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DNA repair has long been considered impossible in human spermatozoa due to the high level of DNA compaction observed in these cells. However, detailed examination of the base excision repair pathway in human spermatozoa has revealed the presence of an enzyme critical to this pathway, 8-oxoguanine DNA glycosylase 1 (OGG1). This glycosylase was associated with the sperm nucleus and mitochondria and could actively excise 8-hydroxy-29-deoxyguanosine (8OHdG), releasing this adduct into the extracellular space. This activity was significantly reduced in the presence of cadmium (II), a recognized inhibitor of OGG1, in a time- and dose-dependent manner (P,0.001). Remarkably, spermatozoa do not possess the downstream components of the base excision repair pathway, apurinic endonuclease 1 (APE1) and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1). The absence of these proteins was particularly significant, as APE1 is required to create a 39-hydroxyl (39-OH) terminus at the apurinic site created by OGG1, which would be recognized by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. As a result, TUNEL was unable to detect oxidatively induced DNA damage in spermatozoa following exposure to hydrogen peroxide. In the same cells, intracellular and extracellular 8OHdG could be clearly detected in a manner that was highly correlated with the outcome of the sperm chromatin structure assay (SCSA). However, incubation of these cells for 48 hours revealed a time-dependent increase in TUNEL positivity, suggesting the perimortem activation of a nuclease. These results emphasize the limited capacity of mature spermatozoa to mount a DNA repair response to oxidative stress, and highlight the importance of such mechanisms in the oocyte in order to protect the embryo from paternally mediated genetic damage. [ABSTRACT FROM AUTHOR]

BACKGROUND Spermatogenesis culminates in production of one of the most highly differentiated cells in biology, the spermatozoon. The gametes that emerge from the testes are, however, functionally immature and only acquire full functionality once they have completed a process of post-testicular maturation in the epididymis and female reproductive tract. Remarkably, this acquisition of sperm function occurs while these cells are transcriptionally and translationally silent and is therefore highly dependent on post-translational modifications to their existing protein complement. In this review, we consider the emerging roles of several prominent molecular chaperone families in orchestrating both the morphological differentiation of male germ cells during spermatogenesis and their functional transformation during sperm maturation. METHODS Journal databases were searched using key words, including chaperone, heat shock protein, testes, spermatogenesis, spermatozoa, epididymal maturation, capacitation and fertilization. RESULTS In the past two decades, molecular chaperones have been acknowledged to play key roles in controlling both the morphological transformation of germ cells during spermatogenesis and the post-testicular maturation of these cells as they transit the male and female reproductive tracts. Furthermore, there is mounting evidence that aberrant chaperone expression may be a major contributing factor to the defective sperm function seen in many cases of male infertility. CONCLUSIONS Molecular chaperones are critically involved in all phases of sperm development. Targeted disruption of these proteins has the ability to arrest spermatogenesis, compromise sperm maturation and inhibit fertilization. These proteins therefore hold considerable promise as targets for novel contraceptive strategies and as diagnostic biomarkers for male infertility. [ABSTRACT FROM PUBLISHER]

Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize. [ABSTRACT FROM AUTHOR]

Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following subchronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8 h at 35 °C followed by 16 h at 25 °C) for a period of 7 d. Neither the testes or epididymides of heat-exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat-exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching, and increased fetal:placental weight ratio. Such changes were causally associated with an altered sperm small noncoding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat-exposed spermatozoa. Such data highlight that even relatively modest excursions in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress. [ABSTRACT FROM AUTHOR]

Background: The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. Objective: The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. Materials and methods: The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub‐chronic (10‐day) corticosterone challenge. Results: The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone‐treated cells. Conclusions: Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases. [ABSTRACT FROM AUTHOR]

