Co-design and evaluation of a digital serious game to promote public awareness about pancreatic cancer.

BACKGROUND: Pancreatic cancer, ranking seventh in global cancer-related deaths, poses a significant public health challenge with increasing incidence and mortality. Most cases are diagnosed at an advanced stage, resulting in low survival rates. Early diagnosis significantly impacts prognosis, making symptom awareness crucial. Symptoms are often subtle, leading to delayed help-seeking behaviour. Patients and their carers prioritise increased public awareness, indicating a need for innovative approaches to promote awareness of the disease. METHODS: This study employed a quasi-experimental pre-test/post-test design to assess the relationship between a serious game and pancreatic cancer awareness. Members of the public (N = 727) were recruited internationally, via social media and with signposting by relevant organisations. Participants completed measures of symptom awareness and help-seeking intentions before and after playing the game. The serious game, co-designed with experts by lived experience, patient advocates and healthcare professionals, presented participants with a human anatomy diagram, with each section linked to a question about pancreatic cancer. RESULTS: The serious game demonstrated a statistically significant improvement on pancreatic cancer awareness based on matched paired t-tests. Due to missing data, paired comparisons were only possible for 489 cases. Symptom awareness scores exhibited a statistically significant increase from pre-test to post-test, with a large effect size (p < 0.001, d = 1.43). Help-seeking intentions also markedly improved, showing a significant increase from pre-test to post-test, with a large effect size (p < 0.001, d = 1.10). Independent-samples t-tests were also conducted to determine if there were any group differences on pre- to post-test changes based on age, gender, and previous knowledge and/or experience of pancreatic cancer. Participants overwhelmingly endorsed the game's usability and educational value, suggesting its potential as an effective tool for enhancing public awareness and proactive health-seeking behaviour. DISCUSSION: This study is the first to explore a serious game's utility in pancreatic cancer awareness. Results suggest that such interventions can effectively increase public awareness and influence help-seeking intentions. The co-design process ensured content relevance, and participant satisfaction was high. Findings highlight the game's potential as an accessible and convenient tool for diverse populations.

Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers.

In mammalian cells, plasma membrane potential plays vital roles in both physiology and pathology and it is controlled by a network of membrane-resident ion channels. There is considerable interest in the use of nanoparticles (NPs) to control biological functions, including the modulation of membrane potential. The photoexcitation of gold NPs (AuNPs) tethered close to the plasma membrane has been shown to induce membrane depolarization via localized heating of the AuNP surface coupled with the opening of voltage-gated sodium channels. Previous work has employed spherical AuNPs (AuNS) with absorption in the 500-600 nm range for this purpose. However, AuNP materials with absorption at longer wavelengths [e.g., near-infrared (NIR)] would enable greater tissue penetration depth in vivo. We show here the use of new anisotropic-shaped AuNPs [gold nanoflowers (AuNFs)] with broad absorption spanning into the NIR part of the spectrum (∼650-1000 nm). The AuNFs are directly synthesized with bidentate thiolate ligands, which preserves the AuNF's shape and colloidal stability, while facilitating conjugation to biomolecules. We describe the characterization of the AuNF particles and demonstrate that they adhere to the plasma membrane when bioconjugated to PEGylated cholesterol (PEG-Chol) moieties. The AuNF-PEG-Chol mediated the depolarization of rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells more effectively than AuNS-PEG-Chol and unconjugated AuNS and AuNF when photoexcited at ∼561 or ∼640 nm. Importantly, AuNF induction of depolarization had no impact on cellular viability. This work demonstrates anisotropic AuNFs as an enabling nanomaterial for use in cellular depolarization and the spatiotemporal control of cellular activity.

A topological refactoring design strategy yields highly stable granulopoietic proteins.

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets.

Design of novel granulopoietic proteins by topological rescaffolding.

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

Pharmacotherapeutics of capecitabine and trastuzumab in the treatment of metastatic breast cancer.

Metastatic HER2-positive breast cancer is an incurable disease with a poor prognosis. This article presents a critical appraisal of two treatments commonly used in the treatment of metastatic HER2-positive breast cancer: the oral chemotherapy drug, capecitabine, and the monoclonal antibody, trastuzumab. What follows is a critical discussion of the pharmacotherapeutics of capecitabine and trastuzumab, which considers their use both as single agents and as a combination regimen in the treatment of metastatic breast cancer. The implications of side effects of these drugs are discussed, both individually and in combination, as are the challenges these bring to the prescriber. The article evaluates the use of these agents and concludes that the combination of capecitabine and trastuzumab is an attractive treatment option for patients and for the prescriber.

