Post-translational modifications (PTMs) are key drivers in the regulation of protein activity. Therefore, the ability to measure and identify them accurately is critical to understanding the function and regulation of these modifications. Nε-carboxyethyllysine (CEL) and lactyllysine (LactylLys) are two modifications that share the same chemical composition and thus mass shift, making traditional LC-MS/MS approaches unsuitable for distinguishing them. Standard LC-MS/MS approaches utilizing HCD show that both modifications display extremely similar fragmentation, with no distinguishing features observed for either modification. Furthermore, cyclic and linear immonium ions, which have recently been reported as unique to LactylLys, were also observed in MS/MS from carboxyethylated peptides. We show that carboxyethylated and lactylated peptides can be chromatographically resolved on in-house packed and commercial C18 columns, and retention time alignment with isotopically labeled peptides can be used for discrimination. Furthermore, we observed differences in the MS/MS spectra obtained from EAD fragmentation of the two PTM-containing peptides. Our results highlight the analytical challenges associated with distinguishing CEL- and LactylLys-modified peptides and provide a proof of concept in which retention time alignment of endogenous peptides with isotopically labeled standards and electron activated dissociation (EAD) fragmentation is applied to accurately assign lactylation at K147 of aldolase A across various cell lines/tissues.

Methylglyoxal (MGO) is a highly reactive metabolite formed from glycolysis that can form advanced glycation endproducts (AGEs) on proteins and DNA. It has been well established that MGO induces DNA double strand breaks as a result of modifications on deoxyguanosine residues. However, recent studies shed new light on the genotoxic properties of MGO by its ability to cause chromosomal mis-segregation events, and other forms of chromosomal instability. These outcomes open a new avenue in which protein modifications, rather than DNA modifications, result in DNA damage. Herein, we present several hypotheses on how modification of proteins by MGO might cause these chromosome mis-segregation events based on identified protein modification sites from proteomic studies. These include various cell cycle proteins, such as those involved in sister chromatid cohesion, centrosome formation and histone proteins. Overall, recent studies implicate MGO in whole chromosome loss events, amongst other chromosomal instability events, suggesting it as a key player in cancer development and progression.

Micronuclei (MN) are a nuclear abnormality that occurs when chromosome fragments or whole chromosomes are not properly segregated during mitosis and consequently are excluded from the main nuclei and wrapped within nuclear membrane to form small nuclei. This maldistribution of genetic material leads to abnormal cellular genomes which may increase risk of developmental defects, cancers, and accelerated aging. Despite the potential importance of MN as biomarkers of genotoxicity, very little was known about the optimal way to measure MN in humans, the normal ranges of values of MN in healthy humans and the prospective association of MN with developmental and degenerative diseases prior to the 1980’s. In the early 1980’s two important methods to measure MN in humans were developed namely, the cytokinesis-block MN (CBMN) assay using peripheral blood lymphocytes and the Buccal MN assay that measures MN in epithelial cells from the oral mucosa. These discoveries greatly increased interest to use MN assays in human studies. In 1997 the Human Micronucleus (HUMN) project was founded to initiate an international collaboration to (i) harmonise and standardise the techniques used to perform the lymphocyte CBMN assay and the Buccal MN assay; (ii) establish and collate databases of MN frequency in human populations world-wide which also captured demographic, lifestyle and environmental genotoxin exposure data and (iii) use these data to identify the most important variables affecting MN frequency and to also determine whether MN predict disease risk. In this paper we briefly describe the achievements of the HUMN project during the period from the date of its foundation on 9th September 1997 until its 26th Anniversary in 2023, which included more than 200 publications and 23 workshops world-wide.

Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated with infertility, DNA damage and fragmentation. Oxidative stress has been shown to affect sperm DNA integrity and telomere length. Sirtuins such as SIRT1 and SIRT3 are involved in aging and oxidative stress response. The aim of the present study is to determine the role of SIRT1 and SIRT3 in regulating oxidative stress, telomere shortening, and their association with oligospermia. Therefore, we assessed the protein levels of SIRT1 and SIRT3, total antioxidant capacity (TAC), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase activity (CAT) in the seminal plasma of 272 patients with oligospermia and 251 fertile men. We also measured sperm telomere length (STL) and leukocyte telomere length (LTL) using a standard real-time quantitative PCR assay. Sperm chromatin and protamine deficiency were also measured as per standard methods. Our results for oligospermic patients demonstrate significant reductions in semen parameters, shorter STL and LTL, lower levels of SOD, TAC, CAT, SIRT1 and SIRT3 levels, and also significant protamine deficiency and higher levels of MDA and DNA fragmentation. We conclude that a shorter TL in sperms and leukocytes is associated with increased oxidative stress that also accounts for high levels of DNA fragmentation in sperms. Our results support the hypothesis that various sperm parameters in the state of oligospermia are associated with or caused by reduced levels of SIRT1 and SIRT3 proteins.

Magnesium is one of the most common elements in the human body and plays an important role as a cofactor of enzymes required for DNA replication and repair and many other biochemical mechanisms including sensing and regulating one-carbon metabolism deficiencies. Low intake of magnesium can increase the risk of many diseases, in particular, chronic degenerative disorders. However, its role in prevention of DNA damage has not been studied fully in humans so far. Therefore, we tested the hypothesis that magnesium deficiency either on its own or in conjunction with high homocysteine (Hcy) induces DNA damage in vivo in humans. The present study was carried out in 172 healthy middle aged subjects from South Australia. Blood levels of magnesium, Hcy, folate and vitamin B12 were measured. Cytokinesis-Block Micronucleus cytome assay was performed to measure three DNA damage biomarkers: micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) in peripheral blood lymphocytes. Data showed that magnesium and Hcy are significantly inversely correlated with each other (r = − 0.299, p < 0.0001). Furthermore, magnesium is positively correlated both with folate (p = 0.002) and vitamin B12 (p = 0.007). Magnesium is also significantly inversely correlated with MN (p < 0.0001) and NPB (p < 0.0001). Individuals with low magnesium and high Hcy exhibited significantly higher frequency of MN and NPBs compared to those with high magnesium and low Hcy (p < 0.0001). Furthermore, there was an interactive effect between these two factors as well in inducing MN (p = 0.01) and NPB (p = 0.048). The results obtained in the present study indicate for the first time that low in vivo levels of magnesium either on its own or in the presence of high Hcy increases DNA damage as evident by higher frequencies of MN and NPBs.

Emerging evidence suggests possible roles of micronutrients in cancer prevention. The study was designed to test the hypothesis that the concentration profile of plasma micronutrients (i.e., the nutriome) in prostate cancer patients is different from that of healthy controls. Plasma samples from 116 Caucasian men diagnosed with late onset of prostate cancer and 132 matched controls from the South Australian population were collected and analysed for their concentration of micronutrients. Plasma concentrations of lutein, lycopene, α-carotene and β-carotene were found to be significantly lower in prostate cancer patients (p = 0.03, 0.008, 0.002 and 0.002, respectively). Plasma levels of elements such as iron, copper, calcium and sulphur were significantly higher (p < 0.0001, <0.0001, <0.0001 and p = 0.0003, respectively) while that of selenium was significantly lower (p = 0.002) in prostate cancer patients. Higher prostate cancer risk is significantly associated with plasma levels below the median of lycopene (OR: 2.24), α-carotene (OR: 2.13), β-carotene (OR: 1.97) and high levels above the median of iron (OR: 2.31), calcium (OR: 4.35) and sulphur (OR: 2.39). The results of this study suggest that the plasma nutriome could be a useful diagnostic of prostate cancer risk.

