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Abstract Background Malaria remains a common course of morbidity in many sub-Saharan African countries. While treatment options have improved in recent times, inappropriate prescription seems conventional among providers, increasing the burden on patients and society. This study examined the cost of inappropriate prescriptions for uncomplicated malaria treatment in Ghana. Methods This study used retrospective data collected from January to December 2016 in 27 selected facilities, under different ownership in three regions of the country, mainly Volta, Upper East and Brong Ahafo. Stratified random sampling technique was used to extract 1625 outpatient folders of patients diagnosed and treated for malaria. Two physicians independently reviewed patient folders according to the stated diagnoses. Malaria prescriptions were described as inappropriate when they do not adhere to the standard treatment guidelines. The economic cost was mainly treatment cost which was sourced as medication cost. Total and average costs for country were calculated using sample estimates and the total number of uncomplicated malaria cases that received inappropriate prescriptions. Results The study revealed that patients received an average of two prescriptions per malaria episode. Artemether-lumefantrine (AL) was the major malaria medication (79.5%) prescribed to patients. Other medications usually antibiotics and vitamins and minerals were included in the prescription. More than 50% of prescribers did not follow the guidelines for prescribing medications to clients. By facility type, inappropriate prescription was high in the CHPS compounds (59.1%) and by ownership, government (58.3%), private (57.5%) and mission facilities (50.7%). Thus, about 55% of malaria prescriptions were evaluated as inappropriate during the review period, which translates into economic cost of approximately US$4.52 million for the entire country in 2016. The total cost of inappropriate prescription within the study sample was estimated at US$1,088.42 while the average cost was US$1.20. Conclusion Inappropriate prescription for malaria is a major threat to malaria management in Ghana. It presents a huge economic burden to the health system. Training and strict enforcement of prescribers’ adherence to the standard treatment guideline is highly recommended.

Less than one-third of HIV-infected pregnant women eligible for combination antiretroviral therapy (ART) globally initiate treatment prior to delivery, with lack of access to timely CD4 results being a principal barrier. We evaluated the effectiveness of an SMS-based intervention to improve access to timely antenatal ART.We conducted a stepped-wedge cluster randomized trial of a low-cost programmatic intervention in 20 antenatal clinics in Gaborone, Botswana. From July 2011-April 2012, 2 clinics were randomly selected every 4 weeks to receive an ongoing clinic-based educational intervention to improve CD4 collection and to receive CD4 results via an automated SMS platform with active patient tracing. CD4 testing before 26 weeks gestation and ART initiation before 30 weeks gestation were assessed.Three-hundred-sixty-six ART-naïve women were included, 189 registering for antenatal care under Intervention and 177 under Usual Care periods. Of CD4-eligible women, 100 (59.2%) women under Intervention and 79 (50.6%) women under Usual Care completed CD4 phlebotomy before 26 weeks gestation, adjusted odds ratio (aOR, adjusted for time that a clinic initiated Intervention) 0.87 (95% confidence interval [CI]0.47-1.63, P = 0.67). The SMS-based platform reduced time to clinic receipt of CD4 test result from median of 16 to 6 days (P

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells, and it is the most frequent form of leukemia diagnosed in Western countries. It is characterized by the proliferation and accumulation of neoplastic B lymphocytes in the blood, lymph nodes, bone marrow and spleen. We report the synthesis and antiproliferative effects of a series of novel ethanoanthracene compounds in CLL cell lines. Structural modifications were achieved via the Diels-Alder reaction of 9-(2-nitrovinyl)anthracene and 3-(anthracen-9-yl)-1-arylprop-2-en-1-ones (anthracene chalcones) with dienophiles, including maleic anhydride and N -substituted maleimides, to afford a series of 9-( E )-(2-nitrovinyl)-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones, 9-( E )-3-oxo-3-phenylprop-1-en-1-yl)-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones and related compounds. Single-crystal X-ray analysis confirmed the structures of the novel ethanoanthracenes 23f , 23h , 24a , 24g , 25f and 27 . The products were evaluated in HG-3 and PGA-1 CLL cell lines (representative of poor and good patient prognosis, respectively). The most potent compounds were identified as 20a , 20f, 23a and 25n with IC 50 values in the ranges of 0.17-2.69 µM (HG-3) and 0.35-1.97 µM (PGA-1). The pro-apoptotic effects of the potent compounds 20a , 20f, 23a and 25n were demonstrated in CLL cell lines HG-3 (82-95%) and PGA-1 (87-97%) at 10 µM, with low toxicity (12-16%) observed in healthy-donor peripheral blood mononuclear cells (PBMCs) at concentrations representative of the compounds IC 50 values for both the HG-3 and PGA-1 CLL cell lines. The antiproliferative effect of the selected compounds, 20a , 20f, 23a and 25n, was mediated through ROS flux with a marked increase in cell viability upon pretreatment with the antioxidant NAC. 25n also demonstrated sub-micromolar activity in the NCI 60 cancer cell line panel, with a mean GI 50 value of 0.245 µM. This ethanoanthracene series of compounds offers potential for the further development of lead structures as novel chemotherapeutics to target CLL.