Paracrine Activin-A Signaling Promotes Melanoma Growth and Metastasis through Immune Evasion.

The secreted growth factor Activin-A of the transforming growth factor ß family and its receptors can promote or inhibit several cancer hallmarks including tumor cell proliferation and differentiation, vascularization, lymphangiogenesis and inflammation. However, a role in immune evasion and its relationship with tumor-induced muscle wasting and tumor vascularization, and the relative contributions of autocrine versus paracrine Activin signaling remain to be evaluated. To address this, we compared the effects of truncated soluble Activin receptor IIB as a ligand trap, or constitutively active mutant type IB receptor versus secreted Activin-A or the related ligand Nodal in mouse and human melanoma cell lines and tumor grafts. We found that although cell-autonomous receptor activation arrested tumor cell proliferation, Activin-A secretion stimulated melanoma cell dedifferentiation and tumor vascularization by functional blood vessels, and it increased primary and metastatic tumor burden and muscle wasting. Importantly, in mice with impaired adaptive immunity, the tumor-promoting effect of Activin-A was lost despite sustained vascularization and cachexia, suggesting that Activin-A promotes melanoma progression by inhibiting antitumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration.

BACKGROUND: Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. METHODS: For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. RESULTS: Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. CONCLUSION: Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.

The role of BRCA1 and BRCA2 mutations in prostate, pancreatic and stomach cancers.

The association of germline mutations in the breast cancer susceptibility gene 1 (BRCA1) and the breast cancer susceptibility gene 2 (BRCA2) with the development of breast and ovarian cancers have been widely researched and recognised. It is known that these genes function at multiple sites in the body. Research has subsequently evolved into the connection of BRCA1/2 with cancers at other sites within the body. This review examines the association of BRCA1/2 germline gene mutations with prostate, pancreatic and stomach cancers. An extensive literature search revealed conflicting findings regarding the association of BRCA1/2 gene mutations with these cancers. Most studies suggest that there is an association between BRCA1/2 mutations and carcinoma of the prostate, pancreas and stomach, but some reports propose that such a correlation may be due to factors other than possessing a mutated BRCA1/2 gene, and other associations may be revealed as further epidemiological information becomes available. The review concludes that as more knowledge arises about the mechanisms of BRCA1/2 gene mutations, it should pave the way for future screening programmes to be applied effectively.

Impact of counselling on quality of life and sleep in cancer patients.

It has been acknowledged that poor quality of sleep significantly correlates with poor quality of life; evidence suggests that counselling has a positive impact not only on the cancer patient's quality of life, but also on family members and friends. The aim of this service evaluation was to determine if there was an improvement in clients' quality of life and sleep patterns following counselling as offered by a local cancer charity. A total of 60 matched pre- and post-counselling questionnaires were completed and subjected to statistical analysis. When considering quality of life, in the domains of Role Emotional, Mental Health and Mental Component Summary Score, it can be concluded that counselling has a positive effect on emotional health and mental wellbeing. The mean total number of hours sleep per night significantly increased from 6 hours sleep per night at baseline to 6.8 hours sleep per night at the completion of counselling (p=0.005) showing clients gained an extra 48 minutes sleep per night. The improved emotional and mental wellbeing alongside the extra 48 minutes sleep per night provides evidence that there is a positive outcome for those patients and families who use counselling services. Nurses and other members of the multidisciplinary team should be encouraged to discuss supportive therapies with patients and those affected by cancer at all stages of the cancer trajectory, regardless of social status, gender or cancer type.

Factors influencing participation in breast cancer screening.

Despite the efficacy of mammography and the widespread promotion of screening programmes, a significant number of eligible women still do not attend for regular breast screening. An integrative review methodology was considered the most appropriate means to critically analyse the available literature pertaining to factors which influence participation in breast cancer screening. From the extensive literature search, 12 selected core research papers met the inclusion criteria and were incorporated in the literature review. Four themes emerged from the literature which impact on participation in mammography screening: psychological and practical issues, ethnicity issues, influence of socioeconomic status and issues related to screening programmes. The recent Independent Review Panel on Breast Cancer Screening endorsed the importance of access to information which clearly communicates the harms and benefits of breast screening to enable women to make informed decisions about their health. The recommendations from the panel and others have been included in this review.

