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PDF file - 107K, In vivo metabolic profile of tumour and liver after administration of ICT2700 to CYP1A1 tumor-bearing mice

We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor α-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in γ-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers. Mol Cancer Ther; 12(1); 27–37. ©2012 AACR.

PDF file - 120K, Representative CYP1A1 activity of the EJ138 bladder cell line and EJ138-1A1 bladder cell line, determined by measurement of Ethoxyresorufin O-de-ethylation

PDF file - 109K, Methodology for analysis of CYP1 gene and protein expression,Detection of DNA-damage induced gamma-H2AX protein expression, and analysis of ICT2700 metabolism in mouse and human liver ex vivo

PDF file - 52K, Lack of CYP1 induction by the chloromethylpyrroloindoline ICT2700

PDF file - 85k, Expression of CYP1A1 protein in human transitional cell carcinoma (TCC) of the bladder by immunohistochemical analyses.

Background A current or recent long-term care facility (LTCF) stay is a strong risk factor for antibiotic-resistant bacterial colonization and infection. However, most electronic health record (EHR) systems do not systematically record LTCF exposure. Absent manual chart review, which is resource-intensive and cannot be incorporated into automated screening algorithms, there is no definitive method for identifying LTCF-exposed inpatients. As a surrogate, researchers often use ‘Admission Source’ to identify LTCF transfers, but this EHR field has not been previously validated, and it may miss the unknown percentage of patients with recent, but not current, LTCF stays. This study evaluated the accuracy of ‘Admission Source’ in identifying LTCF-exposed inpatients. Methods This was a retrospective study of adult admissions from 2018 – 2021 across 12 hospitals in the University of Maryland Medical System. We extracted patient and encounter data and classified patients as LTCF-exposed by ‘Admission Source’ if they were LTCF transfers. For 315 randomly sampled admissions, M.T. and K.G. reviewed the admission ‘History & Physical’ note for mention of LTCF exposure (Fig. 1). Assuming an indication of LTCF in either the ‘Admission Source’ field or clinical note represented true exposure, we estimated each method’s sensitivity with 95% confidence intervals. Results Across 280,581 admissions, 9,476 (3.4%) had an ‘LTCF transfer’ admission source. In the validation sample, 26 (8.3%) were classified as LTCF-exposed by either ‘Admission Source’ or clinical note, of which 12 were identified in the ‘Admission Source’ field and 25 in the notes (Fig. 2). The sensitivity of ‘Admission Source’ for detecting LTCF exposure was 46% (29% – 65%) and for clinical notes was 96% (81% - 99%). Most (12/14) patients missed by ‘Admission Source’ were current LTCF residents (Fig. 3). Conclusion The EHR ‘Admission Source’ field misses the majority of inpatients with recent or current LTCF exposure, risking substantial misclassification in research studies and clinical algorithms that incorporate this variable. Automated techniques for analyzing free-text notes, such as natural language processing, could significantly improve detection of these patients to assist hospital epidemiology and infection control efforts. Disclosures All Authors: No reported disclosures.

Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.

Study hypothesis Study objective: To estimate the frequency of clinically relevant adverse cardiac events (CRACE) in patients admitted to the hospital for chest pain with an intermediate HEART score (4, 5, 6), non-diagnostic EKG, and a negative initial troponin. Methods We conducted a retrospective analysis of all patients admitted to the University of Maryland Medical Center (UMMC) from May 2016 to May 2019 with an intermediate HEART score (4, 5, or 6), a non-diagnostic EKG, and a negative initial troponin. Our primary outcome was the rate of inpatient clinically relevant adverse cardiac events (CRACE), composite of life-threatening dysrhythmia, inpatient STEMI, cardiac or respiratory arrest, and all-cause mortality during hospitalization. Results A total of 1118 patients met our inclusion criteria, 6 of whom had CRACE. Overall the rate of CRACE was 0.5% (95% CI, 0.2–1.2%). Six patients (0.5%, 95% CI, 0.2%–1.2%) experienced inpatient NSTEMIs, 212 patients (19%, 95% CI, 17–21%) underwent provocative testing during their inpatient stay, 5 patients received a stent or CABG, and 5 patients had false positive non-invasive testing and underwent a negative cardiac catheterization. Conclusions In this cohort of admitted patients with a documented intermediate-risk HEART score, nonischemic EKG, and negative initial troponin, the occurrence of CRACE during the index hospitalization was 0.5%.