Ruthenium(ii) complexes are attracting significant research attention as a promising class of photosensitizers (PSs) in photodynamic therapy (PDT). Having previously reported the synthesis of two novel Ru(ii)-polypyridyl-1,8-naphthalimide Tröger's base compounds 1 and 2 with interesting photophysical properties, where the emission from either the Ru(ii) polypyridyl centres or the naphthalimide moieties could be used to monitor binding to nucleic acids, we sought to use these compounds to investigate further and in more detail their biological profiling, which included unravelling their mechanism of cellular uptake, cellular trafficking and cellular responses to photoexcitation. Here we demonstrate that these compounds undergo rapid time dependent uptake in HeLa cells that involved energy dependent, caveolae and lipid raft-dependent mediated endocytosis, as demonstrated by confocal imaging, and transmission and scanning electron microscopy. Following endocytosis, both compounds were shown to localise to mostly lysosomal and Golgi apparatus compartments with some accumulation in mitochondria but no localisation was found to the nucleus. Upon photoactivation, the compounds increased ROS production and induced ROS-dependent apoptotic cell death. The photo-activated compounds subsequently induced DNA damage and altered tubulin, but not actin structures, which was likely to be an indirect effect of ROS production and induced apoptosis. Furthermore, by changing the concentration of the compounds or the laser used to illuminate the cells, the mechanism of cell death could be changed from apoptosis to necrosis. This is the first detailed biological study of Ru(ii)-polypyridyl Tröger's bases and clearly suggests caveolae-dependent endocytosis is responsible for cell uptake - this may also explain the lack of nuclear uptake for these compounds and similar results observed for other Ru(ii)-polypyridyl complexes. These conjugates are potential candidates for further development as PDT agents and may also be useful in mechanistic studies on cell uptake and trafficking., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Chronic lymphocytic leukaemia (CLL) is a malignancy of the immune B lymphocyte cells and is the most common leukaemia diagnosed in developed countries. In this paper, we report the synthesis and antiproliferative effects of a series of ( E )-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds in CLL cell lines and also in Burkitt's lymphoma (BL) cell lines, a rare form of non-Hodgkin's immune B-cell lymphoma. The nitrostyrene scaffold was identified as a lead structure for the development of effective compounds targeting BL and CLL. The series of structurally diverse nitrostyrenes was synthesised via Henry-Knoevenagel condensation reactions. Single-crystal X-ray analysis confirmed the structure of ( E )-9-chloro-10-(2-nitrobut-1-en-1-yl)anthracene ( 19f ) and the related 4-(anthracen-9-yl)-1 H -1,2,3-triazole ( 30a ). The ( E )-9-(2-nitrovinyl)anthracenes 19a, 19g and 19i-19m were found to elicit potent antiproliferative effects in both BL cell lines EBV - MUTU-1 (chemosensitive) and EBV + DG-75 (chemoresistant) with >90% inhibition at 10 μM. Selected ( E )-9-(2-nitrovinyl)anthracenes demonstrated potent antiproliferative activity in CLL cell lines, with IC 50 values of 0.17 μM (HG-3) and 1.3 μM (PGA-1) for compound 19g . The pro-apoptotic effects of the most potent compounds 19a , 19g , 19i , 19l and 19m were demonstrated in both CLL cell lines HG-3 and PGA-1. The ( E )-nitrostyrene and ( E )-9-(2-nitrovinyl)anthracene series of compounds offer potential for further development as novel chemotherapeutics for CLL.

Cationic porphyrins based on the 5,10,15,20- meso -(tetrakis-4- N -methylpyridyl) core (TMPyP4) have been studied extensively over many years due to their strong interactions with a variety of nucleic acid structures, and their potential use as photodynamic therapeutic agents and telomerase inhibitors. In this paper, the interactions of metal-free TMPyP4 and Pt(II)TMPyP4 with guanine-containing nucleic acids are studied for the first time using time-resolved infrared spectroscopy (TRIR). In D 2 O solution (where the metal-free form exists as D 2 TMPyP4) both compounds yielded similar TRIR spectra (between 1450-1750 cm -1 ) following pulsed laser excitation in their Soret B-absorption bands. Density functional theory calculations reveal that vibrations centred on the methylpyridinium groups are responsible for the dominant feature at ca. 1640 cm -1 . TRIR spectra of D 2 TMPyP4 or PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP), double-stranded {d(GC) 5 } 2 or {d(CGCAAATTTGCG)} 2 contain negative-going signals, 'bleaches', indicative of binding close to guanine. TRIR signals for D 2 TMPyP4 or PtTMPyP bound to the quadruplex-forming cMYC sequence {d(TAGGGAGGG)} 2 T indicate that binding occurs on the stacked guanines. For D 2 TMPyP4 bound to guanine-containing systems, the TRIR signal at ca. 1640 cm -1 decays on the picosecond timescale, consistent with electron transfer from guanine to the singlet excited state of D 2 TMPyP4, although IR marker bands for the reduced porphyrin/oxidised guanine were not observed. When PtTMPyP is incorporated into HeLa tumour cells, TRIR studies show protein binding with time-dependent ps/ns changes in the amide absorptions demonstrating TRIR's potential for studying light-activated molecular processes not only with nucleic acids in solution but also in biological cells.

A family of six Ru(II) polypyridyl complexes ( 1 - 6 ) which contain phenanthroline-based ligands functionalized with alkyl chains of different lengths (one methyl group, 10 and 21 carbon alkyl chains) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesized and characterized. The influence of the alkyl chain length on their photophysical and photochemical properties as well as in their photobiological applications has been elucidated by monitoring the changes in their MLCT-centered absorption and emission bands. The presence of one methyl group or 10 carbon alkyl chains does not seem to significantly affect the photophysical and photochemical properties of the resulting Ru(II) complexes when compared to the well-known [Ru(phen) 3 ] 2+ and [Ru(TAP) 2 phen] 2+ . However, an effect on their emission properties and in their ability to photosensitize singlet oxygen is observed for the Ru(II) complexes containing 21 carbon alkyl chains. The binding of these complexes to salmon testes DNA (stDNA) was investigated by observing the changes in the photophysical properties. Complexes 1 , 2 , 4 , and 5 all showed changes in their MLCT bands that could be analyzed using conventional fitting methods, such as the Bard equation. In contrast, complexes 3 and 6 , possessing long aliphatic chains, gave rise to nonclassic behavior. In addition to these analyses, both thermal denaturation and circular dichroism studies of 1 - 6 were carried out in the presence of stDNA which confirmed that these complexes bind to DNA. Confocal microscopy and viability studies in HeLa cervical cancer cells reveal an alkyl chain-length dependence on the cellular uptake and cytotoxicity of the resulting Ru(II) complexes due to an enhancement of their lipophilicity with increasing alkyl chain length. Thus, complexes containing 10 and 21 carbon alkyl chains are rapidly taken up into HeLa cells and, in particular, those with 21 carbon alkyl chains show a significant phototoxicity against the same cell line. Therefore, this study provides further insight into the possible modulation of the photophysical, photochemical, and photobiological properties of Ru(II) polypyridyl complexes by varying the length of the alkyl chains attached to the polypyridyl ligands coordinated to the Ru(II) center and the nature of the auxiliary groups, which we show has a significant effect on photophysical and biological properties.