Characteristics and outcomes of sentinel node-positive breast cancer patients after total mastectomy without axillary-specific treatment.

PURPOSE: Regional failure rates are low in patients with a positive sentinel lymph node biopsy (SLNB) who undergo breast-conserving therapy without axillary lymph node dissection (ALND). The applicability of these findings to total mastectomy (TM) patients is not established. Our aims were to evaluate the characteristics and outcomes of SLNB-positive TM patients who did not receive axillary-specific treatment and to compare them to similar patients who underwent breast-conserving surgery (BCS). METHODS: A total of 535 patients with early-stage breast cancer who underwent definitive breast surgery (210 TM, 325 BCS), had a positive SLNB and did not receive ALND between 1997 and 2009 were identified from an institutional database. Characteristics and outcomes were compared between the TM and BCS groups. RESULTS: Most patients had stage I to IIA, estrogen receptor-positive, progesterone receptor-positive, Her2-negative invasive ductal carcinoma, with minimal nodal disease. Compared to the BCS group, TM patients were younger, had larger tumors, had higher nomogram scores predicting additional axillary disease and were more likely to receive chemotherapy. Ninety-four percent of the BCS cohort and 5 % of the TM cohort received adjuvant radiotherapy. At a median follow-up of 57.8 months, the 4-year local, regional and distant failure rates were 1.7, 1.2 and 0.7 % in the TM group and 1.4, 1.0 and 3.7 % in the BCS group. The 4-year disease-free and overall survival rates were 94.8 and 97.8 % in the TM group and 90.1 and 92.6 % in the BCS group. CONCLUSIONS: Early-stage breast cancer patients with minimal sentinel node disease experience excellent outcomes without ALND, whether they undergo BCS or TM.

Favorable prognosis in patients with T1a/T1bN0 triple-negative breast cancers treated with multimodality therapy.

BACKGROUND: The authors evaluated the clinical characteristics, natural history, and outcomes of patients who had ≤1 cm, lymph node-negative, triple-negative breast cancer (TNBC). METHODS: After excluding patients who had received neoadjuvant therapy, 1022 patients with TNBC who underwent definitive breast surgery during 1999 to 2006 were identified from an institutional database. In total, 194 who had lymph node-negative tumors that measured ≤1 cm comprised the study population. Clinical data were abstracted, and survival outcomes were analyzed. RESULTS: The median follow-up was 73 months (range, 5-143 months). The median age at diagnosis was 55.5 years (range, 27-84 years). Tumor (T) classification was microscopic (T1mic) in 16 patients (8.2%), T1a in 49 patients (25.3%), and T1b in 129 patients (66.5%). Most tumors were poorly differentiated (n = 142; 73%), lacked lymphovascular invasion (n = 170; 87.6%), and were detected by screening (n = 134; 69%). In total, 129 patients (66.5%) underwent breast-conserving surgery, and 65 patients (33.5%) underwent mastectomy. One hundred thirteen patients (58%) received adjuvant chemotherapy, and 123 patients (63%) received whole-breast radiation. The patients who received chemotherapy had more adverse clinical and disease features (younger age, T1b tumor, poor tumor grade; all P < .05). Results from testing for the breast cancer (BRCA) susceptibility gene were available for 49 women: 19 women had BRCA1 mutations, 7 women had BRCA2 mutations, and 23 women had no mutations. For the entire group, the 5-year local recurrence-free survival rate was 95%, and the 5-year distant metastasis-free survival rate was 95%. There was no difference between patients with T1mic/T1a tumors and patients with T1b tumors in the distant recurrence rate (94.5% vs 95.5%, respectively; P = .81) or in the receipt of chemotherapy (95.9% vs 94.5%, respectively; P = .63). CONCLUSIONS: Excellent 5-year locoregional and distant control rates were achievable in patients with TNBC who had tumors ≤1.0 cm, 58% of whom received chemotherapy. These results identified a group of patients with TNBC who had favorable outcomes after early detection and multimodality treatment.

The Oxford shoulder score revisited.

