Parental Strain Research Articles

Prevalence of the Potato virus Y strain composition impacting Michigan seed potato production

Potato virus Y (PVY) is a complex of multiple strains that cause a variety of foliar and tuber symptoms. Several current strains have potential to result in variety-dependent yield and quality losses. Understanding the local PVY strain population and variety responses are critical for detection and management. From 2019-2023, PVY strain composition was monitored across seven Michigan seed potato-producing counties growing prevalent chip, russet, and tablestock market classes. Using multiplex reverse-transcriptase polymerase chain reaction methods, PVY strains were identified from potato tuber or leaf samples collected through the certification program (N = 78 positive lots). PVYN-Wi remained most prevalent in Michigan, however, tuber necrotic strains PVYNTN and PVYE also continued to be detected. These, as well as some PVYN-Wi isolates, are known to cause necrotic symptoms in tubers, causing yield and quality reductions for susceptible potato cultivars. As in other U.S. potato-growing regions, PVYN-Wi and PVYNTN were most prevalent in Michigan seed potato lots. Of note, mild to severe tuber necrosis was observed in diagnostic samples positive with local PVYN-Wi strains. Parental strain PVYO appeared to re-emerge in 2023 and should be considered in certification and management. Mild or asymptomatic reactions may still lead to yield or quality losses and may pose a risk to more susceptible cultivars. Due to the diversity of strains and varieties prevalent in the Great Lakes region, continued uniform certification is recommended. Information gathered from this and future survey efforts will be valuable for growers, inspectors, and future breeding and selection efforts.

8038 Targeting Breast Cancer Stem Cells With Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

8038 Targeting Breast Cancer Stem Cells with Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

Online Parent-mediated Home-based Intervention for Children with Autism During COVID-19 Pandemic, Caregiver’s Experience

Background and Aims: During the COVID-19 pandemic, many caregivers of children with autism faced significant challenges when it came to providing interventions. Studies done so far have proven the efficacy of parent-mediated interventions in children with ASD. COVID pandemic forced to use online platforms for training parents in various parent-mediated interventions. Therefore, we conducted a study on the utility of online parent-mediated home-based intervention for children with autism. Methodology: This is a descriptive qualitative study design. Ten parents of children with autism were included in a series of online interactive meetings addressed by the disability experts. Sensory processing measures (SPM) and sensory profile questionnaires (SP) were used to prepare individualised home programmes. Ongoing support for implementing programme was provided via teleconsultation. Feedback was obtained 6 months after implementing parent-mediated interventions. The perceived utility of was analysed using qualitative data analysis. Results: Nearly all parents reported improvement in skills in language and play areas. 88% parents could administer the programme confidently. The overall teaching format was well-perceived by all parents. The home programme could be easily administered at home as reported by 85% of the parents. In the open-ended question, parents requested a need for more follow-up teleconsultation appointments. Conclusions: The COVID-19 pandemic necessitated significant adjustments for caregivers in providing interventions for children with autism. While it posed challenges, it also spurred innovation in parent-mediated interventions and highlighted the resilience and adaptability of caregivers in supporting their children during uncertain times.

Efficacy of behavioural parent training on attachment security in children with attention deficit hyperactivity disorder: a randomised controlled trial

BackgroundBehavioural parent training (BPT) is a psychosocial intervention designed for children with attention deficit hyperactivity disorder (ADHD). BPT programs teach parents to use effective commands or rules whilst encouraging them to pay careful attention to their child’s appropriate behaviour. In this study, we will investigate the efficacy of BPT on parental stress, mothers’ sense of emotional closeness to their children, and children’s attachment security to their mothers. We will also examine the effects of BPT on children’s internalising and externalising symptoms, ADHD symptoms, and sensitivity to rewards and punishments compared to usual care alone. The use of bias-prone assessment tools limits the ability of previous studies to assess effectiveness. Therefore, in this study, the child’s attachment security will be assessed in a structured interview conducted by assessors blinded to group allocation, and brain changes will be assessed using magnetic resonance imaging.MethodsThis randomised controlled clinical trial will aim to compare the efficacy of BPT to routine clinical care for 60 children with ADHD. Participants will be randomised, with stratification by medication status for ADHD (medicated or non-medicated). The BPT intervention group will receive parent training weekly for 10 weeks in a group of six or less. The primary outcome measure will be changes in parental stress. Furthermore, the key secondary outcome measure will be the child’s attachment security, which will be assessed in an interview conducted by assessors blinded to group allocation. We will also evaluate changes in neural connectivity in both children and mothers using magnetic resonance imaging. Other secondary outcomes will include child behavioural problems, ADHD symptoms, emotional regulation, child sensitivity to rewards and punishments, parental behaviour, and the child and parent’s social support network following the completion of 10 sessions.DiscussionThis study represents the first randomised controlled trial exploring the efficacy of BPT on child attachment security and mothers’ sense of emotional closeness to their children. It aims to provide robust evidence to assist parents of children with ADHD in making appropriate treatment decisions.Trial registrationUMIN000038693. Registered on November 9, 2019.

