In this study, the Marbled Crayfish poses a growing ecological threat due to its rapid reproduction rate. Genetic evidence tied to the Slough Crayfish (Procambarus fallax) confirms a spontaneous mutation arose in a single individual resulting in the new species emerging–the Marbled Crayfish (Procambarus virginalis). The unusual genetic malfunction transformed the original fertilization-based reproductive habits to an entirely asexual mode of reproduction known as parthenogenesis. Despite the Marbled Crayfish being exact female clones of one another, their success as a highly invasive species skyrocketed as they established populations within North America, Europe, Asia, and Africa. Their rapid spread and competition with native species threaten local ecosystems. As a proposition, to control their population, Madagascar is harvesting them as food, as they are a great source of protein and contain a valuable amount of chitin. A study conducted between October 2019 to September 2020 involved hand-collecting 768 Marbled Crayfish, housing them in separate tanks, and feeding them experimental diets. This was done to determine which control feed resulted in the highest weight gain by monitoring their length, weight, weight gain per tank, survival, and overall growth performance. Findings from the study indicate that Marbled crayfish meat is slowly going to be incorporated into European meals because of their suitability for certain dishes and appetizers. This research aims to increase the public and industry awareness of marbled crayfish as a sustainable food resource in hopes to help stabilize the invasive population.

Mouse models are staples of biological research, particularly in immunology and gut microbiota studies, where experimental outcomes may be influenced by stress. However, growing evidence suggests that husbandry choices and protocol design can factor into stress levels in laboratory mouse models. Factors such as housing conditions, enrichment, handling methods, mouse strain and substrain, diet, and bedding can alter experimental markers such as stress hormone levels and gut microbial diversity. These changes may influence experimental findings and contribute to variability between studies and research facilities. Animal husbandry choices and their impact on study results and reproducibility may not be sufficiently considered when designing studies, potentially leaving room for them to contribute unexpected or unwanted influence.

This review examines original research investigating the effects of husbandry and procedural variables on physiological stress markers and microbiome composition in mice. We evaluate commonly used husbandry practices and assess their reported effects on stress-sensitive outcomes, including endocrine, immune, and microbial measures. We also compare methodological differences across studies to identify potential sources of variability that may influence reproducibility.

We hypothesize that variation in husbandry and protocol design represents a significant but underrecognized source of experimental variability in stress-sensitive mouse studies. Greater awareness and improved standardization of these factors may strengthen reproducibility, improve translational relevance, and support the principles of the 3Rs by reducing unnecessary animal use. We recommend the use of reporting and planning frameworks such as ARRIVE and PREPARE to improve consistency and transparency in animal research.

Memory CD8 T cells are critical for long-lasting immunity, vaccine efficacy, and immunotherapies, but the factors controlling their differentiation are incompletely understood. Death-association protein-like 1 (Dapl1) is heterogeneously expressed in naive T cells and cytotoxic T lymphocytes (CTLs). Dapl1+ CTLs are biased to become memory cells, revealing a novel pathway for memory T cell generation. Studying molecular mechanisms which regulate development of Dapl1+ cells could elucidate processes behind memory differentiation. 

Type 1 interferons and IL-12 are cytokines that support differentiation of short-lived effector cells by augmenting Tbet expression in CD8 T cells. Since they promote the effector phenotype rather than memory, we hypothesize that cytokine-mediated Tbet expression during T cell activation decreases the frequency of Dapl1+ cells, therefore limiting memory differentiation. 

To test this hypothesis, CD8 T cells were stimulated with plate-bound antibodies or irradiated splenocytes in the presence of IFNb or IL-12. Frequency of Dapl1+ cells and phenotypes of day 5 CTLs were analyzed by flow cytometry using a Dapl1-reporter mouse model and in-house-made antibodies. As predicted, both cytokines markedly decreased the frequency of Dapl1+ CTLs. Using CD62L as a marker for a memory-like phenotype, we observed that the frequency of CD62L+ cells also decreased. Unexpectedly, Tbet overexpression in CD8 T cells did not substantially decrease Dapl1+ cells, and had no impact on memory-like T cells. Therefore, exposure to IFNb and IL-12 during activation impairs memory formation by suppressing the differentiation of Dapl1+ cells in a Tbet-independent manner. These results can inform future vaccine development against viral infections.

