Sex-specific differences in telomere length of patients with primary knee osteoarthritis.

Accelerated telomere shortening is associated with age-related diseases, including osteoarthritis (OA). We aimed to determine the relative telomere length (TL) in leukocytes and cartilage of patients with primary knee OA and to investigate factors that may affect TL in OA. Relative TL measurements were performed using qPCR in leukocytes of 612 individuals (310 patients with primary knee OA undergoing total knee arthroplasty (TKA) and 302 unaffected controls). We also analysed cartilage in 57 of the 310 OA patients, measuring relative TL in severely affected and less affected (control) cartilage collected from the same knee. Cartilage TLs were compared to leukocyte TLs in all 57 patients. A significant sex-by-disease-status interaction was found in regard to relative TL. Controlling for age, the average difference of leukocyte TL between female OA patients versus female controls was 0.217 units greater than that between male OA patients versus male controls (95% CI; [0.014, 0.421]). Relative TL comparison of severely and less affected cartilage samples from the same joint showed attrition of telomeres corresponding to disease severity (0.345 mean TL difference with 95% CI of [0.151, 0.539]) in the joint. We also noted that both severely and less affected cartilage had shorter telomeres than leukocytes collected from the same patient. Severe and moderate pain in OA patients was associated with shorter TL in leukocytes, but there was no association with depression or smoking in leukocytes and cartilage. Our study indicates that sex is an important factor in OA contributing to leukocyte and cartilage TL and that pain in OA shows an inverse association only with leukocyte TL.

The iPSC secretome is beneficial for in vitro propagation of primary osteoarthritic chondrocytes cell lines.

BACKGROUND: One of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells. METHODS: We added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles. RESULTS: iPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time. CONCLUSION: Our preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

Effect of continuous passive motion on the early recovery outcomes after total knee arthroplasty.

INTRODUCTION: Continuous passive motion (CPM) is a frequently used method in the early post-operative rehabilitation of patients after knee surgery. In this study, the effectiveness of the CPM method was evaluated after primary total knee arthroplasty during an early recovery period. METHODS: Eighty patients undergoing total knee arthroplasty were assigned into two groups. The experimental group received CPM and active exercises, while the control group active exercises only. All subjects were evaluated once before the surgery and at a discharge, in terms of mean active range of motion (AROM), mean Knee Society Score (KSS), and Western Ontario and MacMaster Universities Osteoarthritis Index (WOMAC). RESULTS: The mean AROM for the experimental group was 82.3° ± 14.3° and 76.1° ± 22.2° for the control. The mean KSS score was 136.4 ± 19.3 points for the experimental group, and 135.7 ± 15.1 for the control. There were no statistical differences between the two groups. The KSS functional score was 66.4 ± 8.1 points for the experimental group compared to 62.2 ± 7.3 points for the control, but there was a statistically significant difference between the groups at discharge from the hospital (p = 0.009). A subjective estimation of the pain level, joint stiffness and function also showed a statistically significant difference between the two groups (38.6 ± 14.3 points for the CPM group and 21.2 ± 15.7 for the control). CONCLUSION: These findings show that there is no significant effect of CPM in terms of improving clinical measurements. However, there was a significant beneficial effect on the subjective assessment of pain level, joint stiffness, and functional ability.

Molecular basis for inner kinetochore configuration through RWD domain-peptide interactions.

Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multi-protein assemblies that are evolutionarily conserved between yeasts and humans. One of these assemblies-COMA-consists of subunits Ame1CENP-U, Ctf19CENP-P, Mcm21CENP-O and Okp1CENP-Q A description of COMA molecular organization has so far been missing. We defined the subunit topology of COMA, bound with inner kinetochore proteins Nkp1 and Nkp2, from the yeast Kluyveromyces lactis, with nanoflow electrospray ionization mass spectrometry, and mapped intermolecular contacts with hydrogen-deuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multi-segmented nexus with distinct binding sites for Ame1, Nkp1-Nkp2 and Ctf19-Mcm21. Our crystal structure of the Ctf19-Mcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19-Mcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19-Mcm21 and Chl4CENP-N-Iml3CENP-L Absence of this motif results in dependence on the mitotic checkpoint for viability.

Chondrogenic Differentiation of Pluripotent Stem Cells under Controllable Serum-Free Conditions.

