Normal Cycle Research Articles

Phoenixin knockout mice show no impairment in fertility or differences in metabolic response to a high-fat diet, but exhibit behavioral differences in an open field test.

Phoenixin (PNX) is a conserved secreted peptide that was identified 10 years ago with numerous studies published on its pleiotropic functions. PNX is associated with estrous cycle length, protection from a high-fat diet, and reduction of anxiety behavior. However, no study had yet evaluated the impact of deleting PNX in the whole animal. We sought to evaluate a mouse model lacking the PNX parent gene, small integral membrane protein 20 (Smim20), and the resulting effect on reproduction, energy homeostasis, and anxiety. We found that the Smim20 knockout mice had normal fertility and estrous cycle lengths. Consistent with normal fertility, the hypothalamii of the knockout mice showed no changes in the levels of reproduction-related genes, but the male mice had some changes in energy homeostasis-related genes, such as melanocortin receptor 4 (Mc4r). When placed on a high-fat diet, the wildtype and knockout mice responded similarly, but the male heterozygous mice gained slightly less weight. When placed in an open field test box, the female knockout mice traveled less distance in the outer zone, indicating alterations in anxiety or locomotor behavior. In summary, the homozygous knockout of PNX did not alter fertility and modestly alters a few neuroendocrine genes in response to a high-fat diet, especially in the female mice. However, it altered the behavior of mice in an open field test. PNX therefore may not be crucial for reproductive function or weight, however, we cannot rule out possible compensatory mechanisms in the knockout model. Understanding the role of PNX in physiology may ultimately lead to an enhanced understanding of neuroendocrine mechanisms involving this enigmatic peptide.

Numerical Analysis of the Plantar Pressure Points during the Stance Phases for the Design of a 3D-Printable Patient-Specific Insole

The study of the phenomena occurring in the plantar region is remarkably intriguing, especially when performing a normal gait cycle where the foot is under loading conditions. The effects presented in the foot while walking provide relevant indicators regarding clinical means for enhancing regular performance or rehabilitation therapies. Nevertheless, more than traditional methods are needed to biomechanically evaluate foot structural conditions, leading to an incomplete database for determining the patient’s needs so that advanced methodologies provide detailed medical assessment. Therefore, it is necessary to employ technological engineering tools to optimize biomechanical plantar pressure evaluations to reach suitable personalized treatments. This research initially evaluated numerically the pressure points in the foot sole region in each one of the five stance phases in a normal gait cycle. Medical imaging techniques were utilized to construct an anatomically accurate biomodel of the soft tissues of the right foot. The Finite Element Method was employed to predict peak plantar pressure in barefoot conditions for all stance phases; results from this case study presented a close alignment with gait experimental testing implemented to analyze the feasibility and validation of all mechanical considerations for the numerical analyses. Hence, having a solid foundation in the biomechanical behavior from the first case study close estimates, a 3D-printable patient-specific insole was designed and numerically analyzed to observe the mechanical response in the plantar critical zones utilizing a personalized orthotic device. Results from the second case study notably demonstrated a crucial decrement in excessive pressure values. Employing morphological customization orthopedics modeling combined with 3D-printable materials is revolutionizing assistive device design and fabrication techniques. The fundamental contribution of this research relies on deepening the knowledge of foot biomechanics from an interdisciplinary approach by numerically analyzing pressure distribution in critical regions for all five stances phases; thus, based on the methods employed, the results obtained contribute to the advances of patient-specific foot orthopedics.

Design and synthesis of new quinazolinone derivatives: investigation of antimicrobial and biofilm inhibition effects.