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma - DIPG), is the primary cause of brain tumor-related death in pediatric patients. DIPG is characterized by a median survival of <12 months from diagnosis, harboring the worst 5-year survival rate of any cancer. Corticosteroids and radiation are the mainstay of therapy; however, they only provide transient relief from the devastating neurological symptoms. Numerous therapies have been investigated for DIPG, but the majority have been unsuccessful in demonstrating a survival benefit beyond radiation alone. Although many barriers hinder brain drug delivery in DIPG, one of the most significant challenges is the blood-brain barrier (BBB). Therapeutic compounds must possess specific properties to enable efficient passage across the BBB. In brain cancer, the BBB is referred to as the blood-brain tumor barrier (BBTB), where tumors disrupt the structure and function of the BBB, which may provide opportunities for drug delivery. However, the biological characteristics of the brainstem's BBB/BBTB, both under normal physiological conditions and in response to DIPG, are poorly understood, which further complicates treatment. Better characterization of the changes that occur in the BBB/BBTB of DIPG patients is essential, as this informs future treatment strategies. Many novel drug delivery technologies have been investigated to bypass or disrupt the BBB/BBTB, including convection enhanced delivery, focused ultrasound, nanoparticle-mediated delivery, and intranasal delivery, all of which are yet to be clinically established for the treatment of DIPG. Herein, we review what is known about the BBB/BBTB and discuss the current status, limitations, and advances of conventional and novel treatments to improving brain drug delivery in DIPG. [Display omitted] [ABSTRACT FROM AUTHOR]

Aims: Oxidative stress is causally linked to male reproductive pathologies, driven primarily by lipid peroxidation and an attendant production of highly reactive lipid aldehydes, such as 4-hydroxynonenal (4HNE) within the male germ line. In somatic cells, 4HNE dysregulates proteostasis via targeting of vulnerable proteins for adduction, causing protein misfolding and eventually aggregation. The aims of this study were to explore whether oxidative stress precipitates an equivalent response in the male germ line and determine the protective mechanisms used by germ cells to prevent this cascade of protein damage. Results: We reveal a causative role for oxidative stress in the accumulation of protein deposits in male germ cells. Specifically, 4HNE treatment resulted in a significant increase in cytosolic protein aggregation within pre- and post-meiotic germ cells as measured by the aggregate-detecting fluorophores ProteoStat and Thioflavin T, and the amyloid-specific anti-A11 and anti-OC antibodies. Our data implicate nucleocytoplasmic transport machinery and molecular chaperones as potential mechanisms for the subcellular compartmentalization and/or suppression of aggregating proteins. Thus, the inhibition of karyopherin transport proteins and molecular chaperones resulted in a significant increase in the accumulation of aggregated cellular protein. Innovation: These data establish the novel paradigm that lipid peroxidation is a key contributor to a decline in proteostasis in developing germ cells. These findings will inform the development of novel strategies to protect germ cells from oxidative stress. Conclusion: Together, these results shed light on proteostasis mechanisms that may assist in the management of misfolded proteins in the male germ line under conditions of acute oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukaemia (AML) is a deadly disease characterised by the uncontrolled proliferation of immature myeloid cells within the bone marrow. Altered regulation of DNA methylation is an important epigenetic driver of AML, where the hypoxic bone marrow microenvironment can help facilitate leukaemogenesis. Thus, interactions between epigenetic regulation and hypoxia signalling will have important implications for AML development and treatment. Main body: This review summarises the importance of DNA methylation and the hypoxic bone marrow microenvironment in the development, progression, and treatment of AML. Here, we focus on the role hypoxia plays on signalling and the subsequent regulation of DNA methylation. Hypoxia is likely to influence DNA methylation through altered metabolic pathways, transcriptional control of epigenetic regulators, and direct effects on the enzymatic activity of epigenetic modifiers. DNA methylation may also prevent activation of hypoxia-responsive genes, demonstrating bidirectional crosstalk between epigenetic regulation and the hypoxic microenvironment. Finally, we consider the clinical implications of these interactions, suggesting that reduced cell cycling within the hypoxic bone marrow may decrease the efficacy of hypomethylating agents. Conclusion: Hypoxia is likely to influence AML progression through complex interactions with DNA methylation, where the therapeutic efficacy of hypomethylating agents may be limited within the hypoxic bone marrow. To achieve optimal outcomes for AML patients, future studies should therefore consider co-treatments that can promote cycling of AML cells within the bone marrow or encourage their dissociation from the bone marrow. [ABSTRACT FROM AUTHOR]