The repair of damaged articular cartilage using currently available implantation techniques is not sufficient for the full recovery of patients. Pluripotent stem cells (iPSC)-based therapies could bring new perspectives in the treatment of joint diseases. A number of protocols of in vitro differentiation of iPSC in chondrocytes for regenerative purposes have been recently described. However, in order to use these cells in clinics, the elimination of animal serum and feeder cells is essential. In our study, a strictly defined and controllable protocol was designed for the differentiation of pluripotent stem cells (BG01V, ND 41658*H, GPCCi001-A) in chondrocyte-like cells in serum- and a feeder cell-free system, using the embryoid bodies step. The extension of the protocol and culture conditions (monolayer versus 3D culture) was also tested after the initial 21 days of chondrogenic differentiation. Promotion of the chondrogenic differentiation in 3D culture via the elevated expression of genes related to chondrogenesis was achieved. Using immunofluorescence and immunohistochemistry staining techniques, the increased deposition of the specific extracellular matrix was indicated. As a result, chondrocyte-like cells in the early stages of their differentiation using pellet culture under fully controlled and defined conditions were obtained.

The Role of MicroRNAs in Early Chondrogenesis of Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.

Obstetric fistula policy in Nigeria: a critical discourse analysis.

BACKGROUND: In 2012, Nigeria's Federal Ministry of Health published its National Strategic Framework for the Elimination of Obstetric Fistula (NSFEOF), 2011-2015. The framework has since lapsed and there is no tangible evidence that the goal of eliminating obstetric fistula was met. To further inform future policy directions on obstetric fistula in Nigeria, this paper explores how the NSFEOF conceptualized obstetric fistula and its related issues, including child marriage and early childbearing. METHODS: A critical discourse analysis of the policy was performed. We examined four policies in addition to the strategic framework: the Nigerian constitution; the Marriage Act; the Matrimonial Causes Act; and the National Reproductive Health Policy. We used the three phases of critical discourse analysis: textual analysis, analysis of discourse practice, and analysis of discursive events as instances of sociocultural practice. RESULTS: The analysis demonstrates that, despite its title, the policy document focuses on reduction rather than elimination of obstetric fistula. The overall orientation of the policy is downstream, with minimal focus on prevention. The policy language suggests victim blaming. Furthermore, the extent to which subnational stakeholders in government and civil society were engaged in decision-making process for developing this policy is ambiguous. Although the policy is ostensibly based on principles of social justice and equity, several rhetorical positions suggest that the Nigerian constitutional environment and justice systems make no real provisions to protect the reproductive rights of girls in accordance with the United Nations' "2030 Agenda for Sustainable Development." CONCLUSION: This analysis establishes that the Nigerian constitution, justice environment and the obstetric fistula policy itself do not demonstrate clear commitment to eradicating obstetric fistula. Specifically, a clear commitment to eradicating obstetric fistula would see the constitution and Marriage Act of Nigeria specify an age of consent that is consistent with the agenda to prevent obstetric fistula. Additionally, a policy to end obstetric fistulas in Nigeria must purposefully address the factors creating barrier to women's access to quality maternal healthcare services. Future policies and programs to eliminate obstetric fistulas should include perspectives of nurses, midwives, researchers and, women's interest groups.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies.

Correlations between serum adipocytokine concentrations, disease stage, radiological status and total body fat content in the patients with primary knee osteoarthritis.

PURPOSE: The study was designed to investigate whether serum concentrations of leptin, resistin and adiponectin in obese and normal-weight patients with primary knee osteoarthritis (OA) correlate with clinical and radiological stages of the disease and percentage of total body fat. METHODS: Seventy-three patients with knee OA, divided into obese and normal-weight groups, were clinically evaluated according to the Knee Society Score (KSS), and radiologically assessed using Kellgren and Lawrence scale. The percentage of total body fat and some anthropometric data were also given. Serum leptin, resistin and adiponectin concentrations were measured by Elisa and were correlated with the clinical, radiological and anthropometric parameters. RESULTS: Leptin concentrations were significantly higher (p = 0.001) in the obese patients and positively correlated (R = 0.63) with radiologically assessed OA grade, but only in the normal-weight group. Resistin and adiponectin concentrations were identical in obese and normal-weight patients and negatively correlated (R = -0.41) with the clinical status of obese patients. In both groups, percentage of total body fat positively correlated (R = 0.29 and R = 0.53 for obese and normal-weight respectively) with radiologically assessed OA grade. However, no correlations were found with clinical status of the patients. CONCLUSIONS: It was found that in the obese patients with knee OA, increased percentage of total body fat and elevated serum leptin concentration might favour the advancement of clinical but not radiologically assessed changes in the joint structures, while in normal-weight patients it correlates only with radiologically assessed changes but does not affect to an appreciable extent the clinical status of the patients.