Functional and Proteomic Dissection of the Contributions of CodY, SigB and the Hibernation Promoting Factor HPF to Interactions of Staphylococcus aureus USA300 with Human Lung Epithelial Cells.

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

Research on Engineering the Saccharomyces uvarum for Constructing a High Efficiency to Degrade Malic Acid and Low Yield of Diacetyl Biosynthesis Pathway.

Diacetyl is a flavor compound in wine with a low threshold (1-2 mg/L). It is produced during alcoholic fermentation (AF) Saccharomyces and malolactic fermentation (MLF) initiated by lactic acid bacteria (LAB). Whereas, the environment after AF suppresses the normal metabolism of LAB after AF. Researchs have shown the influence on diacetyl mechanisms of the genes ILV2, ILV6, ILV3, ILV5, BDH1, BDH2, and gene aldB from Lactobacillus plantarum in Saccharomyce uvarum WY1. While we found that the diacetyl contents produced by mutants after MLF (Co-fermentation and Seq-fermentation) were significantly improved compared to AF alone. Moreover, the genes mae1 and mae2 from S. pombe, and gene mleS from L. lactis exhibited significant effect on deacidification in our previous study, but the diacetyl of the mutants showed obvious improvement in this study. Thus the effects of association mutation of genes (ILV2, ILV6, ILV3, ILV5, BDH1, BDH2, aldB, mae1, and mleS) on deacidification, diacetyl and other flavors (organic acids, higher alcohols and esters) metabolism in S. uvarum after AF were detected in the study. Among all the mutants, strains V6AmS, V635mS, and V6B12mS showed the most favorable results. Specifically, the L-malic acid contents decreased to 1.26 g/L, 1.18 g/L, and 1.19 g/L, respectively. Concurrently, diacetyl levels were reduced by 52.56%, 61.84%, and 65.31%. The production of n-propanol increased by 18.84%, 20.89%, and 28.12%, whereas isobutanol levels decreased by 37.01%, 42.36%, and 44.04%, and isoamyl alcohol levels decreased by 19.28%, 19.79%, and 16.74%, compared to the parental strain WY1. Additionally, the concentration of lactate ester in the wine increased to 13.162 mg/L, 14.729 mg/L, and 14.236 mg/L, respectively.

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for all bacteria; however, elevated intracellular free Zn levels can result in mis-metalation of Mn-dependent enzymes. For Mn-centric bacteria such as Streptococcus pneumoniae that primarily use Mn instead of Fe as an enzyme cofactor, Zn is particularly toxic at high concentrations. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae.

Mechanism of enhanced salt tolerance in Saccharomyces cerevisiae by CRZ1 overexpression

Achieving high-gravity fermentation in the industrial production of fuel ethanol, and enhancing the fermentation efficiency of high-salt raw materials, such as waste molasses, can significantly reduce wastewater output and process costs. Therefore, the development of hyperosmotic-tolerant industrial Saccharomyces cerevisiae strains, capable of resisting high-salt stress, offers both environmental and economic benefits. Our previous study highlighted the potential of CRZ1 overexpression as a strategy to improve the yeast strain’s resistance to high-salt stress, however, the underlying molecular mechanisms remain unexplored. The fermentation capabilities of the CRZ1-overexpressing strain, KCR3, and its parental strain, KF7, were evaluated under condition of 1.25 M NaCl at 35 °C. Compared to KF7, KCR3 showed an 81% increase in glucose consumption (129.25 ± 0.83 g/L) and a 105% increase in ethanol production (47.59 ± 0.93 g/L), with a yield of 0.37 g/g. Comparative transcriptomic analysis showed that under high-salt stress, KCR3 exhibited significantly upregulated expression of genes associated with ion transport, stress response, gluconeogenesis, and the utilization of alternative carbon sources, while genes related to glycolysis and the biosynthesis of ribosomes, amino acids, and fatty acids were notably downregulated compared to KF7. Crz1 likely expands its influence by regulating the expression of numerous transcription factors, thereby impacting genes involved in multiple aspects of cellular function. The study revealed the regulatory mechanism of Crz1 under high-salt stress, thereby providing guidance for the construction of salt-tolerant strains.