Fewer than 10 % of children with diffuse midline glioma (DMG) survive 2 years from diagnosis. Radiation therapy remains the cornerstone of treatment and there are no medicinal products with regulatory approval. Although the biology of DMG is better characterized, this has not yet translated into effective treatments. H3K27 -alterations initiate the disease but additional drivers are required for malignant growth. Hence, there is an urgent unmet need to develop new multi-modality therapeutic strategies, including alternative methods of drug delivery. ONC201 (DRD2 antagonist and mitochondrial ClpP agonist) is the most widely evaluated investigational drug. Encouraging early data is emerging for CAR T-cells and oncolytic viruses. GD2, B7-H3 and PI3K signalling are ubiquitous targets across all subtypes and therapeutics directed to these targets would potentially benefit the largest number of children. PI3K, ACVR1, MAPK and PDGFRA pathways should be targeted in rational biological combinations. Drug discovery is a very high priority. New specific and potent epigenetic modifiers (PROTACS e.g. SMARCA4 degraders), with blood-brain penetrance are needed. Cancer neuroscience therapeutics are in early development. Overall survival is the preferred regulatory endpoint. However, the evaluation of this can be influenced by the use of re-irradiation at the time of progression. An efficient clinical trial design fit for regulatory purposes for the evaluation of new therapeutics would aid industry and facilitate more efficient therapy development. Challenges in conducting clinical trials such as the need for comparator data and defining endpoints, could be addressed through an international, first-in-child, randomised, complex innovative design trial. To achieve progress: i) drug discovery; ii) new multi-modality, efficient, collaborative, pre-clinical approaches, possibly including artificial intelligence and, iii) efficient clinical trial designs fit for regulatory purposes are required. • Less than 10 % of children with DMG survive beyond 2 years from diagnosis • GD2, B7-H3 and PI3K/mTOR signalling are ubiquitous targets for all subtypes of DMG • Encouraging early data is emerging for CAR T-cells and oncolytic viruses • Drug discovery is a high priority for epigenetic modifiers e.g. SMARCA4 degraders • An international, randomised, platform, complex innovative design trial is a goal [ABSTRACT FROM AUTHOR]

Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography–tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, phospho-heavy-labeled-spiketide FAIMS Stepped-CV DDA (pHASED), that employs online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients. [ABSTRACT FROM AUTHOR]

Abstract: The recognition and binding of a free-swimming spermatozoon to an ovulated oocyte is one of the most important cellular interactions in biology. While traditionally viewed as a simple lock and key mechanism, emerging evidence suggests that this event may require the concerted action of several sperm proteins. In this study we examine the hypothesis that the activity of such proteins may be coordinated by their assembly into multimeric recognition complexes on the sperm surface. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we tender the first direct evidence that human spermatozoa do indeed express a number of high molecular weight protein complexes on their surface. Furthermore, we demonstrate that a subset of these complexes displays affinity for homologous zonae pellucidae. Proteomic analysis of two such complexes using electrospray ionization mass spectrometry identified several of the components of the multimeric 20S proteasome and chaperonin-containing TCP-1 (CCT) complexes. The latter complex was also shown to harbor at least one putative zona pellucida binding protein, ZPBP2. Consistent with a role in the mediation of sperm–zona pellucida interaction we demonstrated that antibodies directed against individual subunits of these complexes were able to inhibit sperm binding to zona-intact oocytes. Similarly, these results were able to be recapitulated using native sperm lysates, the zona affinity of which was dramatically reduced by antibody labeling of the complex receptors, or in the case of the 20S proteasome the ubiquitinated zonae ligands. Overall, the strategies employed in this study have provided novel, causal insights into the molecular mechanisms that govern sperm–egg interaction. [Copyright &y& Elsevier]

Global changes in DNA methylation are observed in development and disease, and single-cell analyses are highlighting the heterogeneous regulation of these processes. However, technical challenges associated with single-cell analysis of DNA methylation limit these studies. We present single-cell transposable element methylation sequencing (scTEM-seq) for cost-effective estimation of average DNA methylation levels. By targeting high-copy SINE Alu elements, we achieve amplicon bisulphite sequencing with thousands of loci covered in each scTEM-seq library. Parallel transcriptome analysis is also performed to link global DNA methylation estimates with gene expression. We apply scTEM-seq to KG1a acute myeloid leukaemia (AML) cells, and primary AML cells. Our method reveals global DNA methylation heterogeneity induced by decitabine treatment of KG1a cells associated with altered expression of immune process genes. We also compare global DNA methylation estimates to expression of transposable elements and find a predominance of negative correlations. Finally, we observe co-ordinated upregulation of many transposable elements in a sub-set of decitabine treated cells. By linking global DNA methylation heterogeneity with transcription, scTEM-seq will refine our understanding of epigenetic regulation in cancer and beyond. [ABSTRACT FROM AUTHOR]

Background and objective: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. Methods: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. Results: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. Conclusion: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies. [ABSTRACT FROM AUTHOR]