Hematopoietic stem cell- (HSC) and induced pluripotent stem (iPS) cell-derived natural killer (NK) cells containing engineered functions, such as chimeric antigen receptors (CAR), offer great promise for the treatment of seemingly incurable oncological malignancies. Today, some of the main challenges of CAR cell-based therapeutics are the long manufacturing time and safety of the cell sources used. Additional challenges include avoiding graft vs host disease (GVHD) and cytokine release syndrome (CRS). Here, we show compelling evidence for the use of NK cell therapeutics as a reliable off-the-shelf option, as they address key issues. Furthermore, we highlight how iPS cells and directed differentiation toward HSC and NK cells address industrial scalability and safety., (© 2021 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Two water-soluble amphiphilic Ru(ii) polypyridyl complexes containing N-1,10-phenanthrolin-5-yldocosanamide and 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ligands were synthesised and their photophysical and photobiological properties evaluated; both complexes showed a rapid cellular uptake and displayed phototoxicity against HeLa cervical cancer cells.

Lymphomas (cancers of the lymphatic system) account for 12% of malignant diseases worldwide. Burkitt's lymphoma (BL) is a rare form of non-Hodgkin's lymphoma in which the cancer starts in the immune B-cells. We report the synthesis and preliminary studies on the antiproliferative activity of a library of 9,10-dihydro-9,10-ethanoanthracene based compounds structurally related to the antidepressant drug maprotiline against BL cell lines MUTU-1 and DG-75. Structural modifications were achieved by Diels-Alder reaction of the core 9-(2-nitrovinyl)anthracene with number of dienophiles including maleic anhydride, maleimides, acrylonitrile and benzyne. The antiproliferative activity of these compounds was evaluated in BL cell lines EBV - MUTU-1 and EBV + DG-75 (chemoresistant). The most potent compounds 13j , 15 , 16a , 16b , 16c , 16d and 19a displayed IC 50 values in the range 0.17-0.38 μM against the BL cell line EBV - MUTU-1 and IC 50 values in the range 0.45-0.78 μM against the chemoresistant BL cell line EBV + DG-75. Compounds 15 , 16b and 16c demonstrated potent ROS dependent apoptotic effects on the BL cell lines which were superior to the control drug taxol and showed minimal cytotoxicity to peripheral blood mononuclear cells (PBMCs). The results suggest that this class of compounds merits further investigation as antiproliferative agents for BL.

Microtubules are a validated clinical target for the treatment of many cancers. We describe the design, synthesis, biochemical evaluation, and molecular modelling studies of a series of analogues of the microtubule-destabilising agent, combretastatin A-4 (CA-4). Our series of 33 novel compounds contain the CA-4 core structure with modifications to the stilbene linking group, and are predominantly piperazine derivatives. Synthesis was achieved in a two-step process by firstly obtaining the acrylic acid via a Perkin reaction using microwave enhanced synthesis, followed by coupling using either DCC or Mukaiyama's reagent. All target compounds were screened for antiproliferative activity in MCF-7 breast cancer cells. Hydroxyl derivative (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl) propenone (4m) displayed potent antiproliferative activity (IC50 = 190 nM). Two amino-containing derivatives, (E)-3-(3-amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4q) and (E)-3-(3-amino-4-methoxyphenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4x), were the most potent with IC50 values of 130 nM and 83 nM respectively. Representative compounds were shown to depolymerise tubulin, induce G2/M arrest and apoptosis in MCF-7 cells but not peripheral blood mononuclear cells, and induce cleavage of the DNA repair enzyme poly ADP ribose polymerase (PARP) in MCF-7 cells. Modelling studies predict that the compounds bind to tubulin within the colchicine-binding site. These compounds are a valuable addition to the library of CA-4 analogues and 4m, 4q and 4x will be developed further as novel, water-soluble molecules targeting microtubules.

The nitrostyrene scaffold was previously identified as a lead target structure for the development of effective compounds targeting Burkitt's lymphoma. The present study aimed to develop these compounds further in haematological malignancies, including chronic lymphocytic leukaemia (CLL). Cellular viability, flow cytometry and lactate dehydrogenase assays, amongst others, were used to examine the effects of nitrostyrene compounds on CLL cells, including a cell line representing disease with poor prognosis (HG‑3) and in ex vivo CLL patient samples (n=14). The results demonstrated that two representative nitrostyrene compounds potently induced apoptosis in CLL cells. The pro‑apoptotic effects of the compounds were found to be reactive oxygen species and caspase‑dependent, and had minimal effects on the viability of normal donor peripheral blood mononuclear cells. Nitrostyrene compounds exhibited synergistic augmentation of apoptosis when combined with the phosphatidylinositol 3‑kinase inhibitor idelalisib and demonstrated potent toxicity in ex vivo CLL cells, including those co‑cultured with bone marrow stromal cells, making them more resistant to apoptosis (n=8). These compounds also demonstrated activity in samples from patients with poor prognostic indicators; unmutated immunoglobulin heavy chain genes, expression of CD38 and deletions in chromosomes 17p and 11q. These results suggest that this class of pharmaceutically active compounds offer potential in the treatment of CLL.