The Efficacy of Technology-enhanced Behavioral Parent Training for Families with Low-income: Do Parent-centered Profiles Moderate Treatment Outcomes?

Behavior Disorders (BD) in children are common and have long-term impacts. Financially disadvantaged families are at a greater risk of having a child with an early-onset BD, but have more difficulty engaging in and therefore benefiting from Behavior Parent Training (BPT). Building upon the potential of technology-enhanced (TE) treatment approaches in addressing barriers to treatment engagement and effectiveness, TEs to one BPT program, Helping the Noncompliant Child (HNC), were tested in a randomized controlled trial (RCT) with 101 families with low income. The current study aimed to examine for whom TE-HNC vs. standard HNC is optimal at posttreatment by exploring how pretreatment, parent-centered profiles may moderate parenting and child outcomes at posttreatment. Latent profile analyses yielded four distinct parent-centered profiles. The profile membership differentially predicted treatment outcomes by group for positive and negative parenting, as well as child behavior intensity, but not child problem behavior. The heterogeneity of responses to standard relative to TE treatment models among underserved families may provide clues regarding the future personalization of BPT toward improved treatment efficacy.

Predicting the first steps of evolution in randomly assembled communities

Microbial communities can self-assemble into highly diverse states with predictable statistical properties. However, these initial states can be disrupted by rapid evolution of the resident strains. When a new mutation arises, it competes for resources with its parent strain and with the other species in the community. This interplay between ecology and evolution is difficult to capture with existing community assembly theory. Here, we introduce a mathematical framework for predicting the first steps of evolution in large randomly assembled communities that compete for substitutable resources. We show how the fitness effects of new mutations and the probability that they coexist with their parent depends on the size of the community, the saturation of its niches, and the metabolic overlap between its members. We find that successful mutations are often able to coexist with their parent strains, even in saturated communities with low niche availability. At the same time, these invading mutants often cause extinctions of metabolically distant species. Our results suggest that even small amounts of evolution can produce distinct genetic signatures in natural microbial communities.

Provider Perspectives: Feasibility, Acceptability, and Appropriateness of Online Parent Training With Therapist Coaching in a Community Practice Setting

A considerable body of evidence documents the detrimental impact of disruptive behavior disorders (DBDs) in childhood and their longstanding effects across development and into adulthood. While research has yielded evidence-based interventions for DBDs, such as parent management training, disparities in access to care persist. The growing ubiquity of consumer technologies underscores the potential to provide evidence-based treatments through online programs; however, structural barriers continue to impede dissemination in community settings. This paper examines clinic staff’s perspectives on the implementation of an online parent management program in a community mental health center (CMHC) to integrate novel programming with existing therapeutic care to maximize resource efficiency in treatment delivery and increase family adherence and engagement. Quantitative and qualitative data were collected from 16 staff members who were trained to deliver the intervention at a CMHC. Quantitative data indicated positive perceptions across various domains, while qualitative findings highlighted feasibility, acceptability, and appropriateness. Facilitators to feasibility included program ease, integration into sessions, and flexible access. Time constraints and technology access were barriers. Acceptability was high, with videos reinforcing therapy and promoting family connection. The program’s structure and psychoeducation were appreciated, while quiz format and time constraints were concerns. Appropriateness was positive, with broad relevance, insight-building impact, comprehensive coverage, evidence-based strategies, and support. Barriers existed for specific patient characteristics. Implications for implementation in practice and future directions of research are discussed.

Enhancement of mono-acylated MEL-D production in an acyltransferase gene-deleted strain of Pseudozyma tsukubaensis by supplementation with di-acylated MEL-B in culture medium