The validated, patient-reported Oxford shoulder score (OSS) was introduced around 10 years ago, primarily for the assessment of outcomes of shoulder surgery (excluding shoulder stabilisation) in randomised trials. Its uptake has steadily increased in a number of countries and its use has also been extended. Recently a number of issues have been raised in relation to other related patient-reported outcome measures which were devised around the same time as the OSS. This included recommendations to change the scoring system. This paper reviews issues concerning patient-reported outcome measures that apply to the OSS and makes some recommendations (including changes to the scoring system) as to how it should be used.

Clinical and magnetic resonance imaging findings in 92 cats with clinical signs of spinal cord disease.

Medical records of 92 cats presented with clinical signs of spinal cord disease, which had undergone magnetic resonance imaging (MRI), were reviewed. The cats were grouped into seven categories based upon the diagnosis suggested by results of MRI, cerebrospinal fluid analysis and other diagnostic procedures: neoplastic (n=25), inflammatory or infectious (n=13), traumatic (n=8), vascular (n=6), degenerative (n=5), anomalous (n=3) and those with an unremarkable MRI (n=32). There were two independent predictors of abnormal MRI findings: severity of clinical signs and presence of spinal pain. Abnormal MRI findings and speed of onset of disease were significantly associated with survival. For the 32 cats with unremarkable MRI findings, only nine died due to spinal disease and, therefore, the median survival time (MST) was not reached (lower 95% confidence interval (CI)=970 days). For the 60 cats with abnormal MRI findings, 37 died due to their disease and the MST was 138 days (95% CI: 7-807).

The effects of halothane and isoflurane on cardiovascular function in dorsally recumbent horses undergoing surgery.

OBJECTIVE: To determine the haemodynamic effects of halothane and isoflurane with spontaneous and controlled ventilation in dorsally recumbent horses undergoing elective surgery. STUDY DESIGN: Prospective randomized clinical trial. ANIMALS: Twenty-five adult horses, body mass 487 kg (range: 267-690). METHODS: Horses undergoing elective surgery in dorsal recumbency were randomly assigned to one of four treatment groups, isoflurane (I) or halothane (H) anaesthesia, each with spontaneous (SB) or controlled ventilation (IPPV). Indices of cardiac function and femoral arterial blood flow (ABF) and resistance were measured using transoesophageal and transcutaneous Doppler echocardiography, respectively. Arterial blood pressure was measured directly. RESULTS: Four horses assigned to receive isoflurane and spontaneous ventilation (SBI) required IPPV, leaving only three groups for analysis: SBH, IPPVH and IPPVI. Two horses were excluded from the halothane groups because dobutamine was infused to maintain arterial blood pressure. Cardiac index (CI) was significantly greater, and pre-ejection period (PEP) shorter, during isoflurane compared with halothane anaesthesia with both spontaneous (p = 0.04, p = 0.0006, respectively) or controlled ventilation (p = 0.04, p = 0.008, respectively). There was an association between CI and PaCO(2) (p = 0.04) such that CI increased by 0.45 L minute(-1)m(-2) for every kPa increase in PaCO(2). Femoral ABF was only significantly higher during isoflurane compared with halothane anaesthesia during IPPV (p = 0.0006). There was a significant temporal decrease in CI, but not femoral arterial flow. CONCLUSION: The previously reported superior cardiovascular function during isoflurane compared with halothane anaesthesia was maintained in horses undergoing surgery. However, in these clinical subjects, a progressive decrease in CI, which was independent of ventilatory mode, was observed with both anaesthetic agents. CLINICAL RELEVANCE: Cardiovascular function may deteriorate progressively in horses anaesthetized for brief (<2 hours) surgical procedures in dorsal recumbency. Although cardiovascular function is superior with isoflurane in dorsally recumbent horses, the need for IPPV may be greater.

c-FLIP: a key regulator of colorectal cancer cell death.

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4, and DR5 and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. We found that small interfering RNA (siRNA)-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines and that this apoptosis was mediated by caspase-8 and Fas-associated death domain. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands tumor necrosis factor-related apoptosis-inducing ligand and FasL. Overexpression of c-FLIP(L), but not c-FLIP(S), significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIP(L) was the more important splice form in regulating apoptosis in HCT116, H630, and LoVo cells, although specific knockdown of c-FLIP(S) induced more apoptosis in the HT29 cell line. Importantly, intratumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in BALB/c severe combined immunodeficient mice. In addition, the growth of c-FLIP(L)-overexpressing colorectal cancer xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in colorectal cancer cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells.