Modified methods for efficiently differentiating human embryonic stem cells into chondrocyte-like cells.

INTRODUCTION: Human articular cartilage has a poor regenerative capacity. This often results in the serious joint disease- osteoarthritis (OA) that is characterized by cartilage degradation. An inability to self-repair provided extensive studies on AC regeneration. The cell-based cartilage tissue engineering is a promising approach for cartilage regeneration. So far, numerous cell types have been reported to show chondrogenic potential, among others human embryonic stem cells (hESCs). MATERIALS AND METHODS: However, the currently used methods for directed differentiation of human ESCs into chondrocyte-like cells via embryoid body (EB) formation, micromass culture (MC) and pellet culture (PC) are not highly efficient and require further improvement. In the present study, these three methods for hESCs differentiation into chondrocyte-like cells in the presence of chondrogenic medium supplemented with diverse combination of growth factors (GFs) were evaluated and modified. RESULTS: The protocols established here allow highly efficient, simple and inexpensive production of a large number of chondrocyte-like cells suitable for transplantation into the sites of cartilage injury. The most crucial issue is the selection of appropriate GFs in defined concentration. The obtained stem-derived cells reveal the presence of chondrogenic markers such as type II collagen, Sox6 and Sox9 as well as the lack or significantly lower level of pluripotency markers including Nanog and Oct3/4. DISCUSSION: The most efficient method is the differentiation throughout embryoid bodies. In turn, chondrogenic differentiation via pellet culture is the most promising method for implementation on clinical scale. The most useful GFs are TGF-ß1, -3 and BMP-2 that possess the most chondrogenic potential. These methods can also be used to obtain chondrocyte-like cells from differentiating induced pluripotent stem cells (iPSCs).

Application of cell and biomaterial-based tissue engineering methods in the treatment of cartilage, menisci and ligament injuries.

Over 20 years ago it was realized that the traditional methods of the treatment of injuries to joint components: cartilage, menisci and ligaments, did not give satisfactory results and so there is a need of employing novel, more effective therapeutic techniques. Recent advances in molecular biology, biotechnology and polymer science have led to both the experimental and clinical application of various cell types, adapting their culture conditions in order to ensure a directed differentiation of the cells into a desired cell type, and employing non-toxic and non-immunogenic biomaterial in the treatment of knee joint injuries. In the present review the current state of knowledge regarding novel cell sources, in vitro conditions of cell culture and major important biomaterials, both natural and synthetic, used in cartilage, meniscus and ligament repair by tissue engineering techniques are described, and the assets and drawbacks of their clinical application are critically evaluated.

The role of adipocytokines in the pathogenesis of knee joint osteoarthritis.

Osteoarthritis (OA) is one of the most common causes of musculoskeletal disability in the world. Traditionally, it has been thought that obesity contributes to the development and progression of OA by increased mechanical load of the joint structures. Nevertheless, studies have shown that adipose tissue-derived cytokines (adipocytokines) are a possible link between obesity and OA. Furthermore, according to recent findings, not only articular cartilage may be the main target of these cytokines but also the synovial membrane, subchondral bone and infrapatellar fat pad may be encompassed in the process of degradation. This review presents the most recent reports on the contribution of adipocytokines to the knee joint cartilage degradation, osteophyte formation, infrapatellar fat pad alterations and synovitis.

The role of growth factors in stem cell-directed chondrogenesis: a real hope for damaged cartilage regeneration.