New quinazolin-4-ones 9-32 were synthesized in an attempt to overcome the life-threatening antibiotic resistance phenomenon. The antimicrobial screening revealed that compounds 9, 15, 16, 18, 19, 20 and 29 are the most broad spectrum antimicrobial agents in this study with safe profile on human cell lines. Additionally, compounds 19 and 20 inhibited biofilm formation in Pseudomonas aeruginosa, which is regulated by quorum sensing system, at sub-minimum inhibitory concentrations (sub-MICs) with IC50 values 3.55 and 6.86µM, respectively. By assessing other pseudomonal virulence factors suppression, it was found that compound 20 decreased cell surface hydrophobicity compromising bacterial cells adhesion, while both compounds 19 and 20 curtailed the exopolysaccharide production which constitutes the major component of the matrix binding biofilm components together. Also, at sub-MICs Pseudomonas cells twitching motility was impeded by compounds 19 and 20, a trait which augments the cells pathogenicity and invasion potential. Molecular docking study was performed to further evaluate the binding mode of candidates 19 and 20 as inhibitors of P. aeruginosa quorum sensing transcriptional regulator PqsR. The achieved results demonstrate that both compounds bear promising potential for discovering new anti-biofilm and quorum quenching agents against Pseudomonas aeruginosa without triggering resistance mechanisms as the normal bacterial life cycle is not disturbed.

Porcine computational modeling to investigate developmental dysplasia of the hip.

While it is well-established that early detection and initiation of treatment of developmental dysplasia of the hip (DDH) is crucial to successful clinical outcomes, research on the mechanics of the hip joint during healthy and pathological hip development in infants is limited. Quantification of mechanical behavior in both the healthy and dysplastic developing joints may provide insight into the causes of DDHand facilitate innovation in treatment options. In this study, subject-specific three-dimensional finite element models of two pigs were developed: one healthy pig and one pig with induced dysplasia in the right hindlimb. The objectives of this study were: (1) to characterize mechanical behavior in the acetabular articular cartilage during a normal walking cycle by analyzing six metrics: contact pressure, contact area, strain energy density, von Mises stress, principal stress, and principal strain; and (2) to quantify the effect on joint mechanics of three anatomic abnormalities previously identified as related to DDH: variation in acetabular coverage, morphological changes in the femoral head, and changes in the articular cartilage. All metrics, except the contact area, were elevated in the dysplastic joint. Morphological changes in the femoral head were determined to be the most significant factors in elevating contact pressure in the articular cartilage, while the effects of acetabular coverage and changes in the articular cartilage were less significant. The quantification of the pathomechanics of DDH in this study can help identify key mechanical factors that restore normal hip development and can lead to mechanics-driven treatment options.

Will Science Alone Save Your Fertility?

This paper highlights the pressing need for increased awareness and education regarding reproductive health and fertility issues in the Maltese Islands. Despite living in the age of information, misconceptions and misinformation persist, particularly surrounding basic reproductive health knowledge. To address this gap, two conferences on fertility awareness were organized in Malta and Gozo as part of ‘The 3 Missions’ Project, in collaboration with the Malta Council for the Voluntary Sector and Dar Guzeppa Debono Association. The conferences brought together various stakeholders including healthcare professionals, policymakers, and educators to discuss topics such as normal reproductive cycles, infertility treatments, and the legal framework surrounding assisted reproductive technologies (ART). International perspectives were also provided, shedding light on perceptions and practices in other European countries. Key recommendations emerged from these discussions, emphasizing the importance of standardized terminology, user-friendly information dissemination, and the destigmatization of infertility. Furthermore, the paper underscores the need for preconception parenthood preparation and highlights the psychological and social implications of infertility and parenthood. Overall, the conferences served as a platform to raise awareness and initiate collaborative efforts to address fertility-related challenges in the Maltese community.