Decompensation on the medical floor is associated with increased in-hospital mortality.Our aim was to determine the accuracy of the National Early Warning Score (NEWS) in predicting early, unplanned escalation of care in patients admitted to the hospital from the emergency department (ED) compared to the Shock Index (SI) and the quick Sepsis-Related Organ Failure Assessment (qSOFA) score.We conducted a retrospective cohort study of patients admitted directly from the ED to monitored or unmonitored beds (November 9, 2015 to April 30, 2018) in 3 hospitals. Interhospital transfers were excluded. Patient data, vital status, and bed assignment were extracted from the electronic medical record. Scores were calculated using the last set of vital signs prior to leaving the ED. Primary endpoint was in-hospital death or placement in an intermediate or intensive care unit within 24 h of admission from the ED. Scores were compared using the area under the receiver operating curve (AUROC).Of 46,018 ED admissions during the study window, 39,491 (85.8%) had complete data, of which 3.7% underwent escalation in level of care within 24 h of admission. NEWS outperformed (AUROC 0.69; 95% confidence interval [CI] 0.68-0.69) qSOFA (AUROC 0.63; 95% CI 0.62-0.63; p 0.001) and SI (AUROC 0.60; 95% CI 0.60-0.61; p 0.001) at predicting unplanned escalations or death at 24 h.This multicenter study found NEWS was superior to the qSOFA score and SI in predicting early, unplanned escalation of care for ED patients admitted to a general medical-surgical floor.

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. SIMPLE SUMMARY: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

Background: Ras-related nuclear protein (RAN) causses increases in invasion in vitro and is associated with early breast cancer patients deaths in vivo. However, the underlying mechanism is unknown. Methods: Effect of RAN expression on potential targets MMP2, ATF3, CXCR3 was measured by Real-Time PCR / Western blots. Effects of MMP2 and RAN expression on cancer cell lines (e.g. MDA-MB231) was mearured by soft-agar, cell adhesion, and invasion assays. Correlation between MMP2 and RAN and patient survival times was examined in breast cancer patients.Results: Knockdown of RAN lead toreduction of MMP2 and its potential regulators ATF3 and CXCR3 in breast cancer cell lines. Knockdown of ATF3 or CXCR3 downregulates MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Both knockdown of RAN and MMP2 reduced cell adhesion, migration and growth in agar invasion whilst overexpression of MMP2 reversed the knockdown of RAN. Moreover, the level of immunoreactive RAN and MMP2 are positively associated with each other and with patient survival times, respectively in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression. Conclusions: Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN and both RAN and MMP2 in combination can be used for accurate patient stratification of breast cancer metastasis.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Asymptomatic abdominal aortic aneurysms (AAAs) are increasingly common incidental findings and will likely be encountered periodically by the acute care provider. Due to the extremely high morbidity and mortality of AAA rupture, providers must recognize the long-term importance of the asymptomatic AAA, identify risk factors for rupture, and provide patients with education and appropriate follow-up. All patients with an AAA should follow with vascular surgery, but those with AAA > 5.5 cm maximal diameter in men and > 5.0 cm in women should be urgently evaluated by a vascular surgeon, as these aneurysms are high risk for rupture and are frequently considered for elective repair. Those with AAA expansion >0.5 cm in 6 months need the same urgent evaluation. Other risk factors such as female gender, active smoking, COPD, and uncontrolled hypertension also increase the risk of expansion and rupture and should prompt expedited surgical evaluation.

Genetic variability information, which is a heritable difference among maize cultivars, is important to a long-term plant breeding program. In the last decade a number of researchers have studied the correlation between phenotypic and genetic variability in maize by using Deoxyribonucleic Acid (DNA) markers. Maize landraces as maize cultivars grown ‘on farm’ by farmers who reproduce their seed every year, and they can be distinguished by specific traits, so maize landraces are composed of widely variable populations, where the variation can be seen between and within populations. Five white maize landrace seed samples were obtained from five farmers in each of three villages, Nunmafo, Amol and Ajaobaki; five yellow landrace maize seed samples were obtained from five farmers in Nunmafo village only; while the five certified improved open pollinated varieties (Lamuru, Piet kuning, Bisma, Harapan, Kalingga) were obtained from the Indonesia Cereal Research Institute (ICRI) Maros. The banding patterns of the samples’ fingerprint were evaluated in the scoring range of 50-700bp fragment weight using fragment size standard ladder (Figure 6.3). Image data was viewed on the computer screen and saved in the SAGA computer program. The gel images were scored manually in Excel 2010 program for presence (score 1) or absence (score 0) of bands, and the binary data used for further analysis. The results of this study for AFLP analysis of West Timor maize landrace populations from three villages and varieties have confirmed that there are extensivegenetic variations within West Timor yellow and white maize landrace populations and varieties. There were two clusters of genetic variation among all samples. The first cluster was a general West Timor maize landrace population, with more than 80% similarity among individuals, and the second cluster was amuch more diverse grouping with less than 80% similarity. It suggests that even though a high molecular diversity had been found among West Timor maize landrace populations most maize genotypes belong to a West Timor maize landrace cluster. In addition the analysis indicated not only significant difference between maize landraces and maize varieties but also maize landraces from the three villages were genetically significantly different from each other. The molecular marker characterization of West Timor maize landrace populations will be a key step for obtaining an improved understanding of the population so they can be managed carefully for the future. Moreover, due to the genetic diversity captured within local landraces, farmers have the opportunity to perform the phenotypic selections based on plant type and seed yield which are likely to give them significant genetic gain in yield in subsequent seasons. Plant breeders have the additional opportunity of applying molecular marker techniques to assist selection.

