of the world using different dose fractionation of (chemo)radiotherapy. Future randomised control trials are warranted to compare various dose fractionations in these settings.

Primary transoral robotic surgery +/- adjuvant therapy for oropharyngeal squamous cell carcinoma-A large observational single-centre series from the United Kingdom.

OBJECTIVES: To analyse the oncological outcomes following primary Transoral Robotic Surgery (TORS) for oropharyngeal squamous cell carcinoma (OPSCC). DESIGN: Observational case series. SETTING: Tertiary centre; first TORS practice to commence in the UK. PARTICIPANTS: All consecutive patients undergoing primary TORS with curative intent, with or without adjuvant treatment. MAIN OUTCOME MEASURES: Descriptive analysis of patient and tumour pathology variables. Survival outcomes: Overall, Disease-Specific, Progression-Free and Locoregional control. RESULTS: The cohort comprised of 120 patients undergoing TORS with minimum 12-month follow-up data and the following characteristics: mean age 58 years, 91 males (76%), 78 tonsil (65%) and 34 base of tongue primaries (28%), 89% HPV-related OPSCC. The surgical pathology revealed 14 (12%) with positive margins, 19 (16%) had close margins <2mm and 31% with extranodal extension. The treatment was as follows: 39 (33%) treated with TORS alone, 50 (42%) received adjuvant radiotherapy and 31 (26%) received adjuvant radiotherapy with chemotherapy. There were 15 recurrences. Estimated survival for all patients at 3 years (95% CI): overall 85% (78-92), disease-specific 90% (85-96), progression-free 86% (79-92) and locoregional control 90% (84-96). The equivalent survival figures for the HPV-related cases alone were as follows: overall 88% (82-94), disease-specific 93% (87-98), progression-free 88% (81-95) and locoregional control 92% (87-98). CONCLUSIONS: Whilst TORS has become a common practice in the management of OPSCC in the UK, these are the first reported oncological outcomes. For selected patients, TORS with or without adjuvant therapy is an appropriate treatment modality.

In Vivo Modulation of Cervicovaginal Drug Transporters and Tissue Distribution by Film-Released Tenofovir and Darunavir for Topical Prevention of HIV-1.

Clinical trials have demonstrated partial protection against HIV-1 infection by vaginal microbicide formulations based on antiretroviral (ARV) drugs. Improved formulations that will maintain sustained drug concentrations at viral target sites in the cervicovaginal mucosa are needed. We have previously demonstrated that treatment of cervicovaginal cell lines with ARV drugs can alter gene expression of drug transporters, suggesting that the mucosal disposition of ARV drugs delivered vaginally can be modulated by drug transporters. This study aimed to investigate in vivo modulation of drug transporter expression in a nonhuman primate model by tenofovir and darunavir released from film formulations. Cervicovaginal tissues were collected from drug-naïve macaques and from macaques vaginally treated with film formulations of tenofovir or darunavir. Drug release in vaginal fluid as well as drug absorption in cervicovaginal tissues and lymph nodes were verified by mass spectrometry. The effects of exposure to drugs on the expression of transporters relevant to ARV drugs were evaluated by quantitative PCR. We showed expression in cervicovaginal tissue of drug-naïve macaques of transporters important for distribution of ARV drugs, albeit at lower levels compared to human tissue for key transporters including P-glycoprotein. Concentrations of tenofovir and darunavir well above the EC50 values determined in vitro were detected in vaginal fluid and vaginal tissues of macaques treated with drug-dissolving films over 24 h and were also comparable to those shown previously to modulate drug transporter expression. Accordingly, Multidrug Resistance associated Protein 2 (MRP2) in cervicovaginal tissue was upregulated by both tenofovir and darunavir. The two drugs also differentially induced and/or inhibited expression of key uptake transporters for reverse transcriptase inhibitors and protease inhibitors. The lower expression of key transporters in macaques may result in increased retention of ARV drugs at the simian cervicovaginal mucosa compared to the human mucosa and has implications for translation of preclinical data. Modulation of drug transporter expression by tenofovir and darunavir points to the potential benefit of MRP2 inhibition to increase ARV drug penetration through the cervicovaginal epithelium.

Automated High-Throughput Capillary Circular Dichroism and Intrinsic Fluorescence Spectroscopy for Rapid Determination of Protein Structure.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial.