Evaluation of wheat (Triticum aestivium L) diet on reproductive functions of adult female rats

Introduction: The relationships between wheat diet, sex hormone and the estrous cycle are important from the perspective of fertility. Aim: This study aims to investigate the effect of wheat diet on weight, estrous cycle, reproductive hormones and histology of the ovary of female Sprague-Dawley rats. Methods: Prior to the test, vaginal smear was monitored daily for 16 days period to select females with normal (regular) estrous cycle. A total of twenty rats with regular estrous cycle weighing between 170-200 g were randomized into four sets of 5 animals each. Group A is the female control and was fed with rat chow, group B, C and D was given 40%, 60%, 100% of wheat diet respectively. The wheat diet was giving respectively for a period of 4 weeks. Estrous cycle pattern was monitored using the vaginal smear for 16 days. At the end of the experiment, the rats were sacrificed and the blood sample were taken for follicle stimulating hormone, luteinizing hormones, estradiol, and progesterone analysis. The histology of the ovary were carried out. Results: The result showed that there was an increase in the weight of the rats that was fed with wheat and the increase was diet dependent. The wheat diets disrupt the order of appearance of normal estrous cycle stages, hence; there was no constancy in the frequency of all cycle stages in the wheat groups, mostly the 100% wheat group. No significant endocrine or histological differences noticed between control and wheat diets groups. Therefore photomicrograph of wheat diet shows large corpus luteum, thick germinal epithelium, zona granulosa and oocyte. Conclusion: Wheat diets disrupt the estrous cycle of the rats; however no adverse effect was noticed on the circulating reproductive hormones and histology of the ovary. However, high consumption of wheat should be taken with precaution.

Resveratrol activates autophagy and protects from UVA-induced photoaging in human skin fibroblasts and the skin of male mice by regulating the AMPK pathway

Skin photoaging is mostly caused by ultraviolet A (UVA), although active medications to effectively counteract UVA-induced photoaging have not yet been created. Resveratrol, a naturally occurring polyphenol found in the skin of grapes, has been shown to have various biological functions such as anti-inflammatory and antioxidant characteristics. However, the role of resveratrol in UVA-induced photoaging has not been clarified. We investigated the mechanism of action of resveratrol by UVA irradiation of human skin fibroblasts (HSF) and innovatively modified a mouse model of photoaging. The results demonstrated that resveratrol promoted AMP-activated protein kinase (AMPK) phosphorylation to activate autophagy, reduce reactive oxygen species (ROS) production, inhibit apoptosis, and restore normal cell cycle to alleviate UVA-induced photoaging. In addition, subcutaneous injection of resveratrol not only improved the symptoms of roughness, erythema, and increased wrinkles in the skin of UVA photodamaged mice, but also alleviated epidermal hyperkeratosis and hyperpigmentation, reduced inflammatory responses, and inhibited collagen fiber degradation. In conclusion, our studies proved that resveratrol can treat UVA-induced photoaging and elucidated the possible molecular mechanisms involved, providing a new therapeutic strategy for future anti-aging.

Electrographic screening for infantile epileptic spasms syndrome in a single sleep-wake cycle.

Infantile epileptic spasms syndrome (IESS) is a common and urgent diagnosis with seizure and nonseizure mimics. Evaluation with prolonged video-electroencephalography (EEG) can be time-consuming and costly. This study investigated the use of EEG review of a single sleep-wake cycle to exclude IESS. We retrospectively reviewed video-EEG studies to rule out IESS in children between the ages of 2 months and 2 years in the period from January 2019 through June 2020. EEG studies were reviewed from the start of the recording through the first sleep-wake cycle and scored as "normal," "consistent with IESS," or "abnormal but not diagnostic of IESS." Scores were compared to the clinical report created by analysis of the entire video-EEG. Inclusion criteria were met in 238 EEG studies. The mean patient age was 7.6 months. The median duration of the full study was 908 min, compared to 107.5 min for the first sleep-wake cycle only. The median difference in recording time was 801 min, p-value < .01. Scored outcomes were similar. Sixty-eight percent of EEG studies were scored as "normal" on first sleep-wake cycle review as compared to 63% on full study review, 13% scored as "consistent with IESS" compared to 16% and 19% scored as "abnormal but not diagnostic of IESS" compared to 21%. Sensitivity and specificity of the first sleep-wake cycle review for studies "consistent with IESS" was 84% and 100%, respectively. No cases of IESS were scored as normal on first sleep-wake cycle review. A single sleep-wake cycle captured on EEG can triage studies when IESS is suspected. A normal first sleep-wake cycle did not miss cases of IESS and could result in reduced EEG recording time. Because most of these cases presented to an emergency department, a normal first sleep-wake cycle may help providers determine the acuity, or necessity, of further testing.