Telomeres are repetitive nucleotide (TTAGGG) sequences that stabilize the chromosome ends and play an important role in the prevention of cancer initiation and progression. Nucleoplasmic bridges (NPBs) are formed when chromatids remain joined together during mitotic anaphase either due to mis-repair of DNA breaks or due to chromatid end fusion as a result of telomere loss or telomere dysfunction. We tested the hypotheses that (i) telomere length (TL) is shorter in prostate cancer (PC) patients relative to healthy age-matched individuals, (ii) TL differs in different stages of PC and (iii) shorter TL is significantly correlated with NPBs formation in PC cases. TL was measured in whole blood by well-established quantitative PCR method and the frequency of NPBs was measured in lymphocytes using cytokinesis-block micronucleus cytome (CBMNcyt) assay. Our results indicate that TL is shorter and NPBs are increased in PC patients relative to age-matched healthy controls. Furthermore, TL was significantly shorter (p = 0.03) in patients with a Gleason score more than 7 and there was also a significant trend of decreasing TL across all three stages (p trend = 0.01; Gleason score <7, 7 and >7). Furthermore, TL was significantly inversely correlated with NPB frequency in PC patients (r = -0.316; p = 0.001) but not in controls (r = 0.163; p = 0.06) and their relationships became stronger with higher Gleason scores. More studies are required that can confirm our observations and explore mechanistic differences in the role of telomeres in NPB formation in PC cases relative to non-cancer cases.

SummaryIn this study, sea cucumber (Holothuria scabra; Jaegar 1833) extracts and collagens were evaluated for inhibitory properties of protein‐bound advanced glycation end products (AGEs). Processed dried sea cucumber with salt extract showed a significant lower IC50 value for fluorescent AGEs (9.19 ± 7.68 μg mL−1, P < 0.05) and fructosamine (503.47 ± 46.37 μg mL−1, P < 0.05), respectively. Processed dried with and without salt extracts significantly reduced the Nε‐carboxymethyllysine (CML) and methylglyoxal‐derived hydroimidazolone‐1 (MG‐H1) levels tested at 250 μg mL−1. Smoked dried and fresh‐dried extracts significantly reduced the Nε‐carboxyethyllysine (CEL) levels tested at 250 μg mL−1. Pepsin‐solubilised collagen and the crude collagen fibrils significantly reduced CML and MG‐H1 levels whereas CEL levels were unchanged. Pearson's correlation analysis showed that protein‐bound AGE and CML inhibition significantly correlated with the total phenolic and antioxidant activities, respectively. This study provides a strong rationale for further investigation aimed at identifying the active compounds responsible for the observed effects on biomarkers relevant to diabetes.

Almost half of prostate cancer (PC) patients receive radiation therapy as primary curative treatment. In spite of advances in our understanding of both nutrition and the genomics of prostate cancer, studies on the effects of nutrients on the radiation sensitivity of PC patients are lacking. We tested the hypothesis that low plasma levels of selenium and lycopene have detrimental effects on ionising radiation-induced DNA damage in prostate cancer patients relative to healthy individuals. The present study was performed in 106 PC patients and 132 age-matched controls. We found that the radiation-induced micronucleus (MN) and nuclear buds (NBuds) frequencies were significantly higher in PC patients with low selenium (p = 0.008 and p = 0.0006 respectively) or low lycopene (p = 0.007 and p = 0.0006 respectively) levels compared to the controls. The frequency of NBuds was significantly higher (p < 0.0001) in PC patients who had low levels of both selenium and lycopene compared to (i) controls with low levels of both selenium and lycopene and (ii) PC patients with high levels of both selenium and lycopene (p = 0.0001). Our results support the hypothesis that low selenium and lycopene levels increase the sensitivity to radiation-induced DNA damage and suggest that nutrition-based treatment strategies are important to minimise the DNA-damaging effects in PC patients receiving radiotherapy.

Background: The soluble form of receptor for advanced glycation end products (sRAGE) have been implicated in the prevention of numerous pathologic states, and highlights as an attractive therapeutic target. Because diets rich in monounsaturated fatty acids (MUFA) reduce postprandial oxidative stress and inflammation that is related to better health during aging, we investigated the association between red blood cell (RBC) fatty acids with circulatory AGE biomarkers and further stratified this correlation based on GG and GA + AA genotype. Methods: A total of 172 healthy participants (median age = 53.74 ± 0.61 years) were recruited for the study. RBC fatty acid was analysed using gas chromatography and sRAGE was measured using a commercial ELISA kit. Results: The result showed a non-significant correlation between total MUFA with sRAGE however oleic acid (C18:1) exhibited a positive correlation (r = 0.178, p = 0.01) that remained statistically significant (β = 0.178, p = 0.02) after a stepwise multivariate regression analysis after adjusting for age, BMI and gender. In a univariate analysis, a positive significant correlation between C18:1 and sRAGE in GG genotype (r = 0.169, p = 0.02) and a non-significant correlation with GA + AA genotype (r = 0.192, p = 0.21) was evident. When C18:1 was stratified, a significant difference was observed for oleic acid and G82S polymorphism: low C18:1/GA + AA versus high C18:1/GG (p = 0.015) and high C18:1/GA + AA versus high C18:1/GG (p = 0.02). Conclusion: Our study suggests that increased levels of C18:1 may be a potential therapeutic approach in increasing sRAGE in those with GG genotype and play a role in modulating AGE metabolism.

