Background/ObjectiveKidney stones are a globally prevalent urological disease, with calcium oxalate stones being the most common type. Their pathogenesis is complex, and postoperative recurrence rates are high. The calcium-sensing receptor (CaSR) and solute carrier family 26 member 6 (SLC26A6) play key roles in hypercalciuria and hyperoxaluria, respectively. However, the intrinsic relationship and regulatory mechanism between them in kidney stone formation remain unclear. This study aims to investigate whether CaSR regulates SLC26A6 expression through a specific signaling pathway, thereby playing a role in experimental calcium oxalate kidney stone formation in rats.MethodsIn vivo, a calcium oxalate kidney stone model was established in Wistar rats by intragastric administration of 1% ethylene glycol (E.G.,) and 1% ammonium chloride. Rats were divided into six groups: blank control (NC), E.G., model (E.G.,), CaSR agonist (CaSR-a), CaSR inhibitor (CaSR-i), protein kinase A inhibitor (PKA-i), and Forkhead box protein O4 inhibitor (FOXO4-i) groups. Urinary calcium and oxalate levels were measured. Kidney crystal formation was observed via Hematoxylin and Eosin (HE) staining and Pizzolato’s staining. Protein and mRNA expression of CaSR, p-protein kinase A (PKA) substrate, p-FOXO4 (Thr451), and SLC26A6 in kidney tissues were detected by Western blotting, immunohistochemistry, and Real-Time quantitative PCR (RT-qPCR). In vitro, rat renal tubular epithelial cells (NRK-52E) were intervened with calcium oxalate monohydrate (COM) crystals and treated with agonists or inhibitors of CaSR, PKA, and Forkhead box protein O4 (FOXO4). Pathway-related protein expression was detected by Western blotting. A dual-luciferase reporter gene assay was used to validate the transcriptional regulation of the SLC26A6 promoter by FOXO4.ResultsCompared to the NC group, the, E.G., group showed significantly increased urinary calcium and oxalate concentrations, increased renal crystal deposition, and upregulated expression of CaSR, p-PKA substrate, p-FOXO4, and SLC26A6. Activating CaSR (CaSR-a group) further exacerbated these phenomena, whereas inhibiting CaSR (CaSR-i group), PKA (PKA-i group), or FOXO4 (FOXO4-i group) significantly alleviated crystal formation and reduced SLC26A6 expression. Cell experiments confirmed that activating CaSR enhanced PKA and FOXO4 phosphorylation and SLC26A6 expression; activating PKA enhanced FOXO4 phosphorylation and SLC26A6 expression; activating FOXO4 upregulated SLC26A6 expression. The dual-luciferase reporter gene assay showed that FOXO4 functionally regulates SLC26A6 promoter and regulates its transcriptional activity.ConclusionDuring the formation of experimental calcium oxalate kidney stones in rats, CaSR activation promotes PKA-mediated FOXO4 phosphorylation, leading to upregulation of SLC26A6 expression through transcriptional mechanisms. This signaling axis promotes urinary oxalate excretion and contributes to kidney stone formation. This study reveals the potential role of the novel CaSR-PKA-FOXO4-SLC26A6 signaling pathway in the pathogenesis of kidney stones.

Benzohydroxamic acid (BHA) is an important hydroxamate scaffold with potential applications in medicinal chemistry and crop protection, but it has not previously been established as a natural product. Here, we report the discovery and biosynthetic characterization of BHA from Streptomyces angustmyceticus. A conserved TsnB-like gene cassette comprising SaAmOx-AS, SaAmOx, and SaHAT was identified by genome mining, indicating a potential capacity for hydroxamate formation and transfer. BHA was detected as a fermentation product of S. angustmyceticus and structurally confirmed by comparison with an authentic standard, compound purification, and NMR spectroscopic analysis. Benzoic acid was established as the aromatic precursor of BHA through deuterium-labeling experiments with benzaldehyde and cinnamic acid. SaHAT was identified as the key hydroxamate-transfer enzyme that directly converts benzoic acid, rather than benzoyl-CoA, to BHA using glutamyl hydroxamate as the hydroxamate donor in an ATP- and Mg2+-dependent reaction. BHA production was reconstituted in Escherichia coli by heterologous coexpression of SaAmOx-AS, SaAmOx, and SaHAT, confirming that these three enzymes form a minimal biosynthetic module. This work establishes BHA as a microbial natural product and provides a foundation for engineering hydroxamate-containing compounds.