Background: Cancers of the lymphatic cells (lymphomas) account for approximately 12% of malignant diseases worldwide. The nitrostyrene scaffold is identified as a lead target structure for the development of particularly effective compounds targeting Burkitt's lymphoma (BL)., Objectives: The aims of the curent study were to synthesise a panel of nitrostyrene compounds and to evaluate their activity in Burkitt's lymphoma (BL)., Methods: A panel of structurally varied compounds were designed and synthesised using Henry Knoevenagel condensation reactions. Single crystal X-Ray analysis confirmed the E configuration for six examples of these novel structures. A number of nitrostyrene-related compounds were also investigated including 1,3-bis(aryl)-2-nitropropenes together with heterocyclic scaffolds containing the nitrovinyl pharmacophore such as 3-nitro-2-phenyl-2H-chromenes. The antiproliferative activities of the compounds were evaluated using the BL cell lines EBV- MUTU-1 and EBV+ DG- 75 (chemoresistant) to establish preliminary structure-activity relationships., Results: Lead compounds with optimized nitrostyrene scaffolds and 3-nitro-2-phenyl-2Hchromene structures were successfully established with typical IC50 values of 0.45 µM and 0.47 µM in MUTU-1 cells and 1.41 µM and 1.92 µM, respectively, in DG-75 cells. The mechanism of cell death was identified as apoptotic and the lead compound was found to elicit comparable apoptotic effects to Taxol in Burkitt's lymphoma cell lines MUTU-1 and DG-75., Conclusion: This class of pharmaceutically active compounds with potential for the treatment of Burkitt`s lymphoma suggest a potential role for nitrostyrene based agents in chemotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Ruthenium(ii) [Ru(ii)] polypyridyl complexes have been the focus of intense investigations since work began exploring their supramolecular interactions with DNA. In recent years, there have been considerable efforts to translate this solution-based research into a biological environment with the intention of developing new classes of probes, luminescent imaging agents, therapeutics and theranostics. In only 10 years the field has expanded with diverse applications for these complexes as imaging agents and promising candidates for therapeutics. In light of these efforts this review exclusively focuses on the developments of these complexes in biological systems, both in cells and in vivo, and hopes to communicate to readers the diversity of applications within which these complexes have found use, as well as new insights gained along the way and challenges that researchers in this field still face.

Nuclear receptors such as the estrogen receptors (ERα and ERβ) modulate the effects of the estrogen hormones and are important targets for design of innovative chemotherapeutic agents for diseases such as breast cancer and osteoporosis. Conjugate and bifunctional compounds which incorporate an ER ligand offer a useful method of delivering cytotoxic drugs to tissue sites such as breast cancers which express ERs. A series of novel conjugate molecules incorporating both the ER ligands endoxifen and cyclofenil-endoxifen hybrids covalently linked to the antimitotic and tubulin targeting agent combretastatin A-4 were synthesised and evaluated as ER ligands. A number of these compounds demonstrated pro-apoptotic effects, with potent antiproliferative activity in ER-positive MCF-7 breast cancer cell lines and low cytotoxicity. These conjugates displayed binding affinity towards ERα and ERβ isoforms at nanomolar concentrations e.g., the cyclofenil-amide compound 13e is a promising lead compound of a clinically relevant ER conjugate with IC 50 in MCF-7 cells of 187 nM, and binding affinity to ERα (IC 50 = 19 nM) and ERβ (IC 50 = 229 nM) while the endoxifen conjugate 16b demonstrates antiproliferative activity in MCF-7 cells (IC 50 = 5.7 nM) and binding affinity to ERα (IC 50 = 15 nM) and ERβ (IC 50 = 115 nM). The ER binding effects are rationalised in a molecular modelling study in which the disruption of the ER helix-12 in the presence of compounds 11e , 13e and 16b is presented These conjugate compounds have potential application for further development as antineoplastic agents in the treatment of ER positive breast cancers., Competing Interests: The authors declare no conflict of interest.

Two 2-nitroimidazole-1,8-naphthalimide conjugates, 1 and 2, have been synthesised as fluorescence probes for the detection of reductive stress in HeLa cells. The 4-substituted derivative 1 was shown to act as a highly sensitive and selective substrate for nitroreductase where it exhibited a clear blue to green ratiometric fluorescence response visible to the naked eye. Moreover, biological studies demonstrated 1 could be activated in cellulo where the impact of reductive stress was easily monitored using confocal microscopy and flow cytommetry.

Herein we present a supramolecular (delayed luminescent) Eu(III)-based pH-responsive probe/sensor with the ability to detect the urease-mediated hydrolysis of urea in aqueous solution. A series of photophysical titrations show this Eu(III) chelate behaves as an "on-off" luminescent switching probe, with its luminescence being quenched upon urea being enzymatically converted into ammonia and carbon dioxide. Calculation of the rate constant (k) and activation energy (E a ) for this hydrolysis reaction are detailed; the results demonstrate a direct observation of enzymatic activity in solution by the sensor. The potential application of this probe in detecting the onset of catheter-associated urinary tract infections (CAUTIs) is also demonstrated by incorporating 1.Eu into water-permeable hydrogels that can be utilized as an alternative coating for catheters.

Glycosylated 4-amino-1,8-naphthalimide derivatives possess a native glycosidic linkage that can be selectively hydrolysed in situ by glycosidase enzymes to release the naphthalimide as a fluorescent imaging or therapeutic agent. In vitro studies using a variety of cancer cell lines demonstrated that the naphthalimides only get taken up into cells upon enzymatic cleavage from the glycan unit; a mechanism that offers a novel approach for the targeted delivery of probes/drugs.

Four new Ru(ii) polypyridyl complexes that contain an extended aromatic moiety derived from pyrazino[2,3-h]dipyrido[3,2-a:2',3'-c]phenazine and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) have been synthesized, their solid state X-ray crystal structure determined and their photophysical and biological properties evaluated. Their interactions with DNA have been studied, and they have been tested for their potential as photodynamic therapeutic (PDT) agents in the treatment of cancer. A practical modification of a method by Carter, Rodriguez and Bard has been introduced and used to calculate binding parameters for the complexes which show a strong affinity for DNA with binding constants in the order of 10 7 M -1 (in 10 mM phosphate buffer). The complexes containing phen as an ancillary ligand become emissive upon binding to DNA ("light switch effect"), but do not show selective cytotoxicity upon light irradiation. On the other hand, the TAP complexes, which show an inverse "light switch effect" (emission quenched upon binding to DNA), are strongly photo-toxic suggesting their use in Photodynamic Therapy (PDT). In HeLa cells the best PDT agent shows an IC 50 value (light) = 4 μM vs. IC 50 value (dark) = 62 μM.

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC50 values of 2.71μM and 1.86μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86., (Copyright © 2016. Published by Elsevier Ltd.)

The C-KIT receptor tyrosine kinase is constitutively activated in the majority of gastrointestinal stromal tumours (GIST). Imatinib (IM) a selective inhibitor of C-KIT, is indicated for the treatment of KIT-positive unresectable and/or metastatic GIST, and has tripled the survival time of patients with metastatic GIST. However, the majority of patients develop IM-resistance and progress. Although IM elicits strong antiproliferative effects, it fails to induce sufficient levels of apoptosis; acquired IM-resistance and disease recurrence remain an issue, a more effective drug treatment is greatly needed. We examined the effect of a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-15 in combination with IM on GIST cells. PBOX-15 decreased viability and in combination with IM synergistically enhanced apoptosis in both IM-sensitive and IM-resistant GIST cells, decreased the anti-apoptotic protein Mcl-1, and enhanced activation of pro-caspase-3 and PARP cleavage. The combination treatment also led to an enhanced inhibition of C-KIT-phosphorylation and inactivation of C-KIT-dependent signalling in comparison to either drug alone; CDC37, a key regulator of C-KIT in GIST was also dramatically decreased. Furthermore, PBOX-15 reduced CKII expression, an enzyme which regulates the expression of CDC37. In conclusion, our findings indicate the potential of PBOX-15 to improve the apoptotic response of IM in GIST cells and provide a more effective treatment option for GIST patients.

A new series of indole-based analogues were recently identified as potential anticancer agents. The Knoevenagel-type indoles herein presented were prepared via a one-pot condensation of iminium salts with active methylene reagents and were isolated as single geometric isomers. Biological evaluation in different cell-based assays revealed an antiproliferative activity for some analogues already in the nanomolar range against leukaemia, breast and renal cancer cell lines. To explain these effects, the most promising analogues of the series were engaged in further cell-based studies. Compounds 5e, l, p and 6a, b highlighted a pro-apoptotic potential being able to induce apoptosis in HL60, K562 and MCF-7 cell lines in a dose and time-dependent manner. The ability of these compounds to arrest cell cycle at the G2/M phase inspired the immunofluorescence studies which allowed us to identify tubulin as a potential target for compounds 5l and 6b., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Ruthenium polypyridyl complexes show great promise as new photodynamic therapy (PDT) agents. However, a lack of detailed understanding of their mode of action in cells poses a challenge to their development. We have designed a new Ru(II) PDT candidate that efficiently enters cells by incorporation of the lipophilic aromatic pdppz ([2,3-h]dipyrido[3,2-a:2',3'-c]phenazine) ligand and exhibits photoactivity through incorporation of 1,4,5,8-tetraazaphenanthrene ancillary ligands. Its photoreactivity toward biomolecules was studied in vitro, where light activation caused DNA cleavage. Cellular internalization occurred via an energy dependent mechanism. Confocal and transmission electron microscopy revealed that the complex localizes in various organelles, including the mitochondria. The complex is nontoxic in the dark, with cellular clearance within 96 h; however, upon visible light activation it induces caspase-dependent and reactive-oxygen-species-dependent apoptosis, with low micromolar IC50 values. This investigation greatly increases our understanding of such systems in cellulo, aiding development and realization of their application in cancer therapy.

The synthesis and photophysical studies of two cationic Tröger's base (TB)-derived bis-naphthalimides 1 and 2 and the TB derivative 6, characterized by X-ray crystallography, are presented. The enantiomers of 1 and 2 are separated by cation-exchange chromatography on Sephadex C25 using sodium (-)-dibenzoyl-l-tartarate as the chiral mobile phase. The binding of enantiomers with salmon testes (st)-DNA and synthetic polynucleotides are studied by a variety of spectroscopic methods including UV/vis absorbance, circular dichroism, linear dichroism, and ethidium bromide displacement assays, which demonstrated binding of these compounds to the DNA grooves with very high affinity (K ∼ 10(6) M(-1)) and preferential binding of (-)-enantiomer. In all cases, binding to DNA resulted in a significant stabilization of the double-helical structure of DNA against thermal denaturation. Compound (±)-2 and its enantiomers possessed significantly higher binding affinity for double-stranded DNA compared to 1, possibly due to the presence of the methyl group, which allows favorable hydrophobic and van der Waals interactions with DNA. The TB derivatives exhibited marked preference for AT rich sequences, where the binding affinities follow the order (-)-enantiomer > (±) > (+)-enantiomer. The compounds exhibited significant photocleavage of plasmid DNA upon visible light irradiation and are rapidly internalized into malignant cell lines.

The synthesis and characterisation of five bis-1,8-naphthalimide containing Tröger's bases 1–5 formed from their corresponding 3-amino-1,8-naphthalimide precursors 6–10 is described. The photophysical investigations of 1–5 and 6–10 were carried out in several organic solvents as well as in water and as a function of pH using UV-Vis absorption and fluorescence spectroscopies. The DNA binding affinities of 1–5 in aqueous solution at pH 7.4 were also investigated using several UV-Vis absorption and fluorescence experiments by using calf thymus DNA (ct-DNA). These molecules exhibited significant DNA binding affinities; where large binding values (Kb) in the range of 10(6) M(−1) were determined, even in competitive media (50 mM and 160 mM NaCl at pH 7.4). Thermal denaturation measurements also showed that 1–5 significantly stabilised the DNA helix. Using linear and circular dichroism we further demonstrated that the DNA binding interaction occurs both by intercalation and by groove binding. The Tröger's bases were further shown to be rapidly taken up into cells using confocal fluorescence spectroscopy; and cytotoxic studies in HeLa and MCF-7 cells showed that most of the Tröger's bases were effective cytotoxic agents with EC50 values of between 1.1–12 μM and that all the active compounds induced programmed cell death by apoptosis, where up to 70% cellular death was observed after 24 h of incubation for 4.

Neuroblastoma, a malignancy of neuroectoderrmal origin, accounts for 15% of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat. A major obstacle in the effective treatment of neuroblastoma is the development of multidrug resistance (MDR). There is thus a compelling demand for new treatment strategies for this cancer that can bypass such resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various cancer cell lines, ex vivo patient samples and in vivo cancer models. In this study we examined the ability of two members, PBOX-6 and -15, to exhibit anti-cancer effects in a panel of drug sensitive and MDR neuroblastoma cell lines. The PBOX compounds potently reduced the viability of all neuroblastoma cells examined and exhibited a lower fold resistance in MDR cells when compared to standard chemotherapeutics. In addition, the PBOX compounds synergistically enhanced apoptosis induced by etoposide, carboplatin and doxorubicin. Exposure of drug sensitive and resistant cell lines to PBOX-6/carboplatin induced cleavage of Bcl-2, a downregulation of Mcl-1 and a concomitant increase in Bak. Furthermore, activation of caspase-3, -8 and -9 was demonstrated. Finally, gene silencing of Mcl-1 by siRNA was shown to sensitise both drug sensitive and multidrug resistant cells to carboplatin-induced apoptosis demonstrating the importance of Mcl-1 downregulation in the apoptotic pathway mediated by the PBOX compounds in neuroblastoma. In conclusion, our findings indicate the potential of the PBOX compounds in enhancing chemosensitivity in neuroblastoma., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The synthesis of a diverse library of compounds structurally related to maprotiline, a norepinephrine reuptake transporter (NET) selective antidepressant which has recently been identified as a novel in vitro antiproliferative agent against Burkitt's lymphoma (BL) cell lines is reported. A series of 9,10-dihydro-9,10-ethanoanthracenes were synthesised with modifications to the bridge of the dihydroethanoanthracene structure and with alterations to the basic side chain. A number of compounds were found to reduce cell viability to a greater extent than maprotiline in BL cell lines. In addition a related series of novel 9-substituted anthracene compounds were investigated as intermediates in the synthesis of 9,10-dihydro-9,10-ethanoanthracenes. These compounds proved the most active from the screen and were found to exert a potent caspase-dependant apoptotic effect in the BL cell lines, while having minimal effect on the viability of peripheral blood mononuclear cells (PBMCs). Compounds also displayed activity in multi-drug resistant (MDR) cells., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The bi-functional complex [Pt(II)(terpy)(py-naphth)](NO3)2(), incorporating both 2,2':6',2''-terpyridine and 4-N,N'-dimethylamino-1,8-naphthalimide moieties, displays a high binding affinity for DNA as well as displaying cytotoxicity towards and inducing apoptosis in malignant cell lines.

In recent years an extensive series of synthetic combretastatin A-4 (CA-4)-azetidinone (β-lactam) hybrids were designed and synthesised with a view to improve the stability, therapeutic efficacy and aqueous solubility of CA-4. Lead compounds containing a 3,4,5-trimethoxy aromatic ring at position 1 and a variety of substitution patterns at positions 3 and 4 of the β-lactam ring were screened in three adenocarcinoma-derived colon cancer cell lines (CT-26, Caco-2 and the CA-4 resistant cell line, HT-29). In both CT-26 and Caco-2 cells all β-lactam analogues analysed displayed potent therapeutic efficacy within the nanomolar range. Substitution of the ethylene bridge of CA-4 with the β-lactam ring together with the aforementioned aryl substitutions improved the therapeutic efficacy of CA-4 up to 300‑fold in the combretastatin refractory HT-29 cells. The lead compound combretazet-3 (CAZ-3); chemical name [4-(3-hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one] demonstrated improved chemical stability together with enhanced therapeutic efficacy as compared with CA-4 whilst maintaining the natural biological properties of CA-4. Furthermore, CAZ-3 demonstrated significant tumour inhibition in a murine model of colon cancer. Our results suggest that combretastatin-azetidinone hybrids represent an effective novel therapy for the treatment of combretastatin resistant carcinomas.

We here report the synthesis of ethylene glycol N-interlinked imipramine dimers of various lengths from the tricyclic antidepressant desipramine via an amide coupling reaction followed by reduction with lithium aluminium hydride. The target molecules were found to be potent inhibitors of cellular viability while inducing cell type specific death mechanisms in three cancer cell lines including a highly chemoresistant Burkitt's lymphoma cell line. Basic amine analogues were found to be important for increased potency. Imipramine and desipramine were also tested for apoptotic activity and were found to be much less active than the novel dimeric compounds. Imipramine dimers were only found to be moderate inhibitors of the human serotonin transporter (hSERT) having IC(50) values in the micromolar region whilst the induction of cell death occurred independently of hSERT expression. These results demonstrate the potential of newly designed and synthesised imipramines derivatives for use against malignant cells, including those resistant to standard chemotherapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The synthesis and photophysical properties of 1 and 2, two Ru(II)-polypyridyl based-1,8-naphthalimide Tröger's bases, are described; these were found to stabilize double stranded DNA, undergo rapid cellular uptake, displaying good luminescence without affecting cell viability even after 24 hours of incubation., (This journal is © The Royal Society of Chemistry 2012)

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: Imatinib is a direct and potent inhibitor of the constitutively active tyrosine kinase, breakpoint cluster region-Abelson (Bcr-Abl), which is central to the pathogenesis of chronic myeloid leukaemia (CML) patients. As such, imatinib has become the front-line treatment for CML patients. However, the recent emergence of imatinib resistance, commonly associated with point mutations within the kinase domain, has led to the search for alternative drug treatments and combination therapies for CML., Methods: In this report, we analyse the effects of representative members of the novel pro-apoptotic microtubule depolymerising pyrrolo-1,5-benzoxazepines or PBOX compounds on chemotherapy-refractory CML cells using a series of Bcr-Abl mutant cell lines, clinical ex vivo patient samples and an in vivo mouse model., Results: The PBOX compounds potently reduce cell viability in cells expressing the E225K and H396P mutants as well as the highly resistant T315I mutant. The PBOX compounds also induce apoptosis in primary CML samples including those resistant to imatinib. We also show for the first time, the in vivo efficacy of the pro-apoptotic PBOX compound, PBOX-6, in a CML mouse model of the T315I Bcr-Abl mutant., Conclusion: Results from this study highlight the potential of these novel series of PBOX compounds as an effective therapy against CML.

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-(XL) was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.

Four new analogues of the gilvocarcin-type aryl-C-glycoside antitumor compounds, namely 4'-hydroxy gilvocarcin V (4'-OH-GV), 4'-hydroxy gilvocarcin M, 4'-hydroxy gilvocarcin E and 12-demethyl-defucogilvocarcin V, were produced through inactivation of the gilU gene. The 4'-OH-analogues showed improved activity against lung cancer cell lines as compared to their parent compounds without 4'-OH group (gilvocarcins V and E). The structures of the sugar-containing new mutant products indicate that the enzyme GilU acts as an unusual ketoreductase involved in the biosynthesis of the C-glycosidically linked deoxysugar moiety of the gilvocarcins. The structures of the new gilvocarcins indicate substrate flexibility of the post-polyketide synthase modifying enzymes, particularly the C-glycosyltransferase and the enzyme responsible for the sugar ring contraction. The results also shed light into biosynthetic sequence of events in the late steps of biosynthetic pathway of gilvocarcin V.

The Fast Antimicrobial Screen Test (FAST) is a simple and quick screening test developed to detect antibiotic and sulfonamide residues in food animal carcasses in slaughter establishments. This microbial inhibition test detects antimicrobials that are allowed to be used in food animals. It has the ability to detect these antimicrobials at or above the allowable limit in carcass kidney fluids in 6 h. Laboratory evaluations show that the lower limit of detection (LLD) of FAST and the Calf Antibiotic and Sulfa Test (CAST) for antibiotics tested was the same, but the LLD for sulfonamides of FAST was lower than the LLD of CAST. Compared with the Swab Test on Premises (STOP) developed in 1977, the LLD's of FAST for both antibiotics and sulfonamides were significantly better. Under field conditions, the sensitivity of FAST and CAST to antibiotic and sulfonamide residues in animal kidneys was not significantly different, but the time required by FAST was significantly lower than CAST (6 versus 18 h). Compared with the STOP, the sensitivity and the range of detection by FAST for all antimicrobials were significantly higher and the testing time was lower (18 versus 6 h).

Twenty-four Holstein cows, producing at least 21 kg of milk/d, were used in two replicate experiments to determine the effect of presence or absence of pulsation on loss of teat canal keratin during machine milking. Left quarters were milked without pulsation and right quarters were milked with pulsation. On d 0 and 10, keratin was collected from one left and from one right teat canal of each cow prior to milking and from the remaining two teat canals after milking. Milk was collected for assessment of SCC and bacteriological status on d 0 and approximately every 3 d until d 18. Quantity of keratin recovered before milking on d 10 did not differ between teats milked with or without pulsation, but loss of keratin because of milking was greater from teats milked with pulsation. By d 7, 30% (12 of 43) of quarters milked without pulsation had become infected, but no (0 of 47) quarters milked with pulsation were infected. By d 14 to 16, new infections had increased to 68% (28 of 41) of quarters milked without pulsation and 2% (1 of 43) in quarters milked with pulsation; mean SCC in pulsationless quarters increased sevenfold relative to pulsation quarters. Protein and water content of keratin did not differ because of treatment, and changes in lipid composition were minor. Histological analysis of the teats of 4 cows indicated that the mean diameter of the teat canal, within 2 h after milking, was greater without pulsation than with pulsation (680 vs. 483 microns).

Influence of teat canal keratin on susceptibility to intramammary infection was investigated in lactating Jersey cows. In each of two replicate trials, keratin was removed from the left teats of 20 cows immediately before milking. Immediately after milking, all teats were exposed to bacterial challenge by immersion in a suspension of Streptococcus agalactiae (5 x 10(7) cfu/ml). Bacterial challenge was repeated after the next four milkings. Foremilk samples were obtained for 8 d after keratin removal to determine infection status. A mammary quarter was classified as infected based solely upon the bacteriological criteria outlined by the National Mastitis Council. The rate of infection in quarters from which keratin was removed was greater than that in control quarters. Infection rates were 26.3% for keratin-removed quarters and 8.3% for control quarters in trial 1 and 13.5 and 0%, respectively, in trial 2. When more stringent criteria (recovery of greater than 100 cfu of S. agalactiae/ml in three or more successive milk samples and a SCC of greater than 10(6)) were used to identify a subset of infections that were clearly intramammary, infection rates were 9.3% for keratin-removed quarters and 1.4% for control quarters. Thus, partial removal of keratin from the teat canal compromised the ability of the teat to prevent passage of bacterial pathogens from the external environment into the mammary gland.

Two experiments were conducted to investigate lipid composition of teat canal keratin when different conditions of bacterial colonization and quarter inflammation were present. In Experiment 1, 11 multiparous cows with subclinical mastitis (bacteria present but no visible inflammation) in at least one quarter were selected for study. Quarters that were sampled and found negative for bacterial growth were classified as control. In Experiment 2, 10 multiparous cows with clinical mastitis in one or more quarters were selected. Milk samples from inflamed quarters were cultured to identify mastitis pathogens; these quarters were classified as clinical; all other quarters were classified as control. Teat canal keratin was collected from all quarters just before an a.m. milking, samples were weighed, and lipid determinations were conducted by TLC. Keratin from subclinical quarters compared with keratin from control quarters did not differ in either neutral lipid or fatty acid composition. Total lipid was significantly higher in keratin from teats of clinical quarters than in keratin from control quarters (27.8 vs. 21.5 microgram/mg). Neutral lipid composition of keratin was similar between teats from clinical quarters and teats from control quarters. In Experiment 2, quarter foremilk samples were also obtained to determine lipid composition. The FFA in milk from clinical quarters contained fewer short-chain fatty acids, whereas polyunsaturated fatty acids were significantly higher in milk from clinical quarters.