MENSTRUAL CYCLE AND THE INCIDENT OF ANEMIA IN TEENAGE WOMEN

The prevalence of anaemia in Indonesia is still high, especially in adolescent girls. This is due to nutritional status, stress levels, knowledge and menstrual disorders. Menstrual disorders are related to the cycle or duration of menstruation or the amount of blood that comes out. The impact that occurs adolescents will feel weak, tired, lethargic, difficult to concentrate and for the long term related to fertility, and there is a possibility to deliver baby with weight and premature birth. The purpose of the study was to determine the relationship between the menstrual cycle and the incidence of anaemia in adolescent girls. Correlational descriptive research method with cross sectional approach. The sample was 68 respondents with total sampling. Data were collected through questionnaires in the form of fill-in sheets for menstrual cycles and anemia using easy touch to determine Hb levels. The results showed that respondents who experienced a normal cycle as many as 41 respondents (60.3%), 16 respondents (23.5%) experienced a long cycle and as many as 11 respondents (16.2%) experienced a short cycle. Respondents who experienced anemia as many as 36 respondents (52.9%) and did not have anemia as many as 32 respondents (47.1%). The results of the statistical test obtained a ρ value of 0.000. The conclusion of this study is that there is a relationship between the menstrual cycle and the incidence of anemia in adolescent girls. It is recommended for the nursing profession to provide information through health counseling in collaboration with the community and schools about anemia and its causative factors to improve the health status of adolescent girls

Research on the heating effect evaluation of the electromagnetic induction heating system at low temperature based on the electrochemical-thermal coupling model

The extremely fast electromagnetic induction heating system (EIHS) was recently introduced to improve the poor charge and discharge performance of lithium-ion batteries (LiBs) at low temperature. In this work, aimed to investigate the heating effect of EIHS, the accurate electrochemical-thermal coupling model, validated against the charging capacity calibration with various C-rates and HPPC tests at different environments, was developed in depth to reflect the electrochemical and heat transfer behaviors of the LiB exposed to external cool air during the electromagnetic induction heating process. When the copper coil was subjected to a sinusoidal alternating current (SAC) excitation at 10 A, 3 kHz, the LiB can be warmed up to 293.15 K from 243.15 K within 742 s with an even heat generation inside the LiB. The impact of the SAC parameters on the needed heating time was illustrated with time maps, a series of contours suggested that the heating velocity increase with the increase of both the amplitude and frequency. As compared with the low-temperature state, the internal resistance had a more than 3-fold decrease, which can improve the discharging voltage platform and terminal voltage output, thus increasing the pulse power and usable capacity. Neither noticeable capacity retention difference before and after the heating cycles nor obvious capacity loss discrepancy between the normal charge-discharge and heating cycle tests was found at the end of the 200 heating cycles, so the EIHS can achieve a good tradeoff among temperature rising effect, working behaviors and LiB cycle lifespan, which had a considerable potential to enhance the applicability of EVs (electric vehicles) in frigid weather. Moreover, a better heat transfer condition, higher material thermal conductivity and lower specific heat capacity play an important positive role on the improvement of temperature rising performance.

Heavy Metal Pollution of Water Sources "Šićki Brod" and "Studenac" Due to the Exploitation of Coal

The article aims to determine the levels of water pollution by concrete measurements of heavy metals in the water sources "Šićki Brod" and "Studenac". Sampling of water from both sources as well as analysis of the content of the heavy metal elements in the water was carried out. All analyzed elements are classified into the group of heavy metals, which take second place (after pesticides) in terms of harmful effects if found in the environment. Heavy metals are ecologically very important because they are non-degradable and do not disappear, but move through the ecosystem and have a normal biogeochemical cycle. Considering the mining works that were carried out in the area near to source, the impact of mining works on the pollution of water sources was put into comparison.