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that IFN-gamma and doxorubicin cotreatment synergistically induced apoptosis in MDA435 breast cancer cells in a signal transducer and activator of transcription 1-dependent manner. In this study, we found that this synergy was caspase-8 dependent. In addition, we found that IFN-gamma down-regulated the expression of the caspase-8 inhibitor cellular FLICE-inhibitory protein (c-FLIP). Furthermore, IFN-gamma down-regulated c-FLIP in a manner that was dependent on the transcription factors signal transducer and activator of transcription 1 and IFN regulatory factor-1. However, IFN-gamma had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a posttranscriptional level following IFN-gamma treatment. Characterization of the functional significance of c-FLIP modulation by small interfering RNA gene silencing and stable overexpression studies revealed it to be a key regulator of IFN-gamma- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-gamma-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitized MDA435 cells to other chemotherapies, including etoposide, mitoxantrone, and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

A questionnaire-based study of gestation, parturition and neonatal mortality in pedigree breeding cats in the UK.

This study was based on a convenience-sampling questionnaire study of pedigree cat breeding in the UK. Data were collated for the births of 1,056 litters from 14 different pedigree breeds and 942 different households. Significant relationships between various outcomes and relevant predictors were assessed by multiple linear regression or logistic regression as appropriate. The overall mean gestation length of 65.1 days varied significantly between the breeds (P<0.0001), and larger litter sizes were associated with shorter gestation lengths (P=0.04). The mean litter size of 4.6 kittens also varied significantly according to breed (P<0.0001). The weight of kittens born alive (overall mean 93.5 g) increased with longer gestation lengths (P=0.0003), decreased with larger litter sizes (P<0.0001) and varied between the breeds (P<0.0001). A total of 8.0% of pregnancies resulted in a caesarean section, with a higher risk associated with smaller litter sizes (P=0.002). Although the frequency of caesarean sections varied from 0 to 18.5% between individual breeds, breed itself was not shown to have a significant independent effect on this likelihood. A mean of 7.2% of all the kittens were stillborn, which varied according to breed (P=0.0003), and the risk of a stillborn kitten increased with litter size (P=0.0001), and with the presence of congenital defects in the litter (P=0.0002). The mean kitten mortality between birth and 8 weeks of age was 9.1%, and the majority of these occurred in the first week of life. Parturition intervals varied widely. The duration of first stage of labour was less than 2h in 82.9% of cats. The interval between the birth of the first and last kitten was less than 6h in 85.7%, but more than 48 h in three cats. A maximum of 48 h was recorded between the births of individual kittens in unassisted deliveries.

Activated extracellular signal-regulated kinases are necessary and sufficient to initiate tubulogenesis in renal tubular MDCK strain I cell cysts.

A classic in vitro model of renal cyst and tubule formation utilizes the Madin-Darby canine kidney (MDCK) cell line, of which two strains exist. Most cyst and tubule formation studies that utilized MDCK cells have been performed with MDCK strain II cells. MDCK strain II cells form hollow cysts in a three-dimensional collagen matrix over 10 days and tubulate in response to hepatocyte growth factor, which increases levels of active (phosphorylated) ERK1/2. In this study, we demonstrate that MDCK strain I cells also form cysts when grown in a collagen matrix; however, MDCK strain I cell cysts spontaneously initiate the primary steps in tubulogenesis. Analysis of time-lapse microscopy of both MDCK strain I and strain II cell cysts during the initial stages of tubulogenesis demonstrates a highly dynamic process with cellular extensions and retractions occurring rapidly and continuously. MDCK strain I cell cysts can spontaneously initiate tubulogenesis mainly because of relatively higher levels of active ERK in MDCK strain I, compared with strain II, cells. The presence of either of two distinct inhibitors of ERK activation (UO126 and PD09059) prevents tubulogenesis from occurring spontaneously in MDCK strain I cell cysts and, in response to hepatocyte growth factor, in strain II cell cysts. The difference between MDCK strain I and strain II cell lines is likely explained by differing embryological origins, with strain I cells being of collecting duct, and hence ureteric bud, origin. Ureteric bud cells also have high levels of active ERK and spontaneously tubulate in our in vitro collagen gel system, with tubulogenesis inhibited by UO126 and PD09059. These results suggest that a seminal event in kidney development may be the activation of ERK in the mesonephric duct/ureteric bud cells destined to form the collecting tubules.