High intake of red meat and/or dairy products may increase the concentration of iron and calcium in plasma—a risk factor for prostate cancer (PC). Despite our understandings of nutrients and their effects on the genome, studies on the effects of iron and calcium on radiation sensitivity of PC patients are lacking. Therefore, we tested the hypothesis that high plasma levels of iron and calcium could increase baseline or radiation-induced DNA damage in PC patients relative to healthy controls. The present study was performed on 106 PC patients and 132 age-matched healthy individuals. CBMN assay was performed to measure mi-cronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds) in lymphocytes. Plasma concentrations of iron and calcium were measured using inductively coupled plasma atomic emission spectroscopy. MN, NPBs, and NBuds induced by radiation ex vivo were significantly higher in PC patients with high plasma iron (P = .004, P = .047, and P = .0003, respectively) compared to healthy controls. Radiation-induced MN and NBuds frequency were also significantly higher in PC patients (P = .001 and P = .0001, respectively) with high plasma calcium levels relative to controls. Furthermore, radiation-induced frequency of NBuds was significantly higher in PC patients (P < .0001) with high plasma levels of both iron and calcium relative to controls. Our results support the hypothesis that high iron and calcium levels in plasma increases the sensitivity to radiation-induced DNA damage and point to the need of developing nutrition-based strategies to minimize DNA damage in normal tissue of PC patients undergoing radiotherapy.

Nutritional imbalances have been associated with a higher risk for cognitive impairment. This study determined the red blood cell (RBC) fatty acid profile of newly diagnosed mild cognitive impairment (MCI) and Alzheimer’s disease (AD) patients compared to age and gender-matched controls. There was a significant increase in palmitic acid (p < 0.00001) for both MCI and AD groups. Saturated fatty acids were significantly elevated in the MCI group, including stearic acid (p = 0.0001), arachidic acid (p = 0.003), behenic acid (p = 0.0002), tricosanoic acid (p = 0.007) and lignoceric acid (p = 0.001). n-6 polyunsaturated fatty acids (PUFAs) were significantly reduced in MCI, including linoleic acid (p = 0.001), γ-linolenic acid (p = 0.03), eicosatrienoic acid (p = 0.009) and arachidonic acid (p < 0.00004). The n-3 PUFAs, α-linolenic acid and docosahexaenoic acid, were both significantly reduced in MCI and AD (p = 0.0005 and p = 0.00003). A positive correlation was evident between the Mini-Mental State Examination score and nervonic acid in MCI (r = 0.54, p = 0.01) and a negative correlation with γ-linolenic acid in AD (r = −0.43, p = 0.05). Differences in fatty acid profiles may prove useful as potential biomarkers reflecting increased risk for dementia.

The relationship between sleep and micronutrients, including magnesium, is implicated in its regulation. The effects of low magnesium and other micronutrients on sleep disruption and telomere loss are not well understood. The present study was carried out in 172 healthy elderly subjects from South Australia. Plasma micronutrients including magnesium were measured. Each participant provided information about their sleep hours (<7 h or ≥7 h). Lymphocyte telomere length (TL) was measured by real-time qPCR assay. Plasma magnesium level was significantly low in subjects who sleep less than 7 h (p = 0.0002). TL was significantly shorter in people who are low in magnesium and sleep less than 7 h (p = 0.01). Plasma homocysteine (Hcy) is negatively associated with magnesium (r = −0.299; p < 0.0001). There is a significant interaction effect of magnesium and Hcy on sleep duration (p = 0.04) and TL (p = 0.003). Our results suggest that inadequate magnesium levels have an adverse impact on sleep and telomere attrition rate in cognitively normal elderly people, and this may be exacerbated by low levels of vitamin B12 and folate that elevate Hcy concentration.