In three experiments, keratin was collected from individual teats of 40 Holstein and 20 Jersey cows immediately before and after milking. In Experiments 1 and 2, keratin collected from teats of 20 Holstein cows before milking was compared with keratin collected after milking. In Experiment 3, keratin was collected from two teats of 20 Jersey and 20 Holstein cows before milking and compared to the other two teats of the same cows after milking. All three experiments yielded similar results. In Holsteins, keratin weight before milking was 1.6 times greater than keratin weight after milking (3.1 vs. 1.9 mg). In Jerseys, only small amounts of keratin were removed during milking (3.5 mg before vs. 3.1 mg after) In Holsteins and Jerseys, neutral lipid concentration was 1.6 times greater after milking than before, suggesting that when keratin was removed during milking, only moderate amounts of lipid were removed. In Holsteins, total lipid collected per teat was similar before or after milking (59.2 vs. 48.5 micrograms). Results demonstrate that keratin collected after milking had a different lipid composition than keratin collected before milking.

This study was designed to determine the regeneration rate of teat canal keratin in two groups of 10 lactating Holstein cows. The weight of keratin obtained upon successive collections of keratin was determined. Intervals between successive collections were varied between 8 and 72 h. Following initial collection, the quantity of keratin removed was regenerated within 1 to 2.5 d. The rate of keratin regeneration per teat was approximately 1.5 mg of wet weight or .6 mg of dry weight per 24 h. The concentration (microgram lipid/mg keratin, wet weight) of lipid in keratin samples collected during regeneration was almost twice that present initially or after regeneration was complete. With the exception of free cholesterol, composition (%) of lipid components of keratin did not differ between samples collected initially, those collected early in the process of regeneration, and those collected after the mass of keratin removed had been replaced. Although data suggest that keratin matures during the process of regeneration, maturation appears complete by the time mass of keratin is fully regenerated. Additionally, the quantity of keratin recovered at initial sampling was inversely correlated with milk production. We hypothesize that as milk production increases, more keratin is lost during milking.

Methods for collecting keratin from the teat canal were examined to select a procedure to obtain representative samples for lipid analysis. Data obtained by solvent extraction of excised teats were compared with those obtained by scraping keratin from dissected teats of lactating and dry cows. Solvent extraction with petroleum ether or 2:1 chloroform-methanol yielded similar dry weights of material. However, both solvents removed large amounts of material other than keratin from the teat canal. The lipid class and fatty acid profiles of the material extracted by solvent flushing were not similar to profiles obtained by scraping keratin from the teat canal. A metal tapestry needle was suitable for collection of keratin from the teat canal of living cows. About 78% of the keratin present in the teat was collected with the needle. Lipid composition of keratin collected with the needle was the same as in keratin scraped from excised teats. The tapestry needle was suitable as a tool for collecting repeatable, representative samples of keratin for analysis from single teat canals of living cows.

Keratin was obtained by scraping the teat canals of excised teats from 12 lactating and 12 dry cows immediately after slaughter. Teats from four lactating and four dry cows were also stored at -20 degrees C for 2 wk to assess whether keratin composition was affected by frozen storage. Lipids were extracted from keratin of individual teats with 2:1 chloroform-methanol. Neutral lipid classes were determined by TLC and fatty acids by capillary GLC. Total lipid content of keratin was 4% of wet weight. Lipid composition of keratin from fresh and frozen teats was similar. Differences were observed in several lipid classes between keratin from lactating and dry cows. Triglycerides were higher in keratin from lactating cows, 58.4 vs. 28.1%, whereas cholesterol was lower in keratin from lactating cows, 18.5 vs. 37.5%. Short-chain and medium-chain fatty acids were 3x lower in keratin from dry than from lactating cows; 18:2 and polyunsaturated fatty acids were 2x higher in keratin from dry than from lactating cows. Results indicated that large differences exist between the detailed lipid composition of keratin from dry and lactating dairy cows.

Eighteen Holstein cows, free of intramammary infection, were fitted with smooth (n = 9) or abraded (n = 9) intramammary devices (IMD) in 2 diagonally opposed quarters within 4 weeks after calving. The 2 other quarters of each cow were used as controls. Three to 6 weeks after IMD insertion, depending on when milk somatic cell counts returned to a base-line value of less than 4 X 10(5)/ml, all cows were subjected to bacterial challenge exposure in the front or rear quarters by intracisternal injection of about 30 colony-forming units of Escherichia coli/quarter. Challenge exposure was done immediately after milking. Three weeks after the initial bacterial exposure, the other quarter pairs were similarly challenge exposed. Quarter bacteriologic status, concentration of milk somatic cells, and clinical observations (rectal temperature, milk appearance, udder palpation, and general condition of the cow) were monitored. Infection developed in 14 of 16 (88%) quarters with smooth IMD vs 16 of 16 (100%) control quarters and in 7 of 17 (41%) quarters with abraded IMD vs 17 of 17 (100%) control quarters. The difference in infection frequency between quarters with smooth IMD and quarters with abraded IMD was significant (P less than 0.05). Protection against establishment of infection was associated with somatic cell counts greater than 8.0 X 10(5)/ml in milk collected immediately after milking (7 of 12 quarters) or 4 hours later (11 of 12 quarters). In 10 quarters (59%) of cows fitted with abraded IMD, secretory abnormalities appeared before bacterial challenge inoculation. Abnormal milk or visible blood was observed over periods varying from 2 weeks after insertion through the entire lactation.