Analysis of three characterization assays reveals ddPCR of LIN28A as the most sensitive for the detection of residual pluripotent stem cells in cellular therapy products.

BACKGROUND AIMS: With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. DISCUSSION: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.

Examining the Impact of Virtual Animal Stimuli on College Students' Affect and Perception of their Academic Advising Experience.

The benefits of animal-assisted interventions (AAI) involving animals in therapy are widely accepted. The presence of animals in therapy can decrease a patient's reservation about therapy and promote a sense of comfort and rapport during the therapy process. Using survey data from college students (n = 152) attending a large public four-year institution, this study is the first to investigate the benefits of virtual animal stimuli during academic advising appointments. It posits that exposure to virtual animal stimuli can influence positive mental health and well-being in academic advising settings. Specifically, the research questions explored how different types of video content influence students' affect and how virtual animal stimuli impact students' perception of their advisor and university. College students were randomly assigned to watch one of four types of virtual stimuli (wild animals, companion animals, nature, and a control) prior to their advising session. Subjective measures were collected at baseline and after the advising session. Results indicated animal stimuli increase positive affect, and companion animal stimuli influence the student's perception of the advisor. This study supports the notion that companion animal videos positively impact students' well-being and interactions with their advisors and may have broader implications beyond the academic setting.

A class of anti-inflammatory lipids decrease with aging in the central nervous system.

Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

A Study Evaluating Consumer Motivations, Perceptions, and Responses to Direct-to-Consumer Canine Genetic Test Results.

Direct-to-consumer genetic services allow companion animal guardians to purchase a DNA test and receive detailed results about their pet's ancestry, health, and traits results. In collaboration with Wisdom Panel, we present novel findings about consumer motivations, perceptions, and responses to their use of canine genomic services. Wisdom Panel customers were invited to complete an online survey anonymously in which they were asked about their reasons for using a genetic test for their dog, how they perceived the test's results, and how they responded to the results they received. Participant data revealed most utilized a test that provided more ancestry/breed results (75.9%) as compared to health-related results. The majority of participants perceived the breed test results as accurate (52.0% strongly agree, 27.6% somewhat agree) and the genetic services provided as having great value (49.6% strongly agree, 32.7% somewhat agreed). In responding to their dog's results, participants indicated they shared the information with family (88.1%) and friends (84.2%). Collectively, our study indicates consumers are more focused on their dog's ancestry than other test results. Using these findings and previous literature on human direct-to-consumer genetic testing, human-animal dyads, and identity construction, we consider the possibility of "breed options theory" and future areas of research.

Engineering death resistance in CHO cells for improved perfusion culture.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

'Low-normal' motor skills in infants at high risk for poor developmental outcomes: A prevalence and prognostic study.

AIM: To investigate the prevalence and prognostic value of 'low-normal' motor skills in infants at high-risk for poor developmental outcomes. METHOD: Infants born extremely low-birthweight and extremely preterm discharged from neonatal intensive care between 2015 and 2018 completed the Alberta Infant Motor Scale (AIMS), Neuro-Sensory Motor Developmental Assessment (NSMDA) at corrected age 4, 8, and 12 months, and Griffiths Mental Development Scale at corrected age 12 months. RESULTS: Participating infants (n = 191) with a mean gestational age (95% confidence interval [CI]) of 26.80 weeks (26.60, 27.1) and mean birthweight (95% CI) of 869 grams (843, 895) included 45 (23.80%) infants small for gestational age. AIMS rated 50.32%, 35.37%, and 14.86% of infants within the 'low-normal' motor skills range (1-2 SD below the mean for age) at 4, 8, and 12 months respectively. Of the infants within the AIMS 'low-normal' skills range, 55.70%, 88.46%, and 59.10% were classified as having impairment by NSMDA at 4, 8, and 12 months respectively. Griffiths assessment at 12 months identified only 7.33% of infants with 'low-normal' skills and 3.33% with motor disability. Minimal motor impairment rating on the NSMDA at 4 or 8 months significantly predicted general development at 12 months. INTERPRETATION: High-risk infants with 'low-normal' motor skills may warrant referral to early intervention as associated impairment represents increased risk for poorer general development outcomes. WHAT THIS PAPER ADDS: High prevalence of 'low-normal' motor skill exists in high-risk infants. Clinical motor assessment validly identifies infants with motor impairment. Minimal motor impairment in high-risk infants is prognostic of general development. High-risk infants with 'low-normal' motor skills may warrant early intervention. Griffiths Scales of Child Development, Third Edition assessment at 12-months age may under-identify motor difficulties.