The world is undergoing a sixth mass extinction, and the drastic decline of amphibians in the last several decades is a major contributing factor. The spread of the deadly Batrachochytrium dendrobatidis (Bd) fungus is a major causative agent, and captive breeding programs are under way to try and save endangered populations. However, how captivity affects Bd infection and the skin microbiota is not clear. We identified the skin bacteria of the South Australian frog Crinia signifera and showed that their culture-dependent skin microbiota is moderately stable in cohorts sampled from the same location in subsequent years. Their microbiota was also structurally different to that of a sympatric frog species, Limnodynastes tasmaniensis, indicating a specific microbial signature. Twenty-four C. signifera were placed into captivity for four weeks, exposed to a range of water salinity levels, during which time there was a significant reduction in identifiable skin bacterial species diversity, richness, and a change in microbial structure. The infection intensity of Bd was also significantly reduced in captivity, with some frogs becoming completely clear of infection, whilst water salinity level was not a significant determinant of Bd infection or skin microbiota.

Food resources in arid habitats are important for the survival of native fauna, especially where resources provide water or key nutrients during dry periods. However, food resource plants can be susceptible to grazing species or may not be suitable for revegetating arid areas. Pimelea microcephala subsp. microcephala (P.m.microcephala) is an Australian dioecious shrub bearing bright fruit that are likely to attract birds. The plant is also noted for its production of simplexin, a compound toxic to mammals. The aims of our study were 1) to assess the nutrient and simplexin content of P.m.microcephala fruits, and 2) to confirm the identity of native avian species that consume the fruits. With both pieces of information, we could then determine the conservation utility of P.m.microcephala. Combining chemical analysis of fruit nutrients with field observations, we found that ripe fruits contain 60.5% water, 2.8% sugar, and potent antioxidants, which would likely be of nutritional benefit to consuming frugivores. The fruits also contain high levels of the toxin simplexin, which comprised 3.6% of ripe fruits by weight. We identified eight bird species interacting with P.m.microcephala, with at least five of these consuming ripe fruits. Our study demonstrates the potential for P.m.microcephala to contribute to revegetation and provide a food resource for arid zone birds, whilst being protected from grazing by the presence of simplexin. Further studies are needed to establish the species’ absolute significance in terms of fruits as a source of water and nutrients to arid zone bird diets.

Poorer mental health is common in undergraduate students due to academic stress. An interplay between stress and diet exists, with stress influencing food choices. Nutritional interventions may be effective in preventing mental health decline due to complex bidirectional interactions between the brain, the gut and the gut microbiota. Previous studies have shown walnut consumption has a positive effect on mental health. Here, using a randomized clinical trial (Australian New Zealand Clinical Trials Registry, #ACTRN12619000972123), we aimed to investigate the effects of academic stress and daily walnut consumption in university students on mental health, biochemical markers of general health, and the gut microbiota. We found academic stress had a negative impact on self-reported mood and mental health status, while daily walnut consumption improved mental health indicators and protected against some of the negative effects of academic stress on metabolic and stress biomarkers. Academic stress was associated with lower gut microbial diversity in females, which was improved by walnut consumption. The effects of academic stress or walnut consumption in male participants could not be established due to small numbers of participants. Thus, walnut consumption may have a protective effect against some of the negative impacts of academic stress, however sex-dependent mechanisms require further study.