Background: Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers. We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence. Patients and methods: Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation. The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI). Tumour and blood were analysed for prognostic and predictive markers. Results: Patients (n=1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18-88 years). With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation]. OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82-1.16, P = 0.78). At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74-0.99, P = 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78-1.07, P = 0.25). Forty four percent of 682 melanomas assessed had a BRAFV600 mutation. In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P = 0.06). BRAF mutation positivity trended towards better OS with bevacizumab (P = 0.21). Conclusions: Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS. BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab. Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64.

Mutation of M13 Bacteriophage Major Coat Protein for Increased Conjugation to Exogenous Compounds.

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.

Robotic lateral oropharyngectomy following diagnostic tonsillectomy is oncologically safe in patients with high risk human papillomavirus related squamous cell cancer.

PURPOSE: Diagnostic tonsillectomy is rarely an oncologic operation owing to close or positive margins. The standard of care is for further treatment to the primary site, typically with adjuvant radiotherapy. METHODS: 14 patients with close or positive margins following a diagnostic tonsillectomy underwent transoral robotic surgery (TORS) and lateral oropharyngectomy; five patients with the longest follow-up had their excision specimens examined with a step serial sectioning technique (SSS). RESULTS: Conventional histopathological examination of the TORS resection specimens did not demonstrate residual carcinoma in 13 patients, confirmed by examination using SSS in 5 patients. There were no post-operative complications or long-term functional deficit. Seven patients received surgery alone with 100% overall and disease specific survival, respectively (median follow-up 27.5 months; range 5.2-50.4). CONCLUSIONS: This prospective study suggests that TORS lateral oropharyngectomy alone is an oncologically safe treatment when close or positive margins are identified on diagnostic tonsillectomy in HPV-positive SCC.

Extreme long-term voice outcomes after concurrent chemoradiotherapy for advanced non-laryngeal head and neck cancer: Eight-year post-treatment analysis.

BACKGROUND: The long-term effect of concurrent chemoradiation on voice outcomes in the context of non-laryngeal head and neck cancer is not established. METHODS: A prospective, observational study to evaluate the voice quality in disease-free patients receiving concurrent chemoradiation for advanced non-laryngeal squamous cell carcinoma of the upper aerodigestive tract. Voice assessment occurred at four distinct time-points: pretreatment, 3, 12 and 92.6 months (mean) post-treatment in 34, 21 and nine patients, respectively. The authors used a combination of subjective (VoiSS questionnaire), expert rater-assessed (GRBAS scale) and acoustic analysis of the fundamental frequency to assess voice outcomes. Ethical approval was obtained from the United Kingdom National Research Ethics Service. RESULTS: Both the VoiSS impairment and GRBAS domains continued to deteriorate over time from pre-treatment to 92.6 months post-treatment (P = 0.03). There was a strong correlation between increase in total VoiSS and GRBAS scores (r = 0.93). Acoustic analysis demonstrated no statistically significant variation in fundamental frequency. CONCLUSION: Radiation therapy for advanced non-laryngeal head and neck has a significant, deleterious effect on voice, which is apparent up to eight years post-treatment.

Glycoconjugated Site-Selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells.

DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.

In Silico and in Vitro Screening for P-Glycoprotein Interaction with Tenofovir, Darunavir, and Dapivirine: An Antiretroviral Drug Combination for Topical Prevention of Colorectal HIV Transmission.

The aim of the study was to use in silico and in vitro techniques to evaluate whether a triple formulation of antiretroviral drugs (tenofovir, darunavir, and dapivirine) interacted with P-glycoprotein (P-gp) or exhibited any other permeability-altering drug-drug interactions in the colorectal mucosa. Potential drug interactions with P-gp were screened initially using molecular docking, followed by molecular dynamics simulations to analyze the identified drug-transporter interaction more mechanistically. The transport of tenofovir, darunavir, and dapivirine was investigated in the Caco-2 cell models and colorectal tissue, and their apparent permeability coefficient (Papp), efflux ratio (ER), and the effect of transporter inhibitors were evaluated. In silico, dapivirine and darunavir showed strong affinity for P-gp with similar free energy of binding; dapivirine exhibiting a ΔGPB value -38.24 kcal/mol, darunavir a ΔGPB value -36.84 kcal/mol. The rank order of permeability of the compounds in vitro was tenofovir < darunavir < dapivirine. The Papp for tenofovir in Caco-2 cell monolayers was 0.10 ± 0.02 × 10-6 cm/s, ER = 1. For dapivirine, Papp was 32.2 ± 3.7 × 10-6 cm/s, but the ER = 1.3 was lower than anticipated based on the in silico findings. Neither tenofovir nor dapivirine transport was influenced by P-gp inhibitors. The absorptive permeability of darunavir (Papp = 6.4 ± 0.9 × 10-6 cm/s) was concentration dependent with ER = 6.3, which was reduced by verapamil to 1.2. Administration of the drugs in combination did not alter their permeability compared to administration as single agents. In conclusion, in silico modeling, cell culture, and tissue-based assays showed that tenofovir does not interact with P-gp and is poorly permeable, consistent with a paracellular transport mechanism. In silico modeling predicted that darunavir and dapivirine were P-gp substrates, but only darunavir showed P-gp-dependent permeability in the biological models, illustrating that in silico modeling requires experimental validation. When administered in combination, the disposition of the proposed triple-therapy antiretroviral drugs in the colorectal mucosa will depend on their distinctly different permeability, but was not interdependent.