Circulation and Molecular Characterization of Hemorrhagic Enteritis Virus in Commercial Turkey and Meat Chicken Flocks in Australia.

Currently, there is no available vaccine against hemorrhagic enteritis virus (HEV) in Australia. Although it is assumed that subclinical HEV infections occur and may be associated with an increase in colibacillosis in Australian commercial turkey flocks, the prevalence of infection with this virus in the country is largely unknown. The aims of this study were to determine the extent of HEV infection in commercial flocks in Australia and to investigate the diversity of Australian HEV strains. Serum and spleen samples were collected from breeder and grower turkeys and serum was collected from breeder and grower chickens by the two major poultry integrator companies in Australia. Of the turkey samples, 727/849 (86%) sera were positive for anti-HEV antibodies by ELISA. HEV DNA was detected in 215/278 (77%) spleen samples positive by PCR. Of the meat chicken sera, 115/144 (80%) samples were seropositive. Sequencing the whole genome of three HEV field isolates showed that the Australian strains are highly similar and cluster separately from strains from other geographic regions although several point mutations were shared with HEV strains considered to be virulent. In conclusion, HEV infection is ubiquitous in Australian commercial poultry flocks. The impact of the many genomic point mutations detected in Australian HEV strains on virus pathogenicity is unclear.

Circulación y caracterización molecular del virus de la enteritis hemorrágica en parvadas comerciales de pavo y pollos de engorde en Australia. Actualmente, no existe una vacuna disponible contra el virus de la enteritis hemorrágica (HEV) en Australia. Aunque se supone que se producen infecciones subclínicas por el virus de la enteritis hemorrágica y pueden estar asociadas con un aumento de la colibacilosis en las parvadas comerciales de pavos australianos, se desconoce en gran medida la prevalencia de la infección por este virus en el país. Los objetivos de este estudio fueron determinar la diseminación de la infección por el virus de la enteritis hemorrágica en parvadas comerciales en Australia e investigar la diversidad de cepas del virus de la enteritis hemorrágica australianas. Se recolectaron muestras de suero y bazo de pavos reproductores y de engorda y las dos principales empresas integradoras avícolas de Australia recolectaron suero de pollos reproductores y de engorde. De las muestras de pavo, 727/849 (86%) sueros fueron positivos para anticuerpos contra la enteritis hemorrágica por ELISA. Se detectó ADN del virus de la enteritis hemorrágica en 215/278 (77%) muestras de bazo positivas por PCR. De los sueros de carne de pollo, 115/144 (80%) muestras fueron seropositivas. La secuenciación del genoma completo de tres aislados de campo del virus de la enteritis hemorrágica mostró que las cepas australianas son muy similares y se agrupan por separado de las cepas de otras regiones geográficas, aunque se compartieron varias mutaciones puntuales con las cepas del virus de la enteritis hemorrágica consideradas virulentas. En conclusión, la infección por el virus de la enteritis hemorrágica es ubicua en las parvadas avícola comerciales australianas. No está claro el impacto de las diferentes mutaciones puntuales genómicas detectadas en las cepas australianas del virus de la enteritis hemorrágica sobre la patogenicidad del virus.

Modeling apoptosis resistance in CHO cells with CRISPR-mediated knockouts of Bak1, Bax, and Bok.