PURPOSE: The use of stem cells in regenerative medicine offers hope to treat numerous orthopaedic disorders, including articular cartilage defects. Although much research has been carried out on chondrogenesis, this complicated process is still not well understood and much more research is needed. The present review provides an overview of the stages of chondrogenesis and describes the effects of various growth factors, which act during the multiple steps involved in stem cell-directed differentiation towards chondrocytes. METHODS: The current literature on stem cell-directed chondrogenesis, in particular the role of members of the transforming growth factor-ß (TGF-ß) superfamily-TGF-ßs, bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs)-is reviewed and discussed. RESULTS: Numerous studies have reported the chondrogenic potential of both adult- and embryonic-like stem cells and the role of growth factors in programming differentiation of these cells towards chondrocytes. Mesenchymal stem cells (MSCs) are adult multipotent stem cells, whereas induced pluripotent stem cells (iPSC) are reprogrammed pluripotent cells. Although better understanding of the processes involved in the development of cartilage tissues is necessary, both cell types may be of value in the clinical treatment of cartilage injuries or osteoarthritic cartilage lesions. CONCLUSIONS: MSCs and iPSCs both present unique characteristics. However, at present, it is still unclear which cell type is most suitable in the treatment of cartilage injuries.

Optimization of Potent Ligands for the E3 Ligase DCAF15 and Evaluation of Their Use in Heterobifunctional Degraders.

Unlocking novel E3 ligases for use in heterobifunctional PROTAC degraders is of high importance to the pharmaceutical industry. Over-reliance on the current suite of ligands used to recruit E3 ligases could limit the potential of their application. To address this, potent ligands for DCAF15 were optimized using cryo-EM supported, structure-based design to improve on micromolar starting points. A potent binder, compound 24, was identified and subsequently conjugated into PROTACs against multiple targets. Following attempts on degrading a number of proteins using DCAF15 recruiting PROTACs, only degradation of BRD4 was observed. Deconvolution of the mechanism of action showed that this degradation was not mediated by DCAF15, thereby highlighting both the challenges faced when trying to expand the toolbox of validated E3 ligase ligands for use in PROTAC degraders and the pitfalls of using BRD4 as a model substrate.

Conditioned Medium - Is it an Undervalued Lab Waste with the Potential for Osteoarthritis Management?

BACKGROUND: The approaches currently used in osteoarthritis (OA) are mainly short-term solutions with unsatisfactory outcomes. Cell-based therapies are still controversial (in terms of the sources of cells and the results) and require strict culture protocol, quality control, and may have side-effects. A distinct population of stromal cells has an interesting secretome composition that is underrated and commonly ends up as biological waste. Their unique properties could be used to improve the existing techniques due to protective and anti-ageing properties. SCOPE OF REVIEW: In this review, we seek to outline the advantages of the use of conditioned media (CM) and exosomes, which render them superior to other cell-based methods, and to summarise current information on the composition of CM and their effect on chondrocytes. MAJOR CONCLUSIONS: CM are obtainable from a variety of mesenchymal stromal cell (MSC) sources, such as adipose tissue, bone marrow and umbilical cord, which is significant to their composition. The components present in CMs include proteins, cytokines, growth factors, chemokines, lipids and ncRNA with a variety of functions. In most in vitro and in vivo studies CM from MSCs had a beneficial effect in enhance processes associated with chondrocyte OA pathomechanism. GENERAL SIGNIFICANCE: This review summarises the information available in the literature on the function of components most commonly detected in MSC-conditioned media, as well as the effect of CM on OA chondrocytes in in vitro culture. It also highlights the need to standardise protocols for obtaining CM, and to conduct clinical trials to transfer the effects obtained in vitro to human subjects.

Oxidative Stress in Long-Term Exposure to Multi-Walled Carbon Nanotubes in Male Rats.

Multi-walled carbon nanotubes (MWCNTs) serve as nanoparticles due to their size, and for that reason, when in contact with the biological system, they can have toxic effects. One of the main mechanisms responsible for nanotoxicity is oxidative stress resulting from the production of intracellular reactive oxygen species (ROS). Therefore, oxidative stress biomarkers are important tools for assessing MWCNTs toxicity. The aim of this study was to evaluate the oxidative stress of multi-walled carbon nanotubes in male rats. Our animal model studies of MWCNTs (diameter ~15-30 nm, length ~15-20 µm) include measurement of oxidative stress parameters in the body fluid and tissues of animals after long-term exposure. Rattus Norvegicus/Wistar male rats were administrated a single injection to the knee joint at three concentrations: 0.03 mg/mL, 0.25 mg/mL, and 0.5 mg/mL. The rats were euthanized 12 and 18 months post-exposure by drawing blood from the heart, and their liver and kidney tissues were removed. To evaluate toxicity, the enzymatic activity of total protein (TP), reduced glutathione (GSH), glutathione S-transferase (GST), thiobarbituric acid reactive substances (TBARS), Trolox equivalent antioxidant capacity (TEAC), nitric oxide (NO), and catalase (CAT) was measured and histopathological examination was conducted. Results in rat livers showed that TEAC level was decreased in rats receiving nanotubes at higher concentrations. Results in kidneys report that the level of NO showed higher concentration after long exposure, and results in animal serums showed lower levels of GSH in rats exposed to nanotubes at higher concentrations. The 18-month exposure also resulted in a statistically significant increase in GST activity in the group of rats exposed to nanotubes at higher concentrations compared to animals receiving MWCNTs at lower concentrations and compared to the control group. Therefore, an analysis of oxidative stress parameters can be a key indicator of the toxic potential of multi-walled carbon nanotubes.

