Mark Baker

Strategies to Improve the Potency of Oxazolidinones towards Bacterial Biofilms

Publisher: Wiley

Date: 13-04-2022

DOI: 10.1002/ASIA.202200201

Abstract: Biofilms are part of the natural lifecycle of bacteria and are known to cause chronic infections that are difficult to treat. Most antibiotics are developed and tested against bacteria in the planktonic state and are ineffective against bacterial biofilms. The oxazolidinones, including the last resort drug linezolid, are one of the main classes of synthetic antibiotics progressed to clinical use in the last 50 years. They have a unique mechanism of action and only develop low levels of resistance in the clinical setting. With the aim of providing insight into strategies to design more potent antibiotic compounds with activity against bacterial biofilms, we review the biofilm activity of clinically approved oxazolidinones and report on structural modifications to oxazolidinones and their delivery systems which lead to enhanced anti‐biofilm activity.

Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

Publisher: Cold Spring Harbor Laboratory

Date: 30-04-2020

DOI: 10.1101/2020.04.28.067298

Abstract: After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A 1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

Testicular heat stress, a historical perspective and two postulates for why male germ cells are heat sensitive

Publisher: Wiley

Date: 22-11-2022

DOI: 10.1111/BRV.12921

Abstract: Herein, we compare the different experimental regimes used to induce testicular heat stress and summarise their impact on sperm production and male fertility. Irrespective of the protocol used, scrotal heat stress causes loss of sperm production. This is first seen 1–2 weeks post heat stress, peaking 4–5 weeks thereafter. The higher the temperature, or the longer the duration of heat, the more pronounced germ cell loss becomes, within extreme cases this leads to azoospermia. The second, and often underappreciated impact of testicular hyperthermia is the production of poor‐quality spermatozoa. Typically, those cells that survive hyperthermia develop into morphologically abnormal and poorly motile spermatozoa. While both apoptotic and non‐apoptotic pathways are known to contribute to hyperthermic germ cell loss, the mechanisms leading to formation of poor‐quality sperm remain unclear. Mechanistically, it is unlikely that testicular hyperthermia affects messenger RNA (mRNA) abundance, as a comparison of four different mammalian studies shows no consistent single gene changes. Using available evidence, we propose two novel models to explain how testicular hyperthermia impairs sperm formation. Our first model suggests aberrant alternative splicing, while the second model proposes a loss of RNA repression. Importantly, neither model requires consistent changes in RNA species.

Conception and early pregnancy in the mare: lipidomics the unexplored frontier

Publisher: Bioscientifica

Date: 03-2022

DOI: 10.1530/RAF-21-0104

Abstract: Lipids are dynamic biological molecules that play key roles in metabolism, inflammation, cell signalling and structure. They are biologically significant in the physiology of conception and reproduction. Many of the mechanisms surrounding equine conception and the early feto-maternal dialogue are yet to be understood at a biochemical level. Recently, lipidomic technologies have advanced considerably and analytical strategies have been enhanced and ersified. Consequently, in-depth lipidomic exploration now has the potential to reveal new lipid biomarkers and biochemical relationships that improve our understanding of the processes leading to efficient and successful reproduction. This review considers the role of lipids in conception and establishment of pregnancy, providing new insights into the enigmatic pathways governing early reproductive physiology of the mare. This paper discusses the role that lipids play in the very early stages of pregnancy in the mare. Lipids are microscopic non-soluble molecules that are important components of living cells. The manuscript discusses how lipids influence the reproductive cycle of mares, including ovulation and the detailed biological process of becoming pregnant. It explains how lipids are identified in a laboratory setting with a newly developing technology known as ‘lipodomics’. The technology may lead to a more detailed understanding of how mares become pregnant. The focus of the paper is on mare reproduction, but it also draws on similarities with reproduction in other mammals. Remarkably there are gaps in much of our knowledge about the finer details of pregnancy in the horse, and the paper summarises what we already know about lipids, highlighting areas for further research.

p27kip1-dependent gene activity within granulosa cells reveals pathways relevant for control of primordial follicle activation in mice

Publisher: Cold Spring Harbor Laboratory

Date: 24-10-2022

DOI: 10.1101/2022.10.24.513438

Abstract: Female fertility is dictated by the number of oocyte-containing primordial follicles within the ovary. These follicles are established early in development and their selective activation represents the definitive first step towards oocyte maturation and ovulation. The intrinsic molecular mechanisms that regulate ovarian follicle activation are largely uncharacterised. In this study, we report a single-cell RNA sequencing (scRNAseq) dataset to examine mouse embryonic and neonatal ovaries across the developmental window of primordial follicle formation and activation. We identified five distinct clusters of granulosa cells and bioinformatic analysis revealed a population of pregranulosa cells that appeared to be undergoing follicle activation. This cluster was uniquely differentiated by the expression of Tnni3, Slc18a2, Fam13a and Klf2 , all of which are novel to follicle activation. Finally, we examined the transcriptome of a mouse model where all primordial follicles enter folliculogenesis simultaneously ( Cdkn1b -/- ) and showed that the signature genes of activating pregranulosa cells was observed precociously in Cdkn1b -/- ovaries, further demonstrating that p27 kip1 acts as an important repressor of follicle activation. Combined, this data provides insight into how primordial follicle activation is regulated in mammals and could be utilised to identify pathways to target to improve fertility preservation and infertility conditions in the future.

Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception

Publisher: Oxford University Press (OUP)

Date: 07-06-2021

DOI: 10.1093/HUMREP/DEAB134

Abstract: What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. N/A.

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

Publisher: Wiley

Date: 2020

DOI: 10.1002/PMIC.201900205

Abstract: The free-radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid-peroxidation product 4-hydroxynonenal (4-HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4-HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4-HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics-based findings propose a revised understanding of oxidative stress within the male reproductive tract.

Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1074/MCP.RA119.001626

Sialylation of Asparagine 612 Inhibits Aconitase Activity during Mouse Sperm Capacitation; a Possible Mechanism for the Switch from Oxidative Phosphorylation to Glycolysis

Publisher: Elsevier BV

Date: 11-2020

DOI: 10.1074/MCP.RA120.002109

17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, is an effective spermicide and microbicide against Neisseria gonorrhoeae

Publisher: Oxford University Press (OUP)

Date: 02-09-2022

DOI: 10.1093/HUMREP/DEAC188

Abstract: Is 17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, a better spermicide in cervicovaginal fluid (CVF) than its parental peptides, LL-37 and GF-17? At the same mass concentration, 17BIPHE2 exhibited the highest spermicidal activity on human sperm resuspended in CVF-containing medium. LL-37 and its truncated peptide GF-17 exert both spermicidal and microbicidal activities, although they are prone to proteolytic degradation in body fluids. Spermicidal activities of 17BIPHE2 were evaluated in vitro in mouse and human sperm, both resuspended in medium, and then on human sperm incubated in CVF-containing medium in the latter condition, the spermicidal activity and peptide stability in CVF of 17BIPHE2 were compared with that of LL-37 and GF-17. The in vivo contraceptive effects of 17BIPHE2 and the reversibility thereof were then assessed in mice. Finally, in vitro microbicidal effects of 17BIPHE2 on Neisseria gonorrhoeae were determined. Sperm motility and plasma membrane integrity were assessed by videomicroscopy and exclusion of Sytox Green, a membrane-impermeable fluorescent dye, respectively. Successful in vitro fertilization (IVF) was determined by the presence of two pronuclei in oocytes following their coincubation with capacitated untreated or 17BIPHE2-treated sperm. Sperm alone or with 17BIPHE2 were transcervically injected into female mice and successful in vivo fertilization was indicated by the formation of two-cell embryos 42-h postinjection, and by pregnancy through pup delivery 21–25 days afterwards. Peptide intactness was assessed by immunoblotting and HPLC. Reversibility of the contraceptive effects of 17BIPHE2 was evaluated by resumption of pregnancy of the female mice, pretranscervically injected with 17BIPHE2, following natural mating with fertile males. Minimum inhibitory/bactericidal concentrations of 17BIPHE2 on N. gonorrhoeae were obtained through microdilution broth assay. At the same mass concentration, 17BIPHE2 was a more effective spermicide than LL-37 or GF-17 on human sperm resuspended in CVF-containing medium, with the spermicidal concentration of 32.4 µM. This was mainly due to lower susceptibility of 17BIPHE2 to CVF proteases. Importantly, the reproductive tract of mouse females treated three times with 32.4 µM 17BIPHE2 remained normal and their fecundity resumed after stopping 17BIPHE2 treatment. For ethical reasons, the inhibitory effects of 17BIPHE2 on fertilization and pregnancy cannot presently be performed in women. Also, while our study has proven the effectiveness of 17BIPHE2 as a spermicide for mouse and human sperm in vitro, dosage formulation (e.g. in hydrogel) of 17BIPHE2 still needs to be developed to allow 17BIPHE2 to remain in the vagina/uterine cavity with controlled release for its spermicidal action. Since 17BIPHE2 also exerted bactericidal activity against N. gonorrhoeae at its spermicidal concentration, it is a promising candidate to be developed into a vaginal multipurpose prevention technology agent, thus empowering women against unplanned pregnancies and sexually transmitted infections. This work was supported by the Canadian Institutes of Health Research (PJT 173268 to N.T.). There are no competing interests to declare. N/A.

Monash University

Location: Australia

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The University Of Newcastle

Location: Australia

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Linkage Projects - Grant ID: LP160100824

Start Date: 01-2017

End Date: 01-2020

Amount: $560,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100984

Start Date: 07-2011

End Date: 12-2016

Amount: $735,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190102455

Start Date: 06-2019

End Date: 12-2023

Amount: $472,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100191

Start Date: 05-2011

End Date: 12-2011

Amount: $250,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100142

Start Date: 2010

End Date: 07-2011

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989105

Start Date: 2009

End Date: 11-2009

Amount: $495,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200103463

Start Date: 01-2020

End Date: 01-2023

Amount: $510,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100150

Start Date: 2010

End Date: 12-2012

Amount: $500,000.00

Funder: Australian Research Council