Background: Cutaneous ureterostomy (CU), though a simple urinary diversion (UD), is often limited by stomal stenosis and prolonged stenting-particularly for the contralateral ureter, which is prone to tensionrelated ischemia. This study introduces a modified CU technique that minimizes exteriorization of the contralateral ureter, aiming to reduce stomal complications and improve the catheter-free rate (CFR), with a focus on its applicability to contralateral ureters. Methods: We conducted a retrospective review of 19 consecutive patients with histologically confirmed bladder cancer (BC) who underwent radical cystectomy (RC) followed by our modified CU technique for UD at our institution between August 2020 and October 2023. Outcomes assessed included the CFR, serum creatinine levels, estimated glomerular filtration rate (eGFR), surgical results, and surgery-related complications. The relationship between achieving tubeless status and clinicopathological factors was also analyzed. Results: A tubeless state was achieved in 35 out of 38 ureters (92.11%; 19 right and 16 left ureters). No stomal stenosis was observed over a median follow-up period of 25 months (range, 8-46 months). No significant correlation was found between tubeless status and clinicopathological factors. Mean serum creatinine levels showed no significant difference between preoperative values, postoperative day 3, and the last follow-up (P=0.46). The eGFR also showed no significant change following surgery (P=0.69). Aside from one patient who underwent percutaneous nephroscopic lithotripsy for a left renal stone, no postoperative complications exceeding Clavien-Dindo grade III were observed. Conclusions: Our modified CU technique is a safe and straightforward procedure associated with a higher CFR compared to other reported modified CU methods. It may be suitable for nearly all ureters as potential candidates for this approach.

[This corrects the article DOI: 10.3389/fimmu.2023.1252616.].

Tumors of the urinary system primarily encompass prostate, bladder, and kidney cancers, which exhibit high morbidity and mortality rates worldwide and pose a particularly significant threat to men's health. Given the associated high morbidity and mortality, early diagnosis and effective treatment are crucial. Consequently, innovative research is urgently needed to enhance the clinical care of patients with urologic cancers. Recent studies have demonstrated that microRNAs (miRNAs), as key non-coding RNA molecules that regulate gene expression, play a vital regulatory role in malignant tumor development by binding to the mRNA 3'-UTR region. Large-scale genomic analyses (e.g., TCGA Pan-Cancer Atlas) reveal that over 50% of miRNA genes reside in cancer-associated regions, regulating >60% of protein-coding genes. miR-145 exemplifies this paradigm, with its dysregulation causally linked to tumor proliferation, metastasis, and therapy resistance. Among them, miR-145, as a regulatory molecule with significant anticancer properties, presents unique expression characteristics and functional mechanisms in urological tumours. In this review, we summarize the role of miR-145 in specific urological tumors, along with its downstream target molecules and cells, which may enhance our understanding of miR-145 in these cancers. In conclusion, miR-145 is a multifaceted regulator in urological oncology that has strong potential to range from non-invasive biomarker discovery to therapeutic strategies that work synergistically with conventional treatments, ultimately advancing precision medicine in prostate, bladder, and kidney cancers.

Bladder cancer (BC) is a malignant tumor characterized by a high incidence of urinary system diseases. The complex pathogenesis of BC has long been a focal point in medical research. With the robust development of epigenetics, the crucial role of epigenetic modifications in the occurrence and progression of BC has been elucidated. These modifications not only affect gene expression but also impact critical biological behaviors of tumor cells, including proliferation, differentiation, apoptosis, invasion, and metastasis. Notably, DNA methylation, an important epigenetic regulatory mechanism, often manifests as global hypomethylation or hypermethylation of specific gene promoter regions in BC. Alterations in this methylation pattern can lead to increased genomic instability, which profoundly influences the expression of proto-oncogenes and tumor suppressor genes. MiRNAs, as noncoding small RNAs, participate in various biological processes of BC by regulating target genes. Consequently, this work aims to explore the interaction mechanisms between DNA methylation and miRNAs in the occurrence and development of BC. Research has demonstrated that DNA methylation not only directly influences the expression of miRNA genes but also indirectly affects the maturation and functionality of miRNAs by modulating the methylation status of miRNA promoter regions. Simultaneously, miRNAs can regulate DNA methylation levels by targeting key enzymes such as DNA methyltransferases (DNMTs), thereby establishing a complex feedback regulatory network. A deeper understanding of the crosstalk mechanisms between DNA methylation and miRNAs in BC will contribute to elucidating the complexity and dynamics of epigenetic modifications in this disease, and may provide new molecular targets and strategies for the early diagnosis, treatment, and prognostic evaluation of BC.