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts.

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single-celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra-isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co-evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.

Molecular phylogeny and taxonomic revision of the genus Wittrockiella (Pithophoraceae, Cladophorales), including the descriptions of W. australis sp. nov. and W. zosterae sp. nov.

Wittrockiella is a small genus of filamentous green algae that occurs in habitats with reduced or fluctuating salinities. Many aspects of the basic biology of these algae are still unknown and the phylogenetic relationships within the genus have not been fully explored. We provide a phylogeny based on three ribosomal markers (ITS, LSU, and SSU rDNA) of the genus, including broad intraspecific sampling for W. lyallii and W. salina, recommendations for the use of existing names are made, and highlight aspects of their physiology and life cycle. Molecular data indicate that there are five species of Wittrockiella. Two new species, W. australis and W. zosterae, are described, both are endophytes. Although W. lyallii and W. salina can be identified morphologically, there are no diagnostic morphological characters to distinguish between W. amphibia, W. australis, and W. zosterae. A range of low molecular weight carbohydrates were analyzed but proved to not be taxonomically informative. The distribution range of W. salina is extended to the Northern Hemisphere as this species has been found in brackish lakes in Japan. Furthermore, it is shown that there are no grounds to recognize W. salina var. kraftii, which was described as an endemic variety from a freshwater habitat on Lord Howe Island, Australia. Culture experiments indicate that W. australis has a preference for growth in lower salinities over full seawater. For W. amphibia and W. zosterae, sexual reproduction is documented, and the split of these species is possibly attributable to polyploidization.

Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

RADVAN: a randomised phase 2 trial of WBRT plus vandetanib for melanoma brain metastases - results and lessons learnt.

BACKGROUND: Brain metastases occur in up to 75% of patients with advanced melanoma. Most are treated with whole-brain radiotherapy (WBRT), with limited effectiveness. Vandetanib, an inhibitor of vascular endothelial growth factor receptor, epidermal growth factor receptor and rearranged during transfection tyrosine kinases, is a potent radiosensitiser in xenograft models. We compared WBRT with WBRT plus vandetanib in the treatment of patients with melanoma brain metastases. METHODS: In this double-blind, multi-centre, phase 2 trial patients with melanoma brain metastases were randomised to receive WBRT (30 Gy in 10 fractions) plus 3 weeks of concurrent vandetanib 100 mg once daily or placebo. The primary endpoint was progression-free survival in brain (PFS brain). The main study was preceded by a safety run-in phase to confirm tolerability of the combination. A post-hoc analysis and literature review considered barriers to recruiting patients with melanoma brain metastases to clinical trials. RESULTS: Twenty-four patients were recruited, six to the safety phase and 18 to the randomised phase. The study closed early due to poor recruitment. Median PFS brain was 3.3 months (90% confidence interval (CI): 1.6-5.6) in the vandetanib group and 2.5 months (90% CI: 0.2-4.8) in the placebo group (P=0.34). Median overall survival (OS) was 4.6 months (90% CI: 1.6-6.3) and 2.5 months (90% CI: 0.2-7.2), respectively (P=0.54). The most frequent adverse events were fatigue, alopecia, confusion and nausea. The most common barrier to study recruitment was availability of alternative treatments. CONCLUSIONS: The combination of WBRT plus vandetanib was well tolerated. Compared with WBRT alone, there was no significant improvement in PFS brain or OS, although we are unable to provide a definitive result due to poor accrual. A review of barriers to trial accrual identified several factors that affect study recruitment in this difficult disease area.

Drug transporter gene expression in human colorectal tissue and cell lines: modulation with antiretrovirals for microbicide optimization.

OBJECTIVES: The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. METHODS: Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). RESULTS: Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. CONCLUSIONS: These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection.