Biospectroscopy of Nanodiamond-Induced Alterations in Conformation of Intra- and Extracellular Proteins: A Nanoscale IR Study.

The toxicity of nanomaterials raises major concerns because of the impact that nanomaterials may have on health, which remains poorly understood. We need to explore the fate of individual nanoparticles in cells at nano and molecular levels to establish their safety. Conformational changes in secondary protein structures are one of the main indicators of impaired biological function, and hence, the ability to identify these changes at a nanoscale level offers unique insights into the nanotoxicity of materials. Here, we used nanoscale infrared spectroscopy and demonstrated for the first time that nanodiamond-induced alterations in both extra- and intracellular secondary protein structures lead to the formation of antiparallel ß-sheet, ß-turns, intermolecular ß-sheet, and aggregation of proteins. These conformational changes of the protein structure may result in the loss of functionality of proteins and in turn lead to adverse effects.

Lorentz contact resonance spectroscopy for nanoscale characterisation of structural and mechanical properties of biological, dental and pharmaceutical materials.

Scanning probe microscopy has been widely used to obtain topographical information and to quantify nanostructural properties of different materials. Qualitative and quantitative imaging is of particular interest to study material-material interactions and map surface properties on a nanoscale (i.e. stiffness and viscoelastic properties). These data are essential for the development of new biomedical materials. Currently, there are limited options to map viscoelastic properties of materials at nanoscale and at high resolutions. Lorentz contact resonance (LCR) is an emerging technique, which allows mapping viscoelasticity of samples with stiffness ranging from a few hundred Pa up to several GPa. Here we demonstrate the applicability of LCR to probe and map the viscoelasticity and stiffness of 'soft' (biological sample: cell treated with nanodiamond), 'medium hard' (pharmaceutical sample: pMDI canister) and 'hard' (human teeth enamel) specimens. The results allowed the identification of nanodiamond on the cells and the qualitative assessment of its distribution based on its nanomechanical properties. It also enabled mapping of the mechanical properties of the cell to demonstrate variability of these characteristics in a single cell. Qualitative imaging of an enamel sample demonstrated variations of stiffness across the specimen and precise identification of enamel prisms (higher stiffness) and enamel interrods (lower stiffness). Similarly, mapping of the pMDI canister wall showed that drug particles were adsorbed to the wall. These particles showed differences in stiffness at nanoscale, which suggested variations in surface composition-multiphasic material. LCR technique emerges as a valuable tool for probing viscoelasticity of samples of varying stiffness's.

Are mould hepatotoxins responsible for kava hepatotoxicity?

Previous studies with kava components such as kavalactones, pipermethystine and flavokavain B have demonstrated hepatotoxicity from these constituents. Regardless, there has recently been speculation that adulterants or impurities such as the mould hepatotoxin aflatoxin are a more likely cause of kava hepatotoxicity, despite a paucity of supporting evidence. Although there is limited similarity between acute kava hepatotoxicity and acute aflatoxicosis, and background levels of aflatoxin have been detected in kava samples, unless epidemiological investigations can uncover direct evidence implicating mould hepatotoxins, it remains more likely that chemical constituents of kava are the cause of the hepatotoxicity from kava.

Kavalactone metabolism in the isolated perfused rat liver.

Metabolic pathways for kavalactone metabolism in humans and rats have been identified, but more detailed description of the enzyme kinetics involved is lacking. The disposition profiles of three of the six major kavalactones (kavain, methysticin and desmethoxyyangonin) and their respective metabolites (p-hydroxykavain, m,p-dihydroxykavain and p-hydroxy-5,6-dehydrokavain) were examined in the perfusate and bile of the isolated perfused rat liver. The metabolism of the kavalactones is first-order in nature with similar decay half-lives. p-Hydroxykavain and m,p-dihydroxykavain were the only metabolites detected in the perfusate. Kavalactone biliary excretion was negligible.

Kavalactone metabolism in rat liver microsomes.

The specific CYP enzymes involved in kavalactone (KLT) metabolism and their kinetics have not been fully examined. This study used rat liver microsomes (RLM) to determine kavain (KA), methysticin (MTS) and desmethoxyyangonin (DMY) enzyme kinetic parameters, to elucidate the major CYP450 isoforms involved in KLT metabolism and to examine gender differences in KLT metabolism. Formation of the major KLT metabolites was first-order, consistent with classic enzyme kinetics. In both male and female RLM, clotrimazole (CLO) was the most potent inhibitor of KA and MTS metabolism. This suggests CYP3A1/3A23 (females) and CYP3A2 (males) are the main isoenzymes involved in the metabolism of these KLTs in rats, while the roles of CYP1A2, -2 C6, -2 C9, -2E1 and -3A4 are limited. Desmethoxyyangonin metabolism was equally inhibited by cimetidine (CIM) and CLO in females, and CIM and nortriptyline in males. This implies that DMY metabolism involves CYP2C6 and CYP2C11 in males, and CPY2C12 in females. CYP3A1/3A23 may also be involved in females.

Vaccine Hesitancy: Contemporary Issues and Historical Background.

Vaccination, despite being recognized as one of the most effective primary public health measures, is viewed as unsafe and unnecessary by an increasing number of individuals. Anxiety about vaccines and vaccination programs leading to vaccine hesitancy results from a complex mix of social and political influences, cultural and religious beliefs, the availability of and ability to interpret health and scientific information, and personal and population experiences of health systems and government policies. Vaccine hesitancy is becoming a serious threat to vaccination programs, and was identified as one of the World Health Organization's top ten global health threats in 2019. The negative impact of anti-vaccination movements is frequently cited as one of the major reasons for rising vaccine hesitancy amongst the general public world-wide. This review discusses the various issues surrounding vaccine hesitancy and the anti-vaccine movement, starting with the definitions of vaccine hesitancy and the anti-vaccine movement in their early history and in the modern era, before discussing the key drivers of vaccine hesitancy, particularly across different regions of the world, with a focus on various countries with low-, middle-, or high-income economies with different socio-economic populations. The review concludes with the impact of vaccine hesitancy on herd immunity and social, psychological, and public health measures to counter vaccine hesitancy.

Assessment and histological analysis of the IPRL technique for sequential in situ liver biopsy.

BACKGROUND: The isolated perfused rat liver (IPRL) is a technique used in a wide range of liver studies. Typically livers are assessed at treatment end point. Techniques have been described to biopsy liver in the live rat and post-hepatectomy. RESULTS: This paper describes a technique for obtaining two full and one partial lobe biopsies from the liver in situ during an IPRL experiment. Our approach of retaining the liver in situ assists in minimising liver capsule damage, and consequent leakage of perfusate, maintains the normal anatomical position of the liver during perfusion and helps to keep the liver warm and moist. Histological results from sequential lobe biopsies in control perfusions show that cytoplasmic vacuolation of hepatocytes is a sign of liver deterioration, and when it occurs it commences as a diffuse pattern which tends to develop a circumscribed, centrilobular pattern as perfusion progresses. CONCLUSIONS: Liver lobe biopsies obtained using this method can be used to study temporal effects of drug treatments and are suitable for light and electron microscopy, and biochemical analyses.

Native Australian fruit polyphenols inhibit cell viability and induce apoptosis in human cancer cell lines.

Apoptosis is one of the most critical forms of defense against cancer, and the induction of apoptosis by dietary polyphenols represents significant potential for cancer preventive activity. The present study examined polyphenols extracted from selected native Australian fruits--Illawarra plum (Podocarpus elatus Endl., Podocarpaceae), Kakadu plum (Terminalia ferdinandiana Exell, Combretaceae), muntries (Kunzea pomifera F. Muell., Myrtaceae), and native currant (Acrotriche depressa R.Br., Epacridaceae)--for antiproliferative activity against a panel of cancer and normal cell lines. Each fruit selectively inhibited the growth of cancer cell lines in a dose-dependent manner. The mechanism of growth inhibition of the human promyelocytic leukaemia cells (HL-60) was determined to be apoptosis by morphological assessment, DNA fragmentation, flow cytometry, and caspase-3 induction. Furthermore, Kakadu plum was found to activate caspase-7, -9, and poly (ADP-ribose) polymerase (PARP), suggesting it acts via the intrinsic apoptosis pathway. The same fruit also caused direct DNA damage in colon adenocarcinoma cells (HT-29) as detected using the cytokinesis-block micronucleus cytome (CBMN Cyt) assay.

Molecular pathways for cancer chemoprevention by dietary phytochemicals.

Interest in dietary phytochemicals for potential cancer chemoprevention has increased substantially. Screening dietary compounds for chemopreventive activity however, requires a systematic and wide-ranging approach to encompass the complexity of carcinogenesis. We present some of the molecular pathways that underpin the broad biological processes involved in carcinogenesis. Oxidative stress, inflammation, and the evasion of apoptosis are important biological mechanisms by which carcinogenesis occurs. Subsequently, antioxidant, anti-inflammatory, and pro-apoptotic activity represent important activities for preventing, suppressing, or reversing the development of carcinogenesis. Ultimately, these mechanisms of action may provide a useful basis for screening novel phytochemicals for chemopreventive activity. In this review, we identify the important molecular processes that may be targeted in routine screenings of dietary phytochemicals to ultimately select the most effective potential candidates for cancer chemoprevention.

Potential antioxidant, antiinflammatory, and proapoptotic anticancer activities of Kakadu plum and Illawarra plum polyphenolic fractions.

Kakadu plum (Terminalia ferdinandiana Exell, Combretaceae) and Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) extracts were fractionated, using a bioassay-guided approach and screened for antioxidant activity [oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays] and antiinflammatory activity (nitrite concentration and prostaglandin E(2) release in lipopolysaccharide (LPS)-activated murine macrophages). Among 8 fractions obtained from KP and 5 fractions obtained from IP, fraction KPF5 from KP exhibited superior activity in all assays, with an ORAC value of 3,776 ± 603 µmol Trolox/g DW and a CAA value of 52.2 ± 8.6 µmol quercetin equivalents/g DW. In addition, KPF5 further demonstrated an upregulation of the Nrf2/Keap1 ratio in Hep G2 cells. KPF5 also inhibited the expression of COX-2 and iNOS in LPS-activated murine macrophages, potentially through the NF-κB, p44/42 mitogen activated protein kinase and Akt pathways. KPF5 also induced apoptosis and DNA damage in HT-29 cells, as determined by the cytokinesis block micronucleus cytome assay.

Does inflammation play a role in kava hepatotoxicity?

The pathophysiology of kava hepatotoxicity remains inconclusive. There is circumstantial evidence for the roles of toxic metabolites, inhibition of cyclooxygenase (COX) enzymes and depletion of liver glutathione. Pharmacogenomic effects are likely, particularly for Cytochrome P450 genes. Experimental and clinical cases of hepatotoxicity show evidence of hepatitis. The question remains whether this inflammation is caused by components of kava directly, or indirectly due to the downstream effects.

Prescription of compounded ophthalmic medications-a pharmacy perspective.

Compounded ophthalmic products can be helpful for a small number of patients who cannot use a commercially available product or for whom no commercially available product exists. Extemporaneous preparation of medicines for the eye is considered complex compounding and is best undertaken by pharmacists with the appropriate facilities and equipment to ensure a sterile product. When dispensing, the pharmacist evaluates medication appropriateness, effectiveness and safety for each individual patient. For a compounded medicine, the pharmacist will also pay close attention to the formulation, physical, chemical and microbiological stability and utility of the preparation. Optometrists who are considering writing a prescription for any medicine will find that the pharmacist is a useful source of information regarding the availability of alternatives, stock outages and the availability or feasibility of a compounded product. Compounding can be a useful adjunct to the care of some patients and patient safety should always be paramount.

Vaccines and vaccination: history and emerging issues.

Prophylactic vaccines are crucial in modern healthcare and have been used successfully to combat bacterial and viral infectious diseases. Infections like polio and smallpox, which were dreaded historically, and which devastated the human race over many centuries, are now rare. Smallpox has been eradicated completely and polio is nearly eradicated because of vaccines. Vaccines differ fundamentally from other classes of medicines in that they are usually administered as a preventive measure to a healthy individual rather than to a sick person already with an infection, although exceptions to this practice exist. Most currently used prophylactic vaccines are based on established platforms, but many vaccine candidates, in late development stages, including several COVID-19 vaccines, use highly novel vaccine platforms not available historically. History of infectious diseases and prophylactic vaccines are filled with important scientific lessons, and thus provide valuable insights for the future. With hindsight, historically there were some ethically questionable approaches to testing vaccines and the germ warfare against native populations in the Americas and other regions. In this review, we examine key historical lessons learned with prophylactic vaccines with reflections on current healthcare dilemmas and controversies with respect to influenza and COVID-19 vaccines.

A Narrative Review of COVID-19 Vaccines.

The COVID-19 pandemic has shaken the world since early 2020 and its health, social, economic, and societal negative impacts at the global scale have been catastrophic. Since the early days of the pandemic, development of safe and effective vaccines was judged to be the best possible tool to minimize the effects of this pandemic. Drastic public health measures were put into place to stop the spread of the virus, with the hope that vaccines would be available soon. Thanks to the extraordinary commitments of many organizations and individuals from around the globe and the collaborative effort of many international scientists, vaccines against COVID-19 received regulatory approval for emergency human use in many jurisdictions in less than a year after the identification of the viral sequence. Several of these vaccines have been in use for some time; however, the pandemic is still ongoing and likely to persist for the foreseeable future. This is due to many reasons including reduced compliance with public health restrictions, limited vaccine manufacturing/distribution capacity, high rates of vaccine hesitancy, and the emergence of new variants with the capacity to spread more easily and to evade current vaccines. Here we discuss the discovery and availability of COVID-19 vaccines and evolving issues around mass vaccination programs.

The protein corona determines the cytotoxicity of nanodiamonds: implications of corona formation and its remodelling on nanodiamond applications in biomedical imaging and drug delivery.

The use of nanodiamonds for biomedical and consumer applications is growing rapidly. As their use becomes more widespread, so too do concerns around their cytotoxicity. The cytotoxicity of nanodiamonds correlates with their cellular internalisation and circulation time in the body. Both internalisation and circulation time are influenced by the formation of a protein corona on the nanodiamond surface. However, a precise understanding of both how the corona forms and evolves and its influence on cytotoxicity is lacking. Here, we investigated protein corona formation and evolution in response to two classes of nanodiamonds, pristine and aminated, and two types of proteins, bovine serum albumin and fibronectin. Specifically, we found that a corona made of bovine serum albumin (BSA), which represents the most abundant protein in blood plasma, reduced nanodiamond agglomeration. Fibronectin (FN9-10), the second most abundant protein found in the plasma, exhibited a significantly higher nanodiamond binding affinity than BSA, irrespective of the nanodiamond surface charge. Finally, nanodiamonds with a BSA corona displayed less cytotoxicity towards nonphagocytic liver cells. However, regardless of the type of corona (FN9-10 or BSA), both classes of nanodiamonds induced substantial phagocytic cell death. Our results emphasise that a precise understanding of the corona composition is fundamental to determining the fate of nanoparticles in the body.

In vitro and in vivo evaluation of the inhibition potential of risperidone toward clozapine biotransformation.

AIMS: To study the impact of risperidone (RISP) on clozapine (CLZ) biotransformation in vitro in microsomal fractions containing varying expression of CYP oxidases and in vivo in patients. METHODS: Human liver microsomes (n= 11) were assessed for expression of CYPs 1A2, 2D6 and 3A4, because these enzymes mediate RISP and CLZ oxidation. Inhibition of CLZ oxidation by RISP was assessed. Plasma CLZ elimination was estimated in patients with schizophrenia who received either CLZ alone or the CLZ-RISP combination (n= 10 per group). RESULTS: (i) The CYP3A4 and CYP1A2 inhibitors ketoconazole and fluvoxamine inhibited CLZ oxidation to varying extents in individual microsomal fractions. (ii) RISP did not inhibit CLZ oxidation, regardless of variations in CYP expression. (iii) RISP co-administration did not impair CLZ clearance. CONCLUSIONS: No evidence was found for CYP-mediated inhibitory or pharmacokinetic interactions between RISP and CLZ. Occasional literature reports of such interactions may involve other pathways that participate in CLZ disposition.