Chronic prostatitis is a prevalent urological condition that significantly impacts patients’ quality of life. Advances in the study of Extracellular Vesicles (EV) have revealed their close involvement in the pathogenesis of prostatitis. This paper reviews the progress in understanding the role of EV in the pathogenesis of chronic prostatitis type IIIA, particularly their involvement in inflammatory responses, cell signaling, and interactions with immune cells. Additionally, it explores the potential applications of EV as drug delivery vehicles, including the targeted delivery of anti-inflammatory agents and immunomodulators, and highlights the challenges associated with developing exosome-based therapeutic strategies. In-depth research on EV holds promise for offering new insights into the diagnosis and treatment of inflammatory diseases.

Objective:Ovarian cancer is a disease that seriously endangers the health and life safety of women. At present, no effective preventive and therapeutic measures are available. This study probed the impact of cell division cycle-associated 2 (CDCA2) on ovarian cancer development and cisplatin sensitivity, which provides a new research direction for the study of ovarian cancer. Material and Methods:The protein expression level of CDCA2 was tested by Western blot assay. Cell proliferation was evaluated by cell cloning formation assay and Celigo cell counting. Cell invasion and migration were assessed by Transwell assay. An experiment for nude mouse tumor formation was conducted to analyze the influence of CDCA2 knockdown on tumor growth in vivo. We treated CDCA2 knockdown cells with gradient cisplatin and measured cell viability using the cell counting kit-8 assay. Apoptosis and DNA damage induced by CDCA2 knockdown were investigated by flow cytometry and histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX) immunofluorescence, respectively. Results:CDCA2 expression was knocked down in A2780 and SKOV3 cells. After CDCA2 knockdown, cell proliferation, migration, and invasion ability decreased significantly, and tumor growth in vivo was also limited (P < 0.01). The phosphorylation levels of protein kinase B (AKT) and mechanistic target of rapamycin (mTOR) were reduced by CDCA2 knockdown (P < 0.01), but the effect was reversed by the AKT activator SC-79 (P < 0.01). Knockdown of CDCA2 increased the cisplatin sensitivity of ovarian cancer cells by enhancing apoptosis and DNA damage (P < 0.01). Conclusion:CDCA2 knockdown inhibited the development of ovarian cancer through the AKT/mTOR pathway and enhanced cisplatin sensitivity. CDCA2 is a potential target to reverse cisplatin resistance in ovarian cancer. It can also be used as a new research direction for the development of ovarian cancer therapy.

The ubiquitin (Ub) system has been demonstrated to play a crucial role in various cellular processes, including immune responses, cell development, and programmed cell death. Ubiquitination, a form of post-translational modification, occurs in eukaryotic cells and involves several key components, such as Ub-activating enzymes, Ub-binding enzymes, and Ub-protein ligases. Recently, deubiquitinating enzymes—proteases that reverse the modification of proteins by removing Ub or Ub-like molecules, or by remodeling Ub chains on target proteins—have been identified as significant regulators of ubiquitination-mediated degradation. These enzymes profoundly influence cellular pathways and numerous biological processes, including the DNA damage response and DNA repair mechanisms. Recent studies increasingly demonstrate a relationship between ubiquitination, deubiquitination, and urinary diseases. The roles of these processes in urinary diseases are complex, encompassing various aspects of signaling, protein stability, and cellular metabolism. As research advances, the specific mechanisms by which these processes influence urologic diseases will be further clarified. This review examines recent discoveries in this field, aiming to provide new strategies and targets for the diagnosis and treatment of urologic diseases.