Chinese hamster ovary (CHO) cells are the primary platform for the production of biopharmaceuticals. To increase yields, many CHO cell lines have been genetically engineered to resist cell death. However, the kinetics that governs cell fate in bioreactors are confounded by many variables associated with batch processes. Here, we used CRISPR-Cas9 to create combinatorial knockouts of the three known BCL-2 family effector proteins: Bak1, Bax, and Bok. To assess the response to apoptotic stimuli, cell lines were cultured in the presence of four cytotoxic compounds with different mechanisms of action. A population-based model was developed to describe the behavior of the resulting viable cell dynamics as a function of genotype and treatment. Our results validated the synergistic antiapoptotic nature of Bak1 and Bax, while the deletion of Bok had no significant impact. Importantly, the uniform application of apoptotic stresses permitted direct observation and quantification of a delay in the onset of cell death through Bayesian inference of meaningful model parameters. In addition to the classical death rate, a delay function was found to be essential in the accurate modeling of the cell death response. These findings represent an important bridge between cell line engineering strategies and biological modeling in a bioprocess context.

Exploratory content analysis of direct-to-consumer pet genomics: What is being marketed and what are consumers saying?

Mars Petcare introduced the first direct-to-consumer domestic dog genetic test in 2009 and Basepaws introduced the first direct-to-consumer cat genetic test in 2016. Social science research has evaluated numerous aspects of the human direct-to-consumer market, yet no such exploration has evaluated the occurrence of pet owners pursuing pet genetic tests. Using a mixed methods approach, we conducted an exploratory content analysis of direct-to-consumer pet genetic company webpages and consumer reviews shared on Amazon. Initial data reviews indicated some companies may be key industry players, relative to others. Our results present content frequency for each group (key industry players, all other companies), though the primary themes for each remained the same. Analysis showed genetic companies are primarily sharing product and purchasing information, along with trustworthiness to establish the merit of the company and their products. Companies also used statements directed towards pet owners that are suggestive of both pets and "pet parents" benefiting from the test results. The primary themes identified in consumer reviews involved consumers sharing their perception about the tests (e.g., accuracy), what aspects of the test results they focused on (e.g., breed information), and experiences with using the test (e.g., ease of use). Amazon reviews were primarily positive, though the companies with smaller review numbers had higher percentages of negative and ambiguous sentiments. Of interest, reviews most often indicated tests were being used to determine a pet's breed identity, while companies most frequently promoted the health advantages of using their products. Reviews revealed some consumers respond to tests by sharing their pet's results with someone or by altering their pet's care. Considering these results in addition to the growing popularity of this industry and the advancements of genomic technology, further research is needed to determine the role pet genetic testing may have in society and on human-animal relationships.

Exploring women's oxytocin responses to interactions with their pet cats.

BACKGROUND: Extensive research has evaluated the involvement of the neuropeptide oxytocin (OT) in human social behaviors, including parent-infant relationships. Studies have investigated OT's connection to human attachment to nonhuman animals, with the majority of the literature focusing on domestic dogs (Canis lupis familiaris). Utilizing what is known about OT and its role in maternal-infant and human-dog bonding, we apply these frameworks to the study of human-domestic cat (Felis catus) interactions. METHODS: We investigated changes in salivary OT levels in 30 U.S. women of reproductive age before and after two conditions: reading a book (control) and interacting with their pet cat. Participant and cat behavioral patterns during the cat interaction condition were also quantified to determine if differences in women's OT concentrations were associated with specific human and cat behaviors. RESULTS: Our results revealed no changes in women's OT levels during the cat interaction, relative to the control condition, and pre-cat interaction OT levels. However, differences in women's OT concentrations were correlated with some human-cat interactions (e.g., positively with petting cat and cat approach initiation, negatively with cat agonistic behavior) but not all observed behaviors (e.g., use of gentle or baby voice) coded during human-cat interactions. DISCUSSION: This study is the first to explore women's OT in response to interactions with their pet cat and has identified distinct human and cat behaviors that influence OT release in humans.

Whence CRIPTO: The Reemergence of an Oncofetal Factor in 'Wounds' That Fail to Heal.