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. MEL producers produce mainly di-acylated MELs (consisting of two fatty acid chains). Among them, Pseudozyma tsukubaensis is a di-acylated MEL-B (d-MEL-B) producer. In a previous study, we generated an acyltransferase-deleted strain of P. tsukubaensis (ΔPtMAC2), which selectively produced mono-acylated MEL-D (m-MEL-D, consisting of one fatty acid chain), but not d-MEL-B. However, m-MEL-D productivity in ΔPtMAC2 was low, and oil consumption was significantly reduced compared to the parent strain. Based on these findings, we hypothesized that the d-MEL-B produced by the parent strain may act as an emulsifier in the culture medium, leading to easier utilization of the oil. By contrast, the m-MEL-D produced by ΔPtMAC2 may not have the ability to emulsify oil, thus the oil is used inefficiently and productivity of m-MEL-D is low. Therefore, we expected that adding d-MEL-B to the culture medium during ΔPtMAC2 cultivation would increase m-MEL-D production. To enhance the oil consumption and m-MEL-D production of ΔPtMAC2, d-MEL-B and chemical surfactants were added to the culture medium as emulsifiers during ΔPtMAC2 cultivation. Adding d-MEL-B enhanced both the oil consumption and m-MEL-D production of ΔPtMAC2; Tween 20 and Triton X-100 also showed enhancement effects. As expected, d-MEL-B, Tween20 and TritonX-100, showed marked olive oil emulsification activity, whereas m-MEL-D did not. These results strongly support our hypothesis and significantly improve m-MEL-D productivity.

Retrospective investigation into Nigerian response to COVID-19 education crisis as compared to other developed countries

The educational system has been severely impacted by the abrupt closure of schools in numerous nations during the COVID-19 pandemic in an effort to contain the virus. Nonetheless, there is insufficient information available about Nigeria’s response to the education issue and the factors influencing it in comparison to other countries. In light of this, this study examines Nigeria’s response to the COVID-19 educational crisis in comparison to other countries. The research method used was a systematic literature review. Only 40 of the 753 publications that were first found using internet databases like Web of Science, ERIC, Scopus, and Google Scholar satisfied the requirements for inclusion in the research. It was found that concerns with the availability, awareness, accessibility, and utilization of e-learning facilities posed serious obstacles for Nigeria and other developing nations trying to continue remote learning during the COVID-19 pandemic. The differences in the utilization of e-learning during the pandemic were made worse by the digital divide that exists between low- and high-income, rural and urban populations. In order for remote education to be effective, parents, students, and teachers must get system adaptation training. In light of global disruptions, this research offers fresh empirical insights into the critical elements impacting the use of ICTs for teaching and learning. It was recommended that the school sector receive increased funding in order to provide a technical support team, train parents and teachers, and purchase the required equipment.

Abf1 negatively regulates the expression of EPA1 and affects adhesion in Candida glabrata.

Introduction. Adherence is a major virulence trait in Candida glabrata that, in many strains, depends on the EPA (epithelial adhesin) genes, which confer the ability to adhere to epithelial and endothelial cells of the host. The EPA genes are generally found at subtelomeric regions, which makes them subject to subtelomeric silencing. In C. glabrata, subtelomeric silencing depends on different protein complexes, such as silent information regulator and yKu complexes, and other proteins, such as Repressor/activator protein 1 (Rap1) and Abf1. At the EPA1 locus, which encodes the main adhesin Epa1, we previously found at least two cis-acting elements, the protosilencer Sil2126 and the negative element, that contribute to the propagation of silencing from the telomere to the subtelomeric region.Hypothesis. Abf1 binds to the regulatory regions of EPA1 and other regions at the telomere E-R, thereby negatively regulating EPA1 transcription.Aim. To determine whether Abf1 and Rap1 silencing proteins bind to previously identified cis-acting elements on the right telomere of chromosome E (E-R subtelomeric region), resulting in negative regulation of EPA1 transcription and infer Abf1 and Rap1 recognition sites in C. glabrata.Methodology. We used chromatin immunoprecipitation (ChIP) followed by quantitative PCR to determine the binding sites for Abf1 and Rap1 in the intergenic regions between EPA1 and EPA2 and HYR1 and EPA1, and mutants were used to determine the silencing level of the EPA1 promoter region.Results. We found that Abf1 predominantly binds to the EPA1 promoter region, leading to negative regulation of EPA1 expression. Furthermore, the mutant abf1-43, which lacks the last 43 amino acids at its C-terminal end and is defective for subtelomeric silencing, exhibits hyperadherence to epithelial cells in vitro compared to the parental strain, suggesting that EPA1 is derepressed. We also determined the motif-binding sequences for Abf1 and Rap1 in C. glabrata using data from the ChIP assays.Conclusion. Together these data indicate that Abf1 negatively regulates EPA1 expression, leading to decreased adhesion of C. glabrata to epithelial cells.