Abstract 5728: YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors

Abstract Metastatic breast cancer (BC) is the 2nd leading cause of death in women in the US, annually accounting for more than 43,000 deaths and 281,000 new cases of invasive BC. Amongst genetically distinct BC subtypes, those classified as being “triple-negative” (TNBC) are especially devastating due to their highly metastatic behavior, their propensity to recur rapidly, and their low response to standard-of-care therapies. In fact, the acquisition of chemoresistant phenotypes represents the main cause of disease recurrence, metastasis, and death in TNBC patients. Currently, the molecular mechanisms that regulate TNBC progression and metastasis remain unknown, as does the way these metastatic tumors acquire resistance to standard-of-care therapies. We recently established YB1 as a novel driver of these deadly TNBC activities, doing so by stimulating the cancer stem cell phenotype, and by disrupting normal cell cycle progression, therefore promoting therapy resistance and metastasis of TNBC tumors. YB1 is a multifunctional protein that acts as a transcription factor of cancer stem cell genes Nanog, Oct and Sox. Moreover, aberrant activation of YB1 contributes to the metastatic progression of several cancers, including TNBC. Our investigations revealed a major role of YB1 in the regulation of several hallmarks of cancer that drive TNBC tumors progression and metastasis, both in vitro and in preclinical mouse models of TNBC tumors. In extending these discoveries, we now show that aberrant YB1 expression activates oncogenic signaling leading to the dysregulation of cell cycle progression through the regulation of the Cyclin D/CDK4/6 complex signaling and the RB pathway. We also report the identification of a novel small molecule inhibitor, SU056, that specifically targets YB1 and inhibits its oncogenic activity in TNBC cell lines. We further show that the SU056-mediated inhibition of YB1, either as monotherapy or in combination with the CDK4/6 inhibitors, has a significant impact on inhibiting TNBC tumor progression and metastasis. In fact, the SU056-CDK4/6 inhibitors combination showed a synergistic effect when compared to monotherapy, at the same time reducing the toxicity associated with CDK4/6 inhibitors. Mechanistically, our studies revealed that genetic or pharmacologic targeting of YB1 inhibits TNBC tumor progression and metastasis through the regulation cyclin D-CDK4/6-RB pathway and blockade of cell cycle progression. Together, our data support the notion that targeting YB1 represents a potential therapeutic option for the treatment of TNBC, which could be enhanced when combined with standard of care treatment modalities. Citation Format: Wei Wang, Lamyae El Khalki, Neelum Yousaf Zai, Justin Szpendyk, Akram Alkrekshi, Bin Su, Khalid Sossey-Alaoui. YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5728.

Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition

Abstract CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors. Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5705.