The Induced Pluripotent Stem Cells in Articular Cartilage Regeneration and Disease Modelling: Are We Ready for Their Clinical Use?

The development of induced pluripotent stem cells has brought unlimited possibilities to the field of regenerative medicine. This could be ideal for treating osteoarthritis and other skeletal diseases, because the current procedures tend to be short-term solutions. The usage of induced pluripotent stem cells in the cell-based regeneration of cartilage damages could replace or improve on the current techniques. The patient's specific non-invasive collection of tissue for reprogramming purposes could also create a platform for drug screening and disease modelling for an overview of distinct skeletal abnormalities. In this review, we seek to summarise the latest achievements in the chondrogenic differentiation of pluripotent stem cells for regenerative purposes and disease modelling.

Identification of nursing assessment models/tools validated in clinical practice for use with diverse ethno-cultural groups: an integrative review of the literature.

BACKGROUND: High income nations are currently exhibiting increasing ethno-cultural diversity which may present challenges for nursing practice. We performed an integrative review of literature published in North America and Europe between 1990 and 2007, to map the state of knowledge and to identify nursing assessment tools/models which are have an associated research or empirical perspective in relation to ethno-cultural dimensions of nursing care. METHODS: Data was retrieved from a wide variety of sources, including key electronic bibliographic databases covering research in biomedical fields, nursing and allied health, and culture, e.g. CINAHL, MEDline, PUBmed, Cochrane library, PsycINFO, Web of Science, and HAPI. We used the Critical Appraisal Skills Programme tools for quality assessment. We applied Torraco's definition and method of an integrative review that aims to create new knowledge and perspectives on a given phenomena. To add methodological rigor with respect to the search strategy and other key review components we also used the principles established by the Centre for Reviews and Dissemination. RESULTS: Thirteen thousand and thirteen articles were retrieved, from which 53 full papers were assessed for inclusion. Eight papers met the inclusion criteria, describing research on a total of eight ethno-cultural assessment tools/models. The tools/models are described and synthesized. CONCLUSIONS: While many ethno-cultural assessment tools exist to guide nursing practice, few are informed by research perspectives. An increased focus on the efficiency and effectiveness of health services, patient safety, and risk management, means that provision of culturally responsive and competent health services will inevitably become paramount.

From Donor to the Lab: A Fascinating Journey of Primary Cell Lines.

Primary cancer cell lines are ex vivo cell cultures originating from resected tissues during biopsies and surgeries. Primary cell cultures are objects of intense research due to their high impact on molecular biology and oncology advancement. Initially, the patient-derived specimen must be subjected to dissociation and isolation. Techniques for tumour dissociation are usually reliant on the organisation of connecting tissue. The most common methods include enzymatic digestion (with collagenase, dispase, and DNase), chemical treatment (with ethylene diamine tetraacetic acid and ethylene glycol tetraacetic acid), or mechanical disaggregation to obtain a uniform cell population. Cells isolated from the tissue specimen are cultured as a monolayer or three-dimensional culture, in the form of multicellular spheroids, scaffold-based cultures (i.e., organoids), or matrix-embedded cultures. Every primary cell line must be characterised to identify its origin, purity, and significant features. The process of characterisation should include different assays utilising specific (extra- and intracellular) markers. The most frequently used approaches comprise immunohistochemistry, immunocytochemistry, western blot, flow cytometry, real-time polymerase chain reaction, karyotyping, confocal microscopy, and next-generation sequencing. The growing body of evidence indicates the validity of the usage of primary cancer cell lines in the formulation of novel anti-cancer treatments and their contribution to drug development.

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication.

The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.