Gamma Delta (γδ) T cells are critical mediators of immunity against numerous zoonotic pathogens for which effective vaccines remain limited. In ruminants, γδ T cells constitute a major population of circulating and tissue-resident lymphocytes and coordinate downstream immune responses upon activation. WC1, a multigenic receptor array expressed on ruminant γδ T cells, functions as a hybrid pattern recognition receptor (PRR) and T cell co-receptor. In cattle, thirteen WC1 genes (WC1-1 to WC1-13) encode proteins containing 6–11 scavenger receptor cysteine-rich (SRCR) domains capable of binding unprocessed antigen. We hypothesized that diversification within the WC1 multigenic array reflects pathogen-driven co-evolution and sought to characterize SRCR domain binding specificity toward Mycobacterium spp. and Leptospira spp.

SRCR-encoding sequences from bovine WC1 genes were cloned into a mammalian expression vector and transiently expressed in suspension HEK293F cells. Recombinant SRCR proteins were purified and quantified prior to functional analysis. Binding interactions were assessed using ELISA and whole-cell bacterial pull-down assays with fractionated or fixed Mycobacterium spp. and Leptospira spp.

Distinct SRCR domains demonstrated pathogen-specific binding profiles. Multiple btWC1-12 domains exhibited high-affinity binding to Mycobacterium spp., whereas btWC1-3 preferentially bound Leptospira spp. Protease K treatment of Mycobacterium bovis BCG Danish and Pasteur strains did not reduce btWC1-12 binding, suggesting that the ligand recognized by these SRCR domains is likely non-proteinaceous. These findings support selective pathogen recognition by WC1 SRCR domains and reinforce their role as hybrid co-receptors and PRRs in γδ T cell-mediated immunity.

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RELATED ABSTRACTS

• Investigating the Nitric Oxide Production of Alveolar Macrophages at the Levels of Gene Expression, Protein Production, and Reactive Nitrogen Intermediates, Phan, Mai Ha, UMass Amherst, Poster Session 6, 4:15 PM - 5:00 PM, 165, D2
• Investigating the Role of RXLR Effectors in the Basil Downy Mildew Pathogen Peronospora belbahrii, Moinzadeh, Kiyan Michael, UMass Amherst, Poster Session 6, 4:15 PM - 5:00 PM, 163, C8

Sugt1 (also known as Sgt1) is an essential protein-coding gene that participates in multiple fundamental cellular processes, including ubiquitination, proteasomal degradation, kinetochore assembly, centrosome maturation, and protein homeostasis through its role as a co-chaperone of Hsp90. Conventional Sugt1 knockout results in early embryonic lethality in mice, making tissue-specific functions of this gene difficult to study. To investigate the role of Sugt1 during oocyte development, we established an oocyte-specific Sugt1 conditional knockout (cKO) model using the Cre/LoxP system driven by Gdf9-Cre to restrict deletion of Sugt1 to oocytes.

Oocytes are central to female reproduction and undergo a tightly regulated maturation process that is essential for generating developmentally competent eggs. Preliminary findings indicate that Sugt1 cKO females are functionally infertile under our experimental conditions. While no overt defects were observed in germinal vesicle (GV) oocytes with respect to oocyte number or gross morphology, metaphase II (MII) oocytes collected from the oviducts exhibited markedly reduced numbers, decreased survival rates, and pronounced morphological abnormalities. cKO oocytes frequently lacked a first polar body and showed abnormal microtubule organization and chromosome clustering, suggesting defects in meiotic progression.

Immunofluorescence analyses using markers for microtubules, apoptosis, DNA damage, and chromatin revealed clear structural differences between control and cKO oocytes. Based on these observations, we hypothesize that loss of Sugt1 in oocytes disrupts spindle and cytoskeletal organization, chromatin integrity, and chromosome separation required for successful oocyte maturation. This study aims to comprehensively phenotype MII oocyte abnormalities caused by Sugt1 deletion and to clarify the role of Sugt1 in mammalian oocyte development.

