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Abstract Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is clinically defined and characterised by persistent disabling tiredness and exertional malaise, leading to functional impairment. Methods This study introduces the weighted standing time (WST) as a proxy for ME/CFS severity, and investigates its behaviour in an Australian cohort. WST was calculated from standing time and subjective standing difficulty data, collected via orthostatic intolerance assessments. The distribution of WST for healthy controls and ME/CFS patients was correlated with the clinical criteria, as well as pathology and cytokine markers. Included in the WST cytokine analyses were activins A and B, cytokines causally linked to inflammation, and previously demonstrated to separate ME/CFS from healthy controls. Forty-five ME/CFS patients were recruited from the CFS Discovery Clinic (Victoria) between 2011 and 2013. Seventeen healthy controls were recruited concurrently and identically assessed. Results WST distribution was significantly different between ME/CFS participants and controls, with six diagnostic criteria, five analytes and one cytokine also significantly different when comparing severity via WST. On direct comparison of ME/CFS to study controls, only serum activin B was significantly elevated, with no significant variation observed for a broad range of serum and urine markers, or other serum cytokines. Conclusions The enhanced understanding of standing test behaviour to reflect orthostatic intolerance as a ME/CFS symptom, and the subsequent calculation of WST, will encourage the greater implementation of this simple test as a measure of ME/CFS diagnosis, and symptom severity, to the benefit of improved diagnosis and guidance for potential treatments.

Nuclear receptors (NRs) and their coregulators play fundamental roles in initiating and directing gene expression influencing mammalian reproduction, development and metabolism. SRA stem Loop Interacting RNA-binding Protein (SLIRP) is a Steroid receptor RNA Activator (SRA) RNA-binding protein that is a potent repressor of NR activity. SLIRP is present in complexes associated with NR target genes in the nucleus; however, it is also abundant in mitochondria where it affects mitochondrial mRNA transcription and energy turnover. In further characterisation studies, we observed SLIRP protein in the testis where its localization pattern changes from mitochondrial in diploid cells to peri-acrosomal and the tail in mature sperm. To investigate the in vivo effects of SLIRP, we generated a SLIRP knockout (KO) mouse. This animal is viable, but sub-fertile. Specifically, when homozygous KO males are crossed with wild type (WT) females the resultant average litter size is reduced by approximately one third compared with those produced by WT males and females. Further, SLIRP KO mice produced significantly fewer progressively motile sperm than WT animals. Electron microscopy identified disruption of the mid-piece/annulus junction in homozygous KO sperm and altered mitochondrial morphology. In sum, our data implicates SLIRP in regulating male fertility, wherein its loss results in asthenozoospermia associated with compromised sperm structure and mitochondrial morphology.

PURPOSE:Fibrosis can be a disabling, severe side effect of radiotherapy that can occur in patients, and for which there is currently no effective treatment. The activins, proteins which are members of the TGFβ superfamily, have a major role in stimulating the inflammatory response and subsequent fibrosis. Follistatin is an endogenous protein that binds the activins virtually irreversibly and inhibits their actions. These studies test if follistatin can attenuate the fibrotic response using a murine model of radiation-induced fibrosis. EXPERIMENTAL DESIGN:C57BL/6 mice were subcutaneously injected with follistatin 24 hours prior to irradiation. Mice were irradiated in a 10 x 10 mm square area of the right hind leg with 35 Gy and were given follistatin 24 hours before radiation and three times a week for six months following. Leg extension was measured, and tissue was collected for histological and molecular analysis to evaluate the progression of the radiation-induced fibrosis. RESULTS:Leg extension was improved in follistatin treated mice compared to vehicle treated mice at six months after irradiation. Also, epidermal thickness and cell nucleus area of keratinocytes were decreased by the follistatin treatment compared to the cells in irradiated skin of control mice. Finally, the gene expression of transforming growth factor β1 (Tgfb1), and smooth muscle actin (Acta2) were decreased in the irradiated skin and Acta2 and inhibin βA subunit (Inhba) were decreased in the irradiated muscle of the follistatin treated mice. CONCLUSIONS:Follistatin attenuated the radiation-induced fibrotic response in irradiated mice. These studies provide the data to support further investigation of the use of follistatin to reduce radiation-induced fibrosis in patients undergoing radiotherapy for cancer.

Lung transplantation exposes the donated lung to a period of anoxia. Re-establishing the circulation after ischemia stimulates inflammation causing organ damage. Since our published data established that activin A is a key pro-inflammatory cytokine, we assessed the roles of activin A and B, and their binding protein, follistatin, in patients undergoing lung transplantation.Sera from 46 patients participating in a published study of remote ischemia conditioning in lung transplantation were used. Serum activin A and B, follistatin and 11 other cytokines were measured in samples taken immediately after anaesthesia induction, after remote ischemia conditioning or sham treatment undertaken just prior to allograft reperfusion and during the subsequent 24 hours.Substantial increases in serum activin A, B and follistatin occurred after the baseline sample, taken before anaesthesia induction and peaked immediately after the remote ischemia conditioning/sham treatment. The levels remained elevated 15 minutes after lung transplantation declining thereafter reaching baseline 2 hours post-transplant. Activin B and follistatin concentrations were lower in patients receiving remote ischemia conditioning compared to sham treated patients but the magnitude of the decrease did not correlate with early transplant outcomes.We propose that the increases in the serum activin A, B and follistatin result from a combination of factors; the acute phase response, the reperfusion response and the use of heparin-based anti-coagulants.

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

Follistatin is a potent regulator of the inflammatory response and binds to and inhibits activin A action. Activin A is a member of the TGFβ protein superfamily which has regulatory roles in the inflammatory response and in the fibrotic process. Fibrosis can occur following cell injury and cell death induced by agents such as ionizing radiation (IR). IR is used to treat cancer and marked fibrotic response is a normal tissue (non-tumour) consequence in a fraction of patients under the current dose regimes. The discovery and development of a therapeutic to abate fibrosis in these radiosensitive patients would be a major advance for cancer radiotherapy. Likewise, prediction of which patients are susceptible to fibrosis would enable individualization of treatment and provide an opportunity for pre-emptive fibrosis control and better tumour treatment outcomes. The levels of activin A and follistatin were measured in fibroblasts derived from patients who developed severe radiation-induced fibrosis following radiotherapy and compared to fibroblasts from patients who did not. Both follistatin and activin A gene expression levels were increased following IR and the follistatin gene expression level was lower in the fibroblasts from fibrosis patients compared to controls at both basal levels and after IR. The major follistatin transcript variants were found to have a similar response to IR and both were reduced in fibrosis patients. Levels of follistatin and activin A secreted in the fibroblast culture medium also increased in response to IR and the relative follistatin protein levels were significantly lower in the samples derived from fibrosis patients. The decrease in the follistatin levels can lead to an increased bioactivity of activin A and hence may provide a useful measurement to identify patients at risk of a severe fibrotic response to IR. Additionally, follistatin, by its ability to neutralise the actions of activin A may be of value as an anti-fibrotic for radiation induced fibrosis.

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.