A high-throughput assay using liquid chromatography-tandem mass spectrometry for simultaneous in vivo phenotyping of 5 major cytochrome p450 enzymes in patients.

The phenotyping cocktail is a practical approach for phenotyping of cytochrome P450 (CYP) enzymes in vivo. In this study, a liquid chromatography-tandem mass spectrometry method using a dual-extraction approach was developed and validated to quantify 5 selective substrates and their metabolites for the simultaneous phenotyping CYPs 1A2, 2C19, 2C9, 2D6, and 3A4 in patient blood samples. The assay was applied in a pilot study of 11 patients with schizophrenia. Five blood samples were collected before and at 1, 2, 4, and 6 hours after administration of a phenotyping cocktail consisting of 100 mg caffeine, 20 mg omeprazole, 25 mg losartan, 30 mg dextromethorphan, and 2 mg midazolam. The method successfully quantitated the CYP enzyme activities without serious side effects in patients. The ratios of metabolite to parent area under the concentration-time curve values were calculated over the 6-hour postdosage to reflect CYP2D6, CYP3A4, and CYP2C9 activities. The ratios of metabolite to parent plasma concentrations were calculated at 4-hour postdosage for CYP1A2 and at 4- or 6-hour postdose for CYP2C19, respectively. The plasma concentration of midazolam at 4 hours was also estimated as another phenotyping index for CYP3A4 activity. The simultaneous assay of all these analytes in a single matrix (plasma) will increase the feasibility of CYP phenotyping in patients.

High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites.

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.

Compounded medicines and 'off label' prescribing - a case for more guidance.

BACKGROUND: Interest by prescribers and pharmacists in the provision of individualised pharmaceutical therapy in the form of compounded medicines has grown in recent times. However, there have also been a number of case reports of patient harm associated with these medicines. OBJECTIVE: To highlight areas for clinicians and pharmacists to consider when prescribing or dispensing compounded medicines, which are consistent with quality use of medicines principles. DISCUSSION: Regulators of pharmaceutical products have expressed concerns with the production, marketing and use of compounded medicines dispensed by pharmacists. This has prompted debate over the need for more regulation of these products. We propose an expansion of off label prescribing guidelines to include a risk based assessment of pharmaceutical quality, a consumer information/education strategy and the development of a code of practice for pharmacists engaging in compounding. These strategies recognise a shared responsibility among prescribers, dispensers and regulators to achieve contemporary quality, safety, and efficacy standards and support the quality use of compounded medicines.

Biological impact of nanodiamond particles - label free, high-resolution methods for nanotoxicity assessment.

Current methods for the assessment of nanoparticle safety that are based on 2D cell culture models and fluorescence-based assays show limited sensitivity and they lack biomimicry. Consequently, the health risks associated with the use of many nanoparticles have not yet been established. There is a need to develop in vitro models that mimic physiology more accurately and enable high throughput assessment. There is also a need to set up new assays that offer high sensitivity and are label-free. Here we developed 'mini-liver' models using scaffold-free bioprinting and used these models together with label-free nanoscale techniques for the assessment of toxicity of nanodiamond produced by laser-assisted technology. Results showed that NDs induced cytotoxicity in a concentration and exposure-time dependent manner. The loss of cell function was confirmed by increased cell stiffness, decreased cell membrane barrier integrity and reduced cells mobility. We further showed that NDs elevated the production of reactive oxygen species and reduced cell viability. Our approach that combined mini-liver models with label-free high-resolution techniques showed improved sensitivity in toxicity assessment. Notably, this approach allowed for label-free semi-high throughput measurements of nanoparticle-cell interactions, thus could be considered as a complementary approach to currently used methods.