CircRNAs are an important class of non-coding RNAs, which are produced via back-splicing of exons and/or intron sequences of precursor mRNAs and generally cannot be translated into proteins as they do not bind to ribosomes. There is varying evidence supporting the claim that circRNAs are abnormally expressed in cancer and play a crucial role in cancer initiation and progression. Ubiquitin is a highly stable protein that can be conjugated to target proteins. The most crucial role of ubiquitination is to mediate the degradation of substrate proteins by the proteasome. An increasing amount of evidence indicates that circRNAs are involved in the precise degradation of proteins via the ubiquitin-proteasome system. This review systematically summarizes the intricate mechanisms by which circRNAs regulate target protein ubiquitination, modulate cancerous signaling pathways, and control tumorigenesis and tumor development. Although studies are continuously uncovering additional complex interactions between circRNAs and proteins, we believe that circRNAs are promising but challenging molecules that have the potential to facilitate precise cancer therapies in the future.

In renal cell carcinoma (RCC) patients with inferior vena cava (IVC) tumor thrombus, neoadjuvant therapy could alleviate the burden of tumor thrombus, enhance the safety and feasibility of surgical resection, and improve patient prognosis. The combination of tislelizumab and axitinib has demonstrated efficacy in the treatment of advanced RCC. Our study aimed to evaluate the efficacy and safety in the neoadjuvant therapy setting of tislelizumab and axitinib in RCC patients with IVC tumor thrombus. In this retrospective study, seven patients of nonmetastatic RCC with IVC tumor thrombus who received 3 cycles of neoadjuvant therapy with tislelizumab plus axitinib at the First Affiliated Hospital of Xiamen University from May 2020 to December 2023 were included. The main outcomes included objective response rate (ORR), reduction of tumor thrombus size and level, surgical outcomes, and adverse events (AEs). The median age was 66 (range, 50-72) years, and five (71.4%) patients were male. Five (71.4%) patients were diagnosed with clear cell carcinoma, and two (28.6%) patients were papillary type I carcinoma. Four (57.1%) patients had level II tumor thrombus and three (42.9%) patients had level III. The ORR of patients was 57.1%. The mean decrease in thrombus diameter and length was 5.8 (1.8-17.2) mm and 18.5 (4.4-41.5) mm, respectively. All patients showed a decrease in IVC tumor thrombus. The mean time from the end of neoadjuvant therapy to radical nephrectomy and thrombectomy was 31.7(range, 22-45) days. No intraoperative complications or postoperative Clavien-Dindo grade>3 complications occurred. The most common AEs were all grade 1-2, and only one patient had grade 4 hepatic impairment. No AEs delayed the surgery schedule. This study of RCC patients receiving neoadjuvant combination with tislelizumab and axitinib effectively reduced primary tumor and IVC tumor thrombus with the absence of serious AEs, demonstrating a promising neoadjuvant therapy.

The management of locally advanced prostate cancer (PCa) and oligometastatic prostate cancer (OMPCa) remains a clinical challenge. The heterogeneous nature of PCa prompts a need for precision treatment. This study aims to verify whether genomic biomarker-guided neoadjuvant therapy for locally advanced PCa and OMPCa can result in an improvement in the pathological responses and survival outcomes in a Chinese population. In this open-label prospective phase II umbrella clinical trial, 40 patients will be enrolled. Next-generation sequencing data analysis of PCa tissues from the diagnostic needle biopsies will be performed. The genomically evaluable patients will be divided into 4 groups on the basis of genomic testing results, and receive 6 cycles of patient-tailored neoadjuvant systemic therapy targeted to alternative molecular pathways (including parmiparib, cisplatin, tislelizumab or docetaxel, respectively), and both in combination with rezvilutamide and goserelin microspheres. The primary endpoint is the rate of pathologic complete response. Secondary endpoints include rates of clinical complete response and pathological minimal residual disease (defined as residual tumor 5 mm or less), overall survival, progression-free survival and safety outcomes. SEGNO, to the best of our knowledge, is the first umbrella clinical trial designed to provide high-level evidence to support the implementation of genomic biomarker-guided neoadjuvant therapy for locally advanced PCa and OMPCa. Clinicaltrial.gov, NCT06387056.