Are there unique facets of therapeutic alliance for users of digital mental health interventions? An examination with the eHealth Therapeutic Alliance Inventory

Therapeutic alliance (TA) is a well-established predictor of clinical outcomes in traditional psychotherapy. However, its association with outcomes in eHealth interventions has been inconsistent, which might be due to the absence of measurements specifically designed to capture TA in eHealth settings. The eHealth Therapeutic Alliance Inventory (ETAI) incorporates conventional as well as unique eHealth TA subscales, enabling to examine the contribution of new facets of TA beyond traditional concepts. This study investigates the predictive contribution of eHealth TA subscales compared to conventional TA subscales on clinical outcomes and evaluates the concurrent criterion validity of the ETAI. The study was conducted within the framework of a randomized controlled trial involving a 10-week digital parent training program aimed at addressing child disruptive behaviors. Parents were randomly assigned to either an enhanced-quality or a standard-quality program. Parents from 68 families completed the ETAI at five weeks' post-program initiation and at the post-intervention phase. The primary outcome was the improvement in child behavior, measured by the Eyberg Child Behavior Inventory. Positive Pearson correlations were found between all ETAI subscales covering unique eHealth TA aspects, measured at the 5-week time-point, and improvement in child behavior at post-intervention (rs ≥ 0.23, ps < 0.03). The conventional TA subscale showed no significant Pearson correlation with improvement in child behavior. When examining the unique contributions of ETAI-subscales to explain the improvement in child behavior, only ETAI-Perceived Emotional Investment subscale was found to have a unique contribution (β = 0.29, p = 0.019). In addition, scores on most ETAI subscales were significantly higher among parents using the enhanced-quality program compared with the standard program (Cohen's ds > 0.48), reinforcing ETAI's criterion validity. The development of TA scales that incorporates unique eHealth TA subscales show initial promise in predicting outcomes. Further research is needed to better understand how different factors of eHealth TA relate to clinical outcomes across diverse clinical targets and programs.

Satisfaction and Effectiveness of Telepractice for Parents of Young Children with Language Delay

Objectives: This study aims to provide parent teaching techniques to mothers of 2-3-yearold children with language developmental delays via telepractice and to analyze the effectiveness and participant satisfaction. The training focuses on techniques to promote language development in their children.Methods: The study involved training and coaching seven mothers in parallel talk, self-talk, mand-model and time delay techniques via telepractice. The interactions between mothers and children were recorded at home and analyzed to observe changes in mothers’ use of the techniques and the characteristics of children’s speech. After completing the training-coaching procedure, a satisfaction survey was conducted among the participants.Results: Most participants showed improvement in the production rate per utterance and the total frequency of the techniques after the telelpractice for parent training and coaching. The children also exhibited more diverse types of utterances and increased production rates and frequencies following their mothers’ improvements. The satisfaction with remote parent training was reported to be high.Conclusion: The results of this study suggest that for parent intervention, training and coaching via telepractice can be an effective alternative for educating parents of young children with language developmental delays, enabling them to engage in interactions that promote their children’s language development.

Rhesus rotavirus NSP1 mediates extra-intestinal infection and is a contributing factor for biliary obstruction.

We previously demonstrated that in Ifnar1-/-Ifngr1-/- or Stat1-/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues.

Generation and Genetic Stability of a PolX and 5′ MGF-Deficient African Swine Fever Virus Mutant for Vaccine Development

The African swine fever virus (ASFV) causes fatal disease in pigs and is currently spreading globally. Commercially safe vaccines are urgently required. Aiming to generate a novel live attenuated vaccine (LAV), a recombinant ASFV was generated by deleting the viral O174L (PolX) gene. However, during in vitro generation, an additional spontaneous deletion of genes belonging to the multigene families (MGF) occurred, creating a mixture of two viruses, namely, Arm-ΔPolX and Arm-ΔPolX-ΔMGF. This mixture was used to inoculate pigs in a low and high dose to assess the viral dynamics of both populations in vivo. Although the Arm-ΔPolX population was a much lower proportion of the inoculum, in the high-dose immunized animals, it was the only resulting viral population, while Arm-ΔPolX-ΔMGF only appeared in low-dose immunized animals, revealing the role of deleted MGFs in ASFV fitness in vivo. Furthermore, animals in the low-dose group survived inoculation, whereas animals in the high-dose group died, suggesting that the lack of MGF and PolX genes, and not the PolX gene alone, led to attenuation. The two recombinant viruses were individually isolated and inoculated into piglets, confirming this hypothesis. However, immunization with the Arm-ΔPolX-ΔMGF virus did not induce protection against challenge with the virulent parental ASFV strain. This study demonstrates that deletion of the PolX gene alone neither leads to attenuation nor induces an increased mutation rate in vivo.