Conservation efforts to secure the long-termsurvival of crocodilian specieswould benefit fromthe establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodileCrocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this studyweexamined the development of motility during spermtransport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasingmotility (percentagemotile and rate of movement) as they transit themale reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMPlevels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals. [ABSTRACT FROM AUTHOR]

Kinase-Substrate analysis of pHASED data?set for resistant cells in comparison to FLT3-ITD (log2 fold change±0.5). Kinase activity inferred by KSEA analysis of phosphorylation changes in pHASED dataset (log2±0.5, p<=0.05) for resistant cells in comparison to FLT3-ITD. [Extracted from the article]

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with the development of hypermutant pediatric high-grade glioma, and confers a poor prognosis. While therapeutic histone deacetylase (HDAC) inhibition of diffuse intrinsic pontine glioma (DIPG) has been reported; here, we use a clinically relevant biopsy-derived hypermutant DIPG model (PBT-24FH) and a CRISPR-Cas9 induced genetic model to evaluate the efficacy of HDAC inhibition against hypermutant DIPG. We screened PBT-24FH cells for sensitivity to a panel of HDAC inhibitors (HDACis) in vitro , identifying two HDACis associated with low nanomolar IC50s, quisinostat (27 nM) and romidepsin (2 nM). In vivo , quisinostat proved more efficacious, inducing near-complete tumor regression in a PBT-24FH flank model. RNA sequencing revealed significant quisinostat-driven changes in gene expression, including upregulation of neural and pro-inflammatory genes. To validate the observed potency of quisinostat in vivo against additional hypermutant DIPG models, we tested quisinostat in genetically-induced mismatch repair (MMR)-deficient DIPG flank tumors, demonstrating that loss of MMR function increases sensitivity to quisinostat in vivo. Here, we establish the preclinical efficacy of quisinostat against hypermutant DIPG, supporting further investigation of epigenetic targeting of hypermutant pediatric cancers with the potential for clinical translation. These findings support further investigation of HDAC inhibitors against pontine high-grade gliomas, beyond only those with histone mutations, as well as against other hypermutant central nervous system tumors. [ABSTRACT FROM AUTHOR]

While immunotherapy for pediatric cancer has made great strides in recent decades, including the FDA approval of agents such as dinutuximab and tisgenlecleucel, these successes have rarely impacted children with pediatric central nervous system (CNS) tumors. As our understanding of the biological underpinnings of these tumors evolves, new immunotherapeutics are undergoing rapid clinical translation specifically designed for children with CNS tumors. Most recently, there have been notable clinical successes with oncolytic viruses, vaccines, adoptive cellular therapy, and immune checkpoint inhibition. In this article, the immunotherapy working group of the Pacific Pediatric Neuro-Oncology Consortium (PNOC) reviews the current and future state of immunotherapeutic CNS clinical trials with a focus on clinical trial development. Based on recent therapeutic trials, we discuss unique immunotherapy clinical trial challenges, including toxicity considerations, disease assessment, and correlative studies. Combinatorial strategies and future directions will be addressed. Through internationally collaborative efforts and consortia, we aim to direct this promising field of immuno-oncology to the next frontier of successful application against pediatric CNS tumors. [ABSTRACT FROM AUTHOR]

The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract. [ABSTRACT FROM AUTHOR]

Abstract: Fat1 is a single pass transmembrane protein and the largest member of the cadherin superfamily. Mouse knockout models and in vitro studies have suggested that Fat1 influences cell polarity and motility. Fat1 is also an upstream regulator of the Hippo pathway, at least in lower vertebrates, and hence may play a role in growth control. In previous work we have established that FAT1 cadherin is initially cleaved by proprotein convertases to form a noncovalently linked heterodimer prior to expression on the cell surface. Such processing was not a requirement for cell surface expression, since melanoma cells expressed both unprocessed FAT1 and the heterodimer on the cell surface. Here we further establish that the site 1 (S1) cleavage step to promote FAT1 heterodimerisation is catalysed by furin and we identify the cleavage site utilised. For a number of other transmembrane receptors that undergo heterodimerisation the S1 processing step is thought to occur constitutively but the functional significance of heterodimerisation has been controversial. It has also been generally unclear as to the significance of receptor heterodimerisation with respect to subsequent post-translational proteolysis that often occurs in transmembrane proteins. Exploiting the partial deficiency of FAT1 processing in melanoma cells together with furin-deficient LoVo cells, we manipulated furin expression to demonstrate that only the heterodimer form of FAT1 is subject to cleavage and subsequent release of the extracellular domain. This work establishes S1-processing as a clear functional prerequisite for ectodomain shedding of FAT1 with general implications for the shedding of other transmembrane receptors. [Copyright &y& Elsevier]