RELATED ABSTRACTS

• RBBP4 in Early Mammalian Oogenesis: Implications for DNA Damage Repair, Rivard, Vienna, UMass Amherst, Poster Session 5, 3:15 PM - 4:00 PM, 163, C24
• Investigating Skewed Floral Morph Ratios in Mitchella repens Through the Assessment of Germination Rates and Seedling Viability, Arciniega, Daisy R., Worcester State University, Poster Session 3, 1:15 PM - 2:00 PM, Auditorium, A53
• Seasonal Variations in Reproductive Hormones: An Analysis of the EAGeR and BioCycle Studies, Manchikanti, Nidhi, UMass Amherst, Poster Session 5, 3:15 PM - 4:00 PM, Concourse, B7
• Optimizing Early Embryo Culture Toward ART/IVF Efficiency, Bell, Emilie, UMass Amherst, Poster Session 4, 2:15 PM - 3:00 PM, Auditorium, A86

What role does the histone-binding protein RBBP4 serve in repairing DNA damage during mammalian oogenesis? This question can be answered by looking at the female oocyte. The female oocyte is unique in its lifespan and ability to preserve DNA integrity over decades. By investigating how it protects against DNA damage caused by stress and inheritance pressures, the understanding of female reproductive biology can be expanded.

To investigate the role of RBBP4, this research used an oocyte-specific conditional-knockout (cKO) mouse model. The desired genotypes of control and cKO were determined via ear tissue lysis and PCR. Oocytes were collected, cultured, and treated with etoposide (0 𝜇𝑀 and 10 𝜇𝑀 for 3 hours), a chemotherapy agent that induces DNA double-stranded breaks. After overnight recovery, the oocytes were washed and stained with DNA damage markers (DAPI, TUNEL, P-53, and 𝛾-H2AX) before being imaged with fluorescence microscopy.

Results showed that the cKO group exhibited greater DNA damage than the control group after etoposide exposure and during the recovery period. These findings suggest that RBBP4 is involved in the DNA damage pathway of early oocytes. Broader implications for these knowledge advancements of the oocyte role in long-term genomic maintenance are important within the context of women’s health. By better understanding how the genome ages, scientists and clinicians can better understand the implications for fertility, pregnancy outcomes, reproductive aging, and how women’s reproductive systems respond to various stressors.

RELATED ABSTRACTS

• Functional Analysis of SUGT1 During Mouse Oocyte Development, Lee, Hsin, UMass Amherst, Poster Session 5, 3:15 PM - 4:00 PM, 163, C23
• Investigating Skewed Floral Morph Ratios in Mitchella repens Through the Assessment of Germination Rates and Seedling Viability, Arciniega, Daisy R., Worcester State University, Poster Session 3, 1:15 PM - 2:00 PM, Auditorium, A53
• Seasonal Variations in Reproductive Hormones: An Analysis of the EAGeR and BioCycle Studies, Manchikanti, Nidhi, UMass Amherst, Poster Session 5, 3:15 PM - 4:00 PM, Concourse, B7
• Optimizing Early Embryo Culture Toward ART/IVF Efficiency, Bell, Emilie, UMass Amherst, Poster Session 4, 2:15 PM - 3:00 PM, Auditorium, A86

In this study, we investigate the outcomes of the Dbf4 gene knockout on female mice infertility. Dbf4, also known as Dbf4p, was initially identified in Saccharomyces cerevisiae. DBF4 was recognized as a regulatory subunit of the Cdc7p-Dbf4p kinase complex (DDK), which is required for Cdc7 kinase activity and initiation of DNA replication by phosphorylating Mcm2-7 proteins during the cell cycle. After activation with DBF4 binding, DDK phosphorylates the MCM2-7 helicase to fire the replication origin, committing the cell to S-phase transition. Additionally, Dbf4-dependent kinase protein is a major cell cycle regulator of DNA end resection and is required for homologous recombination (HR) activation. Early mammalian embryo development relies on a successful transition in the S-phase of the cell cycle, which is enabled almost entirely by maternal replication factors stored during oocyte development. In the mouse model, immature germinal vesicle (GV) oocytes had high levels of Cdc7 with a lack of kinase activity due to low levels of the regulatory subunit, DBF4. As oocytes mature, DBF4 accumulates, which leads to the activation of Cdc7 kinase. This activation is crucial for initiating DNA replication in zygotes post-fertilization. By using a conditional knockout model, our research demonstrated that cKO of Dbf4 results in embryonic arrest at the one-cell stage, with no significant difference in ovulation rate between control and cKO mice. We assess defects in DNA replication, DNA damage, and chromatin remodeling using Immunofluorescence staining and EdU incorporation assay. These findings highlight Dbf4 as a critical factor in early embryogenesis and infertility.