Hypoxia is a characteristic feature of the tumor microenvironment that significantly influences cancer progression and treatment responses. Hypoxia-inducible factor (HIF), a key regulator of hypoxic adaptation, has been demonstrated to modulate hypoxic gene expression profiles and signaling networks, thereby serving as a potential therapeutic target. Long-stranded non-coding RNAs (lncRNAs), defined as non-coding RNAs exceeding 200 nucleotides in length, regulate various cellular processes by modulating gene expression at transcriptional, post-transcriptional, and epigenetic levels. Evidence suggests that lncRNAs can be regulated by HIF at the transcriptional level. Conversely, HIF itself can be modulated by numerous lncRNAs, with alterations in these lncRNAs being associated with tumorigenesis, resulting in a reciprocal regulatory network. Recently, the critical role of lncRNAs in hypoxia-driven cancer progression has been elucidated in digestive tumors, including colorectal, pancreatic, gastric, and hepatocellular carcinomas. An increasing number of studies have revealed the complex interplay between lncRNAs and HIF in regulating various processes such as proliferation, metastasis, apoptosis, and drug resistance. In this paper, we aim to provide a comprehensive summary of recent advances regarding the roles of hypoxia and lncRNAs in digestive system tumors and to illustrate the mechanisms through which lncRNAs interact with hypoxia in tumor cells. This will enhance our understanding of the regulatory roles of lncRNAs in modulating the microenvironment of digestive system tumors, thereby facilitating the development of novel anticancer drugs.

Urologic oncology is a significant public health concern on a global scale. Recent research indicates that long chain non-coding RNAs (lncRNAs) and autophagy play crucial roles in various cancers, including urologic malignancies. This article provides a summary of the latest research findings, suggesting that lncRNA-mediated autophagy could either suppress or promote tumors in prostate, kidney, and bladder cancers. The intricate network involving different lncRNAs, target genes, and mediated signaling pathways plays a crucial role in urological malignancies by modulating the autophagic process. Dysregulated expression of lncRNAs can disrupt autophagy, leading to tumorigenesis, progression, and enhanced resistance to therapy. Consequently, targeting particular lncRNAs that control autophagy could serve as a dependable diagnostic tool and a promising prognostic biomarker in urologic oncology, while also holding potential as an effective therapeutic approach.

Recently, the non-protein-coding functional elements in the human genome have been identified as key regulators in postgenomic biology, and a large number of pseudogenes as well as long non-coding RNAs (lncRNAs) have been found to be transcribed in multiple human cancers. However, only a small proportion of these pseudogenes has been functionally characterized. In this study, we screened for pseudogenes associated with human non-small-cell lung cancer (NSCLC) by comparative analysis of several independent datasets from the GEO. We identified a transcribed pseudogene named DUXAP8 that is upregulated in tumor tissues. Patients with higher DUXAP8 expression exhibited shorter survival, suggesting DUXAP8 as a new candidate prognostic marker for NSCLC patients. Knockdown of DUXAP8 impairs cell growth, migration, and invasion, and induces apoptosis both in vitro and in vivo. Mechanistically, DUXAP8 represses the tumor suppressors EGR1 and RHOB by recruiting histone demethylase LSD1 and histone methyltransferase EZH2, thereby promoting cell proliferation, migration, and invasion. These findings indicate that the pseudogene DUXAP8 may act as an oncogene in NSCLC by silencing EGR1 and RHOB transcription by binding with EZH2 and LSD1, which may offer a novel therapeutic target for this disease.

tRNA-derived fragments (tRFs) play crucial roles in cancer progression. Among them, tRF-27 has been identified as a key factor in promoting naïve trastuzumab resistance in HER2-positive breast cancer. However, the origin of tRF-27 remains uncertain. In this study, we propose that the upregulated expression of specific cysteine tRNAs may lead to the increased accumulation of tRF-27 in trastuzumab-resistant JIMT1 cells. Mechanistically, the reduced inhibitory H3K27me3 modification at the promoter regions of tRF-27-related tRNA genes in JIMT1 cells, potentially resulting from decreased EZH2 and increased KDM6A activity, may be a critical factor stimulating the transcriptional activity of these tRNA genes. Our research offers fresh insights into the mechanisms underlying elevated tRF-27 levels in trastuzumab-resistant breast cancer cells and suggests potential strategies to mitigate trastuzumab resistance in clinical treatments.