R4212 --> 867.2 --> Background: Altered duodenal microbiota profiles have been identified in patients with functional dyspepsia (FD), however the nature of altered host‐microbe interactions is not understood. Given that increased duodenal epithelial permeability is a feature of FD, we hypothesised that FD patients would be seroreactive to constituents of the duodenal microbiota. We aimed to investigate whether specific microbial species elicited immune responses in FD patients. Methods: Three novel Streptococcusisolates cultured from duodenal biopsies of FD patients were selected for analysis. These isolates were taxonomically affiliated with S. salivarius57.I (designated strain AGIRA0001), S. gordonii Challis CH1(AGIRA0002) and S. salivarius CCHSS3 (AGIRA0003) by genome‐based analysis. Microbial lysates were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane for immunoblotting. Immunoblots were probed with plasma from 17 controls and 40 FD patients. Mass spectrometry (MS) used to identify seroreactive bacterial peptide fragments. Peripheral blood gut‐homing T cells (CD4+ integrina4+integrin ß7+chemokine receptor 9+) were quantified by flow cytometry. Results: Screening of Streptococci isolates AGIRA0001‐0003 revealed AGIRA0003 possesses no less than two polypeptides (~75‐100kDa and ~30‐35kDa) that were seroreactive, with positivity observed for 90% of FD and 35% control sera, with a response at either molecular weight significantly associated with Rome III FD diagnosis (p<0.0001). Further, seroreactive (but not seronegative) patients had greater proportions of gut‐homing T cells compared to controls (0.33±0.77 vs 0.84±4.26, p=0.04). Matching of multiple tryptic peptide masses from each seroreactive band to the AGIRA0003 genome annotation allowed for prediction of the seroreactive bacterial proteins as GBS Bsp‐repeat domain protein (75‐100kDa) and 30S ribosomal subunit S2 protein (30‐35kDa). Conclusions: Our data demonstrates that there are strain‐specific bacterial‐immune interactions associated with FD. Importantly, our findings are linked to previous reports of increased gut‐homing T cells in FD, indicating that repeated exposure to microbial antigens may drive symptom onset in these patients. [ABSTRACT FROM AUTHOR]

Breast cancer is the leading cause of cancer death in women. The majority of these deaths are due to disease metastasis, in which cancer cells disseminate to multiple organs and disrupt vital physiological functions. It is widely accepted that breast cancer cells secrete extracellular vesicles (EVs), which contain dynamic molecular cargo that act as versatile mediators of intercellular communication. Therefore, Evs. secreted by breast cancer cells could be involved in the development of metastatic disease and resistance to treatment. Moreover, changes in EV cargo could reflect the effects of therapy on their parent tumor cells. The aim of this feasibility study was to quantitatively profile the proteomes of Evs. isolated from blood samples taken from treatment sensitive and resistant metastatic breast cancer patients to identify proteins associated with responses. Three serial blood samples were collected from three patients with metastatic breast cancer receiving systemic therapy including a responder, a non-responder, and a mixed-responder. Evs. were isolated from plasma using size exclusion chromatography and their protein cargo was prepared for tandem mass tag (TMT)-labelling and quantitative analyses using two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. After filtering, we quantitatively identified 286 proteins with high confidence using a q value of 0.05. Of these, 149 were classified as EV associated candidate proteins and 137 as classical, high abundant plasma proteins. After comparing EV protein abundance between the responder and non-responder, we identified 35 proteins with unique de-regulated abundance patterns that was conserved at multiple time points. We propose that this proof-of-concept approach can be used to identify proteins which have potential as predictors of metastatic breast cancer response to treatment. [ABSTRACT FROM AUTHOR]

Competition to achieve paternity has coerced the development of a multitude of male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes as they transit the male (epididymal maturation) and female (capacitation) reproductive tracts prior to realizing their potential to fertilize an ovum. However, the origin and adaptive advantage afforded by these intricate processes of post-testicular sperm maturation remain to be fully elucidated. Here, we review literature pertaining to the nature and the physiological role of epididymal maturation and subsequent capacitation in comparative vertebrate taxa including representative species from the avian, reptilian, and mammalian lineages. Such insights are discussed in terms of the framework they provide for helping to understand the evolutionary significance of post-testicular sperm maturation. Image 1 • Eutherian sperm undergo complex post-testicular maturation prior to fertilization. • The need for post-testicular sperm maturation varies in sub-therian vertebrates. • We discuss evolutionary forces that have shaped these divergent maturation strategies. [ABSTRACT FROM AUTHOR]