Abstract 4641: Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells

Abstract Among the myriad of molecular alterations associated with cancer, loss of the normal tumor suppressive functions of p53, the “guardian of the genome,” is the most consistent. Thus, the possibility of targeting oncogenic, mutant p53 proteins has long been a therapeutic goal. However, that aspiration has not been fully realized, leaving patients vulnerable to aggressive disease that is resistant to chemotherapy and to targeted agents such as PARP inhibitors and immunotherapy owing to the loss of wild type p53 functionality and the gain of new oncogenic functions when mutant p53 is expressed. An effective therapeutic strategy is urgently needed for p53 mutant cancers, and our team is developing novel analogues of curcumin with the potential to convert mutant p53 to a wild type conformation and restore its tumor suppressive functions. In this study, we investigated the effects of AKT-100, which was developed from the parent molecule and curcumin analogue HO-3867. Effects were compared to another drug believed to reactivate p53 functionality, APR-246, using CyQUANT IC50 cell proliferation assays and bulk and single cell RNA sequencing. Structural analyses are underway investigating the specific sites of interaction of AKT-100 with various mutant forms of p53. Our findings indicate that, first, AKT-100 exhibits impressive cell killing in multiple ovarian and serous endometrial cancer cells at concentrations ranging from 100-300 nM and is synergistic with the PARP inhibitor olaparib. Second, RNA sequencing demonstrates the reactivation of wild type p53 genes associated with normal cell cycle regulation (induction of CDKN1A encoding p21 and GADD45A), apoptosis (induction of PMAIP1 encoding Noxa and DR5 encoding Death Receptor 5), and the inhibition of DNA replication and multiple alternative mechanisms of DNA repair employed by malignant cells. Third, RNA sequencing indicates that AKT-100 acts through both p53-dependent and independent pathways to inhibit cancer cell proliferation. Gene expression is impacted in ways that portend a therapeutic effect as well as highlight potential resistance pathways (e.g. significant induction of PLK5 encoding polo like kinase 5) that can be further targeted to create synthetic lethal drug combinations with AKT-100 chosen specifically to target the various mutant forms of p53. Taken together, these results identify novel curcumin analogues in early development with the potential to improve therapy for p53 mutant cancers. Through this strategy it is also possible to identify and block resistance pathways that are activated by different forms of mutant p53 in response to AKT-100 and to create synthetic lethal precision drug combinations. Citation Format: Kimberly K. Leslie, Jamie L. Padilla, Geneva L. Williams, Hua-Ying Fan, Cristian G. Bologa, Eric R. Prossnitz, Jun-Yong Choe, Lane E. Smith, Alexander Goss, Robert J. Lake, Avinash D. Sahu. Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4641.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3347.

Characteristics and functions of memory regulatory T cells in normal pregnancy cycle and pregnancy complications

Regulatory T cells (Tregs) are activated and expanded after exposure to fetal-specific (paternal) antigens. A proportion of Tregs differentiate into memory Tregs (mTregs), exhibiting immune memory function and exerting more potent immunosuppression than naive Tregs (nTregs). However, it is unclear how mTregs are regulated during normal and pathological pregnancies (e.g., gestational diabetes mellitus (GDM) and preeclampsia (PE)). In this study, PD-1, HLA-G, and HLA-DR expressions on memory CD4+ T cells, naive CD4+ T cells, Tregs, mTregs, and nTregs in healthy non-pregnant women (n=20), healthy first (n=20), second (n=20), and third-trimester women (n=20), postpartum women (n=20), GDM (n=20), and PE patients (n=20) were analyzed. The proportion of mTregs out of Tregs was increased (P<0.05) in the first trimester compared with that in non-pregnancy and reduced in the second and third trimesters. The proportions of PD-1+ Tregs and mTregs were significantly increased during the first trimester compared to those of non-pregnancy (P<0.01), reached their maximum in the second trimester. Moreover, the proportions of HLA-G+ memory CD4+ T cells, Tregs, and mTregs were increased in the first and second trimesters (P<0.01), reached their maximum in the third trimester. GDM patients were characterized by significantly lower percentages of PD-1+ and HLA-G+ mTregs (P<0.01), while PE patients were characterized by significantly lower percentages of HLA-G+ mTregs (P<0.01), compared with the healthy third-trimester women. In general, as demonstrated by this study, mTregs increase in number and enhance maternal-fetal immunoregulation during pregnancy, and their dysfunction can result in pregnancy complications such as GMD or PE.

Breastmilk auto-expression and self-suckling in a Zoo-bred Japanese macaque (Macaca fuscata)

Breastmilk auto-expression, also called “self-suckling", is a rarely observed behaviour in mammals. We present detailed observations of this behaviour in a Zoo-bred Japanese macaque (Macaca fuscata), in the context of extended nursing by the offspring. This is in contrast to prior reports of self-suckling in primates, which were associated with the death of the infant. We followed mother Miya (MI) and infant Shoyo (BB) for the first two years of the infant’s life, documenting changes in his activity budget during development. After first observing that MI was consuming her own breastmilk independent of nursing bouts with BB, we began measuring instances of this behaviour within MI’s activity budget. We describe this self-suckling behaviour in the context of ongoing nursing interactions and a normal oestrus cycle and speculate whether this behaviour is the cause or a consequence of MI’s failure to become pregnant after BB’s birth.