RELATED ABSTRACTS

• Seasonal Variations in Reproductive Hormones: An Analysis of the EAGeR and BioCycle Studies, Manchikanti, Nidhi, UMass Amherst, Poster Session 5, 3:15 PM - 4:00 PM, Concourse, B7
• Quantitative Comparison of the Soil Chemical Characteristics of No-Till and Conventional-Till Agricultural Practices, Knechtel, Connor Lee, UMass Amherst, Poster Session 4, 2:15 PM - 3:00 PM, Auditorium, A36

Women with a pathogenic germline mutation in the BRCA1 or BRCA2 gene have an elevated lifetime risk of developing breast cancer. Additionally, the risk of developing increases for all women during pregnancy and up to several years post-partum. However, mammograms and MRIs –breast cancer detection tests, the latter for BRCA mutation carriers– work poorly on lactating breasts. The use of breast milk as a liquid biopsy could mitigate this gap by assessing individuals’ risk and helping to develop strategies for prevention. Breast milk collection is a non-invasive, easy, and reliable way to assess breast health in postpartum women. By analyzing breast milk from BRCA mutation carriers, key differences in breast milk composition associated with their risk can be identified.

Mesoscale Discovery (MSD) technology was used to evaluate pro-inflammatory cytokines and osteoprotegerin (OPG) in breast milk samples from BRCA1 and BRCA2 mutation carriers and controls with the wildtype allele. A total of 40 participants, 18 with germline BRCA mutations and 22 controls, were studied. Analysis will include comparisons between BRCA and control groups, between BRCA1 and BRCA2 groups, and analyte correlations with other breast cancer risk factors including but not limited to age, body mass index, alcohol consumption and baby’s age at time of milk donation. It is hypothesized that IL-10, IL-13, IL-2, and IL-8 will have higher levels in BRCA mutation carriers as compared to controls, while OPG levels will be lowered in BRCA mutation carriers as compared to controls.

RELATED ABSTRACTS

• Disparities in Incidence of Cervical/Breast Cancer in Female Incarcerated Individuals in California, Quinn, Jamie Leah, UMass Amherst, Poster Session 5, 3:15 PM - 4:00 PM, Auditorium, A7
• The Influence of Mechanically Stimulated Osteocytes on Metastatic Breast Cancer Cell Behavior, Choubah, Leah Y., UMass Amherst, Poster Session 6, 4:15 PM - 5:00 PM, 163, C4

The permeability of the mammary gland plays an important role in lactation. Usually within the first week after a woman gives birth, tight junctions form between the epithelial cells lining her mammary gland, reducing permeability and allowing for the production of a copious milk supply. This reduced permeability limits the transfer of constituents in blood from passing between the epithelial cells. Low mammary permeability, characteristic of established lactation, is essential for the production of high-quality milk, which impacts infant health, including the development of the infant gut microbiome. It is generally assumed that permeability remains low throughout established lactation, however, in a retrospective study we found that 15% of women with established lactation had elevated levels of mammary epithelium permeability (MEP) associated with altered nutrient content of the milk. These findings prompted us to design a prospective study, the Breastfeeding Child Health and Mammary Permeability Study (Breastfeeding CHAMPS), to investigate the relationship between MEP and milk production, milk nutrient content, and infant gut microbiome. In CHAMPS, 400 participants will provide milk and infant fecal samples at four time periods during lactation: 7 days (transitional), 21 days (early), and 3 & 5 months postpartum (established). MEP was assessed by measuring the concentration of sodium in milk using ion selective probes, and nutrient content (fat, carbohydrates, and protein) was assessed with infrared analysis. We expect that a greater understanding of the relationship between MEP and milk production will help mothers reach their personal lactation goals and improve infant health.