This chapter explores the nature and role of the pastoral counsellor in the lives of people whom psychiatry diagnoses as suffering from mental illness. The pastoral counsellor will share many of the attitudes concerning mental disturbances which form the psychiatric view of madness as illness. Religious conversion had often been accompanied by the phenomenon of mental and emotional upheaval. The psychiatrist viewing the process identifies the characteristics of pathology, or abnormal mental functioning. The ‘crisis’ may be one which involves the complete breakdown of a person’s sense of meaning and rational functioning. For many psychiatrists, psychosis is a sign of deep and disturbing illness. The Christian tradition conveyed to Simon a view of psychological disturbance as an expression of inner conflict. In viewing madness from a religious perspective, pastoral counsellors see it as part of a range of experience within which madness and sanity differ only in their creative fruitfulness and the social context in which they occur.

A deactivated alkene precursor (IC50 = 81 μM) to the azinomycin epoxide natural product can be bioactivated by several cytochromes P450 (CYP) to generate antiproliferative metabolites with increased potency (IC50 = 1–30 μM) in CHOwt cells. CYP1A1 and 3A4 were shown to generate exclusively the unnatural and the natural-configured azinomycin epoxide diastereoisomer respectively, while CYP1B1 produced both epoxides in a 3 : 1 mixture. The antiproliferative activity is linked to DNA damage as demonstrated using the comet assay.

Rising atmospheric carbon dioxide concentration ([CO

The polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB–DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity.

Cytochromes P450 (CYPs) are a superfamily of mixed function oxidases, which in the liver have great significance to the pharmaceutical industry because their expression will determine the fate of most clinical agents. CYPs are also targets for inhibitors of hormone-dependent diseases and conversion of prodrugs to active agents in normal and cancer tissues. We have applied simple modifications to established methods of isolating CYPs, using 8 M urea to solubilise microsomal proteins and specific molecular weight gel bands for in-gel digestion in combination with nanoHPLC MALDI MS to acquire peptide MS/MS spectra for database searching. As a consequence of the changes we significantly improved the yield of proteomic data, identifying 26 mouse CYPs (CYP1a2, 2a4, 2a5, 2a12, 2b9, 2c29, 2c37, 2c39, 2c40, 2c50, 2c54, 2c70, 2d9, 2d10, 2d26, 2e1, 2f2, 2j5, 3a11, 3a13, 3a25, 3a41, 4a14, 4f14, 8b1 and 27a1) with an average sequence coverage of 30.1%, including some previously undetected highly homologous isoforms. In addition, other important enzymes in drug metabolism are also identified. There is a divergence of opinion over the expression of CYP1a1 in liver and we could not detect the presence of this isoform. In order to provide definitive evidence of the ability to detect CYP1a1, we analysed CHO cells transfected with human CYP1A1 and identified unique peptides that differentiated this isoform from human CYP1A2.

Background: Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT.Methods: Solid phase–bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD+ as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti–his-tag antibody using an LAS-1000 System.Results: The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase.Conclusions: The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane–permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a nonpermeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC50) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.

New drug delivery technologies have an important niche in treatments as they enable drugs to be more effective. Remarkably, drug delivery is still considered a poor relation to drug discovery with greater than 95% of all new potential therapeutics having poor pharmacokinetics. The greatest obstruction for cytosolic release of therapeutic molecules is the membrane barrier of target cells. The use of protein transduction domains (PTD), capable of transporting effector molecules, such as compounds, proteins and DNA, into cells has become increasingly attractive in the design of drugs as they promote the cellular uptake of cargo molecules. The PTDs are also often referred to as Trojan peptides, membrane translocating sequences or cell permeable proteins. They are generally 10-16 amino acids in length. The mechanism of internalisation remains unclear, however, recent evidence supports an energy dependant process involving endocytosis. PTDs may be grouped according to their composition, for example peptides rich in arginine and/or lysine (Tat protein from HIV-1), or secondary structure, for example α-helix (HA2 from influenza virus hemagglutinin) and β- sheet (protegrins derived from antimicrobial peptides).