Significant alterations in sleep duration and/or quality of sleep become more pronounced as people get older. Poor sleep in elderly people is associated with adverse health outcomes and cellular aging. We examined the relationship between telomere length (TL) and sleep duration, Health Promotion Index (HPI), and tested whether the presence of Apolipoprotein-E4 (ApoE-ε4) allele affects both sleep and TL. The present study was carried out in 174 healthy participants (21% male; mean age 53.79 years) from South Australia. Lymphocyte TL was measured by real-time quantitative PCR (qPCR) and ApoE genotype was determined by TaqMan assay. HPI was calculated from a questionnaire regarding 8 lifestyle habits, including sleeping hours. Multivariate regression analysis was used to establish these associations adjusted for specified confounders. TL was found to be inversely associated with age (r = -0.199; p = .008) and body mass index (r = -0.121; p = .11), and was significantly shorter in participants who slept for less than 7 hours (p = .001) relative to those sleeping ≥7 hours. TL was positively correlated with HPI (r = 0.195; p = .009). ApoE-ε4 allele carriers who slept for less than 7 hours had shortest TL (p = .01) compared to noncarriers. Plasma soluble receptor for advanced glycation end product (sRAGE) level was significantly (p = .001) lower in individuals who sleep less than 7 hours and ApoE-ε4 carriers. Our results suggest that inadequate sleep duration or poor HPI is associated with shorter TL in cognitively normal people and that carriage of APOE-ε4 genotype may influence the extent of these effects.

There is much interest on the impact of nutrition on integrity of the genome and its stability because of the deleterious health consequences of DNA damage. One of the main components of the genome is telomeres which are TTAGGG repeats at the ends of chromosomes which are essential for maintenance of chromosome stability by preventing telomere end fusions. This important function of telomeres is impaired when either the telomere sequence length becomes critically shortened or if it is damaged by adducts caused by reactive metabolites. Several studies have explored the association of fatty acids with telomere length in humans but, so far, no clear picture has emerged regarding whether specific dietary fatty acids have a substantially significant impact on telomere integrity and function. Furthermore, knowledge on potential mechanisms is speculative due to a paucity of well-designed studies and great heterogeneity between studies. We provide some suggestions on improving observational and randomized controlled study design to make progress in this field.

The accurate segregation of sister chromatids is complex, and errors that arise throughout this process can drive chromosomal instability and tumorigenesis. We recently showed that methylglyoxal (MGO), a glycolytic by-product, can cause chromosome missegregation events in lymphocytes. However, the underlying mechanisms of this were not explored. Therefore, in this study, we utilised shotgun proteomics to identify MGO-modified proteins, and label-free quantitation to measure changes in protein abundance following exposure to MGO. We identified numerous mitotic proteins that were modified by MGO, including those involved in the separation and cohesion of sister chromatids. Furthermore, the protein abundance of Securin, an inhibitor of sister chromatid separation, was increased following treatment with MGO. Cytological examination of chromosome spreads showed MGO prevented sister chromatid separation, which was associated with the formation of complex nuclear anomalies. Therefore, results from this study suggest MGO may drive chromosomal instability by preventing sister chromatid separation.

Type 2 diabetes (T2D) is associated with elevated frequencies of micronuclei (MNi) and other DNA damage biomarkers. Interestingly, individuals with T2D are more likely to be deficient in micronutrients (folic acid, pyridoxal-phosphate, cobalamin) that play key roles in one-carbon metabolism and maintaining genomic integrity. Furthermore, it has recently been shown that deficiencies in these nutrients, in particular folic acid leaves cells susceptible to glucose-induced DNA damage. Therefore, we sought to investigate if the B lymphoblastoid WIL2-NS cell line cultured under folic acid-deficient conditions was more sensitive to DNA damage induced by glucose, or the reactive glycolytic byproduct methylglyoxal (MGO) and subsequent advanced glycation endproduct formation. Here, we show that only WIL2-NS cultured under folic acid-deficient conditions (23 nmol/l) experience an increase in MNi frequency when exposed to high concentrations of glucose (45 mmol/l) or MGO (100 µmol/l). Furthermore, we showed aminoguanidine, a well-validated MGO and free radical scavenger was able to prevent further MNi formation in folic acid-deficient cells exposed to high glucose, which may be due to a reduction in MGO-induced oxidative stress. Interestingly, we also observed an increase in MGO and other dicarbonyl stress biomarkers in folic acid-deficient cells, irrespective of glucose concentrations. Overall, our evidence shows that folic acid-deficient WIL2-NS cells are more susceptible to glucose and/or MGO-induced MNi formation. These results suggest that individuals with T2D experiencing hyperglycemia and folic acid deficiency may be at higher risk of chromosomal instability.