There exists a set of factors termed oncofetal proteins that play key roles in ontogeny before they decline or disappear as the organism's tissues achieve homeostasis, only to then re-emerge in cancer. Although the unique therapeutic potential presented by such factors has been recognized for more than a century, their clinical utility has yet to be fully realized1. This review highlights the small signaling protein CRIPTO encoded by the tumor derived growth factor 1 (TDGF1/Tdgf1) gene, an oft cited oncofetal protein whose presence in the cancer literature as a tumor promoter, diagnostic marker and viable therapeutic target continues to grow. We touch lightly on features well established and well-reviewed since its discovery more than 30 years ago, including CRIPTO's early developmental roles and modulation of SMAD2/3 activation by a selected set of transforming growth factor ß (TGF-ß) family ligands. We predominantly focus instead on more recent and less well understood additions to the CRIPTO signaling repertoire, on its potential upstream regulators and on new conceptual ground for understanding its mode of action in the multicellular and often stressful contexts of neoplastic transformation and progression. We ask whence it re-emerges in cancer and where it 'hides' between the time of its fetal activity and its oncogenic reemergence. In this regard, we examine CRIPTO's restriction to rare cells in the adult, its potential for paracrine crosstalk, and its emerging role in inflammation and tissue regeneration-roles it may reprise in tumorigenesis, acting on subsets of tumor cells to foster cancer initiation and progression. We also consider critical gaps in knowledge and resources that stand between the recent, exciting momentum in the CRIPTO field and highly actionable CRIPTO manipulation for cancer therapy and beyond.

Patient-derived xenografts and organoids model therapy response in prostate cancer.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.

'Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells.

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. 'Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis-related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.

CRIPTO antagonist ALK4L75A-Fc inhibits breast cancer cell plasticity and adaptation to stress.

BACKGROUND: CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. METHODS: We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. RESULTS: ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. CONCLUSIONS: Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Salivary aldosterone and cortisone respond differently to high- and low-psychologically stressful soccer competitions.

Aldosterone and cortisone are released in response to physical and psychological stress. However, aldosterone and cortisone responses in children engaged in physical competition have not been described. We examined salivary aldosterone and salivary cortisone responses among Hong Kongese boys, aged 8-11 years, during (1) a soccer match against unknown competitors (N = 84, high psychological stress condition) and (2) an intrasquad soccer scrimmage against teammates (N = 81, low psychological stress condition). Aldosterone levels increased during the soccer match and intrasquad soccer scrimmage conditions, consistent with the view that aldosterone responds to physical stress. During the soccer match, winning competitors experienced larger increases in aldosterone compared to losing competitors, indicating that the degree of aldosterone increase was attenuated by match outcome. Cortisone increased during the soccer match and decreased during the intrasquad soccer scrimmage. Competitors on teams that resulted in a tie had larger cortisone increases compared to winners or losers. These findings highlight that the degree of cortisone change is related to boy's cognitive appraisal of the competitor type (i.e., teammates vs. unknown competitors) and the competitive nature of the game (e.g., tie). These results shed new light on adrenal hormone mediators of stress and competition during middle childhood.

A Multidisciplinary Review of the Roles of Cripto in the Scientific Literature Through a Bibliometric Analysis of its Biological Roles.

Cripto is a small glycosylphosphatidylinisitol (GPI)-anchored and secreted oncofetal protein that plays important roles in regulating normal physiological processes, including stem cell differentiation, embryonal development, and tissue growth and remodeling, as well as pathological processes such as tumor initiation and progression. Cripto functions as a co-receptor for TGF-ß ligands such as Nodal, GDF1, and GDF3. Soluble and secreted forms of Cripto also exhibit growth factor-like activity and activate SRC/MAPK/PI3K/AKT pathways. Glucose-Regulated Protein 78 kDa (GRP78) binds Cripto at the cell surface and has been shown to be required for Cripto signaling via both TGF-ß and SRC/MAPK/PI3K/AKT pathways. To provide a comprehensive overview of the scientific literature related to Cripto, we performed, for the first time, a bibliometric analysis of the biological roles of Cripto as reported in the scientific literature covering the last 10 years. We present different fields of knowledge in comprehensive areas of research on Cripto, ranging from basic to translational research, using a keyword-driven approach. Our ultimate aim is to aid the scientific community in conducting targeted research by identifying areas where research has been conducted so far and, perhaps more importantly, where critical knowledge is still missing.