ObjectivesTo investigate the safety and cost analysis of oral propranolol treatment for high-risk infantile hemangiomas starting from the outpatient setting.MethodsA total of 41 high-risk infantile hemangioma patients from outpatient settings and 43 from inpatient settings were selected for the study. After routine pre-treatment examinations, patients were administered propranolol in a stepwise incremental dosing regimen over three consecutive days in the outpatient clinic. Changes in heart rate, blood pressure and PR interval before and after medication were compared. On the 10th day post-medication, liver and kidney functions, fasting blood glucose, tumor ultrasonography, and electrocardiogram were re-evaluated. The costs of treatment starting from the outpatient clinic (including pre-treatment examinations and the first three days of treatment) were calculated and compared with those of similarly managed inpatient cases.ResultsThe majority of patients exhibited a reduction in heart rate and blood pressure, as well as an extended PR interval after treatment of medication (P < 0.05), which remained within normal limits without clinical symptoms. On the 10th day post-medication, statistical differences in blood biochemistry and electrocardiograms were observed when compared to pre-treatment values (P < 0.05), but all values remained within normal ranges. No severe adverse reactions such as hypoglycemia occurred. Additionally, the cost of treatment from the outpatient clinic was significantly lower than that of inpatient care.ConclusionOral propranolol treatment for high-risk infantile hemangiomas starting from the outpatient setting is associated with few adverse reactions and significantly reduced treatment costs. It is worthy of broader application in hospitals without dermatology wards.

BackgroundThe calcium-sensitive receptor (CaSR) has been identified as a key factor in the formation of kidney stones. A substantial body of research has illuminated the function of CaSR in stone formation with respect to oxidative stress, epithelial injury, crystal adhesion, and stone-associated proteins. Nevertheless, as a pivotal molecule in renal calcium excretion, its pathway that contributes to stone formation by regulating calcium supersaturation remains underexplored.MethodsAn in vitro rat calcium oxalate kidney stone model was established through the co-cultivation of calcium oxalate monohydrate (COM) with NRK-52E cells, while an in vivo model was constructed using the ethylene glycol method. Subsequently, the level of the CaSR-claudin-14 pathway was determined. To further elucidate the molecular pathway of CaSR-mediated regulation of claudin-14, drugs were selectively added to the in vitro and ex vivo kidney stone models, and the expression of claudin-14 and the levels of stone formation were detected. Moreover, the direct regulation of claudin-14 by CaSR with STAT3 serving as a transcription factor was examined via the dual luciferase assay. Eventually, a Cldn-14 knockout rat model and a model of kidney stone induction by ethylene glycol were generated using CRISPR-Cas9 technology to further clarify the role of claudin-14 in the CaSR-regulated formation of kidney stones.ResultsIn vitro and in vivo observations revealed that calcium oxalate induces high expression of CaSR-claudin-14. Specifically, CaSR regulates claudin-14 expression through phosphorylation modification of STAT3 via protein kinase A (PKA). In vitro, the intervention of PKA and STAT3 reversed the elevated claudin-14 levels and stone formation induced by CaSR. Finally, we generated cldn-14 knockout rats using CRISPR-Cas9 technology and observed that ethylene glycol still induced stone formation in these animals. Nevertheless, the specific activation or inhibition of CaSR demonstrated no notable impact on stone formation.ConclusionThe results of our study indicate that calcium oxalate crystals induce the activation of the pro-stone pathway of CaSR. That is, activated CaSR regulates claudin-14 levels via the PKA-STAT3 pathway, which further promotes calcium salt stone formation. The role of CaSR in the regulation of stone homeostasis is further enriched.

In most types of erectile dysfunction, particularly in advanced stages, typical pathological features observed are reduced parenchymal cells coupled with increased tissue fibrosis. However, the current treatment methods have shown limited success in reversing these pathologic changes. Recent research has revealed that changes in autophagy levels, along with alterations in apoptosis and fibrosis-related proteins, are linked to the progression of erectile dysfunction, suggesting a significant association. Autophagy, known to significantly affect cell fate and tissue fibrosis, is currently being explored as a potential treatment modality for erectile dysfunction. However, these present studies are still in their nascent stage, and there are limited experimental data available. This review analyzes erectile dysfunction from a pathological perspective. It provides an in-depth overview of how autophagy is involved in the apoptotic processes of smooth muscle and endothelial cells and its role in the fibrotic processes occurring in the cavernosum. This study aimed to develop a theoretical framework for the potential effectiveness of autophagy in preventing and treating erectile dysfunction, thus encouraging further investigation among researchers in this area.