Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library

Dubious effects of methadone as an "anticancer" drug on ovarian cancer cell-lines and patient-derived tumor-spheroids

Background. The opioid agonist D, L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label "anticancer" drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored. Methods. We analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models. Results. OPRM1 was transcriptionally expressed in 69% of OC-tissues and in three of eight OC cell-lines. D, L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D, L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four sphero id models, treatment with D, L-methadone significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D, L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive. Conclusions. Our study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D, L-methadone function. As such, D,L-methadone may exert also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect. (C) 2022 The Authors. Published by Elsevier Inc.Peer reviewe

Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin

Non-contact universal sample presentation for room temperature macromolecular crystallography using acoustic levitation

Macromolecular Crystallography is a powerful and valuable technique to assess protein structures. Samples are commonly cryogenically cooled to minimise radiation damage effects from the X-ray beam, but low temperatures hinder normal protein functions and this procedure can introduce structural artefacts. Previous experiments utilising acoustic levitation for beamline science have focused on Langevin horns which deliver significant power to the confined droplet and are complex to set up accurately. In this work, the low power, portable TinyLev acoustic levitation system is used in combination with an approach to dispense and contain droplets, free of physical sample support to aid protein crystallography experiments. This method facilitates efficient X-ray data acquisition in ambient conditions compatible with dynamic studies. Levitated samples remain free of interference from fixed sample mounts, receive negligible heating, do not suffer significant evaporation and since the system occupies a small volume, can be readily installed at other light sources

Dubious effects of methadone as an “anticancer” drug on ovarian cancer cell-lines and patient-derived tumor-spheroids

BackgroundThe opioid agonist D,L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label “anticancer” drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored.MethodsWe analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models.ResultsOPRM1 was transcriptionally expressed in 69% of OC-tissues and in three of eight OC cell-lines. D,L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D,L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four spheroid models, treatment with D,L-methadone significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D,L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive.ConclusionsOur study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D,L-methadone function. As such, D,L-methadone may exert also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect.</p

Polycystic ovary syndrome

The document attached has been archived with permission from the editor of the Medical Journal of Australia. An external link to the publisher’s copy is included.Polycystic ovary syndrome (PCOS) affects 5-20% of women of reproductive age worldwide. The condition is characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology (PCOM) - with excessive androgen production by the ovaries being a key feature of PCOS. Metabolic dysfunction characterized by insulin resistance and compensatory hyperinsulinaemia is evident in the vast majority of affected individuals. PCOS increases the risk for type 2 diabetes mellitus, gestational diabetes and other pregnancy-related complications, venous thromboembolism, cerebrovascular and cardiovascular events and endometrial cancer. PCOS is a diagnosis of exclusion, based primarily on the presence of hyperandrogenism, ovulatory dysfunction and PCOM. Treatment should be tailored to the complaints and needs of the patient and involves targeting metabolic abnormalities through lifestyle changes, medication and potentially surgery for the prevention and management of excess weight, androgen suppression and/or blockade, endometrial protection, reproductive therapy and the detection and treatment of psychological features. This Primer summarizes the current state of knowledge regarding the epidemiology, mechanisms and pathophysiology, diagnosis, screening and prevention, management and future investigational directions of the disorder.Robert J Norman, Ruijin Wu and Marcin T Stankiewic

Acoustic Injectors For Drop-On-Demand Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples

Age-related brain structural alterations as an intermediate phenotype of psychosis

Background: There is only limited agreement with respect to location, directionality and functional implications of brain structural alterations observed in patients with schizophrenia. Additionally, their link to occurrence of psychotic symptoms remains unclear. A viable way of addressing these questions is to examine populations in an at-risk mental state (ARMS) before the transition to psychosis. Methods: We tested for structural brain alterations in individuals in an ARMS compared with healthy controls and patients with first-episode psychosis (FEP) using voxel-based morphometry and measures of cortical thickness. Furthermore, we evaluated if these alterations were modified by age and whether they were linked to the observed clinical symptoms. Results: Our sample included 59 individuals with ARMS, 26 healthy controls and 59 patients with FEP. We found increased grey matter volume and cortical thickness in individuals with ARMS and a similar pattern of structural alterations in patients with FEP. We further found stronger age-related reductions in grey matter volume and cortical thickness in both patients with FEP and individuals with ARMS, linking these alterations to observed clinical symptoms. Limitations: The ARMS group comprised subgroups with heterogeneous levels of psychosis risk and medication status. Furthermore, the cross-sectional nature of our study and the reduced number of older patients limit conclusions with respect to observed interactions with age. Conclusion: Our findings on consistent structural alterations in individuals with ARMS and patients with FEP and their link to clinical symptoms have major implications for understanding their time of occurrence and relevance to psychotic symptoms. Interactions with age found for these alterations may explain the heterogeneity of findings reported in the literature