Effect of geometrical, operational and material parameters in the lubrication regime of hard-on-hard hip implants

The lubrication regime of a hip implant is influenced by various parameters, including material, geometry, roughness, relative angular movement, and loading circumstances. These factors impact friction and wear performance, ultimately limiting the implant's longevity. In this study, a ball-on-plane model is considered to predict the lubrication regime across the entire gait cycle for the metal-on-metal and ceramic-on-ceramic hip implants. According to ISO 14242-1, a normal walking gait cycle is considered for the loads and rotations in the hip implant for this study. A comprehensive analysis is done to estimate the lubrication regime by considering different material combinations, body weights, femoral head sizes, clearances, and roughness across the entire gait cycle. The correlation coefficients show that the geometrical parameters are dominant in affecting the lubrication film thickness compared to the operational and material parameters. The femoral head size and body weight are found to be the most dominant and least dominant parameter respectively. Among the hard-on-hard tribo-pairs, ceramics operate predominantly in the full-film lubrication regime compared to metals due to its fine surface finish. The present research suggests that in order to maximise the longevity of a hip implant, an orthopaedic surgeon should choose one with a larger femoral head diameter, less clearance, and an ultra-fine surface finish, regardless of any material tribo-pair.

Changes in fecal microbiota during estrous cycle in healthy thoroughbred mares

Gut microbiota plays a crucial role in various physiological processes, including the regulation of the reproductive system and steroid sex hormones. Throughout the normal estrous cycle of healthy mares, the levels of estradiol-17β (E2) and progesterone (P4) in the blood exhibit periodic changes. To investigate the relationship between cyclic changes in steroid sex hormones and the gut microbiome of mares, we analyzed the fecal microbiota composition in healthy mares during the typical estrous cycle. Blood and fecal samples from five healthy mares were collected, E2 and P4 levels in serum were analyzed using radioimmunoassay (RIA), and the gut microbiome was analyzed by 16S rRNA sequencing. The overall richness and composition of the gut microbiota remained relatively stable during the normal estrous cycle in mares. The Linear Discriminant Analysis Effect Size analysis of the microbial composition during the follicular and luteal phases identified the Rhodococcus genus as differentially abundant. These findings indicate that the mare's gut microbiota's significant composition remains consistent throughout the estrous cycle. At the same time, specific low-abundance pathogenic bacteria exhibit changes that align with sexual hormonal fluctuations.

Self-correction of cycle threshold values by a normal distribution-based process to improve accuracy of quantification in real-time digital PCR.

Thedigital polymerase chain reaction (dPCR) is a new and developing nucleic acid detection technology with high sensitivity that can realize the absolute quantitative analysis of samples. In order to improve the accuracy of quantitative results, real-time digital PCR emphasizes the kinetic information during amplification to identify prominent abnormal data. However, it is challenging to use a unified standard to accurately classify the amplification curve of each well as negative and positive, due to the interference caused by various factors in the experiment. In this work, a normal distribution-based cycle threshold value self-correcting model (NCSM) was established, which focused on the feature of the cycle threshold values in amplification curves and conducted continuous detection and correction on the whole. The cycle threshold value distribution was closer to the ideal normal distribution to avoid the influence of interference. Thus, the model achieves a more accurate classification between positive and negative results. The corrective process was applied to plasmid samples and resulted in an accuracy improvement from 92 to 99%. The coefficient of variation was below 5% when considering the quantitation of a range between 100 and 10,000 copies. At the same time, by utilizing this model, the distribution of cycle threshold values at the endpoint can be predicted with fewer thermal cycles, which can reduce the cycling time by around 25% while maintaining a consistency of more than 98%. Therefore, using the NCSM can effectively enhance the quantitative accuracy and increase the detection efficiency based on the real-time dPCR platform.