Cannabis has been used to relieve the symptoms of disease for thousands of years. However, social and political biases have limited effective interrogation of the potential benefits of cannabis and polarised public opinion. Further, the medicinal and clinical utility of cannabis is limited by the psychotropic side effects of ∆9-tetrahydrocannabinol (∆9-THC). Evidence is emerging for the therapeutic benefits of cannabis in the treatment of neurological and neurodegenerative diseases, with potential efficacy as an analgesic and antiemetic for the management of cancer-related pain and treatment-related nausea and vomiting, respectively. An increasing number of preclinical studies have established that ∆9-THC can inhibit the growth and proliferation of cancerous cells through the modulation of cannabinoid receptors (CB1R and CB2R), but clinical confirmation remains lacking. In parallel, the anti-cancer properties of non-THC cannabinoids, such as cannabidiol (CBD), are linked to the modulation of non-CB1R/CB2R G-protein-coupled receptors, neurotransmitter receptors, and ligand-regulated transcription factors, which together modulate oncogenic signalling and redox homeostasis. Additional evidence has also demonstrated the anti-inflammatory properties of cannabinoids, and this may prove relevant in the context of peritumoural oedema and the tumour immune microenvironment. This review aims to document the emerging mechanisms of anti-cancer actions of non-THC cannabinoids. [ABSTRACT FROM AUTHOR]

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a poor overall survival. Reactive oxygen species (ROS) have been shown to be elevated in a wide range of cancers including AML. Whilst previously thought to be mere by-products of cellular metabolism, it is now clear that ROS modulate the function of signalling proteins through oxidation of critical cysteine residues. In this way, ROS have been shown to regulate normal haematopoiesis as well as promote leukaemogenesis in AML. In addition, ROS promote genomic instability by damaging DNA, which promotes chemotherapy resistance. The source of ROS in AML appears to be derived from members of the "NOX family" of NADPH oxidases. Most studies link NOX-derived ROS to activating mutations in the Fms-like tyrosine kinase 3 (FLT3) and Ras-related C3 botulinum toxin substrate (Ras). Targeting ROS through either ROS induction or ROS inhibition provides a novel therapeutic target in AML. In this review, we summarise the role of ROS in normal haematopoiesis and in AML. We also explore the current treatments that modulate ROS levels in AML and discuss emerging drug targets based on pre-clinical work. [ABSTRACT FROM AUTHOR]

Background: The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. Results: Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. Conclusion: Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation. [ABSTRACT FROM AUTHOR]

The identification of recurrent driver mutations in genes encoding tyrosine kinases has resulted in the development of molecularly-targeted treatment strategies designed to improve outcomes for patients diagnosed with acute myeloid leukemia (AML). The receptor tyrosine kinase FLT3 is the most commonly mutated gene in AML, with internal tandem duplications within the juxtamembrane domain (FLT3-ITD) or missense mutations in the tyrosine kinase domain (FLT3-TKD) present in 30–35% of AML patients at diagnosis. An established driver mutation and marker of poor prognosis, the FLT3 tyrosine kinase has emerged as an attractive therapeutic target, and thus, encouraged the development of FLT3 tyrosine kinase inhibitors (TKIs). However, the therapeutic benefit of FLT3 inhibition, particularly as a monotherapy, frequently results in the development of treatment resistance and disease relapse. Commonly, FLT3 inhibitor resistance occurs by the emergence of secondary lesions in the FLT3 gene, particularly in the second tyrosine kinase domain (TKD) at residue Asp835 (D835) to form a 'dual mutation' (ITD-D835). Individual FLT3-ITD and FLT3-TKD mutations influence independent signaling cascades; however, little is known about which divergent signaling pathways are controlled by each of the FLT3 specific mutations, particularly in the context of patients harboring dual ITD-D835 mutations. This review provides a comprehensive analysis of the known discrete and cooperative signaling pathways deregulated by each of the FLT3 specific mutations, as well as the therapeutic approaches that hold the most promise of more durable and personalized therapeutic approaches to improve treatments of FLT3 mutant AML. [ABSTRACT FROM AUTHOR]