A Comparative Analysis of Orexins in the Physio-Pathological Processes of the Male Genital Tract: New Challenges? A Review.

Orexins A (OXA) and B (OXB) and their specific receptors, receptor 1 (OX1R) and 2 (OX2R) for orexins, are hypothalamic peptides involved in orchestrating several functions in the central nervous system and peripheral organs, including sleep, excitement, nutrition, reward, circadian rhythm, anxiety, cognition, and reproduction. The aim of this narrative review is, in particular, to speculate the role of orexins in the male genital tract of animal species and human beings. The experimental evidence collected in recent years assumed that in the testes of the animal species here described, orexins are directly involved in steroidogenesis and spermatogenesis regulation. In the epididymis, these peptides are locally synthesized, thus suggesting their role governing the fertilizing capability of the immature male gamete. In addition to playing a physiological role, orexins are involved in numerous inflammatory and/or neoplastic pathologies too. The expression of the orexinergic system in prostate cancer suggests that they might play a potential therapeutic function. Overall, the future directions of this literature review allow us to hypothesize a role of the orexinergic complex not only as a marker for the diagnosis of certain tumors affecting the male genital tract but also for the treatment of hypo/infertility condition.

Challenges and Promise for Glioblastoma Treatment through Extracellular Vesicle Inquiry.

Glioblastoma (GB) is a rare but extremely aggressive brain tumor that significantly impacts patient outcomes, affecting both duration and quality of life. The protocol established by Stupp and colleagues in 2005, based on radiotherapy and chemotherapy with Temozolomide, following maximum safe surgical resection remains the gold standard for GB treatment; however, it is evident nowadays that the extreme intratumoral and intertumoral heterogeneity, as well as the invasiveness and tendency to recur, of GB are not compatible with a routine and unfortunately ineffective treatment. This review article summarizes the main challenges in the search for new valuable therapies for GB and focuses on the impact that extracellular vesicle (EV) research and exploitation may have in the field. EVs are natural particles delimited by a lipidic bilayer and filled with functional cellular content that are released and uptaken by cells as key means of cell communication. Furthermore, EVs are stable in body fluids and well tolerated by the immune system, and are able to cross physiological, interspecies, and interkingdom barriers and to target specific cells, releasing inherent or externally loaded functionally active molecules. Therefore, EVs have the potential to be ideal allies in the fight against GB and to improve the prognosis for GB patients. The present work describes the main preclinical results obtained so far on the use of EVs for GB treatment, focusing on both the EV sources and molecular cargo used in the various functional studies, primarily in vivo. Finally, a SWOT analysis is performed, highlighting the main advantages and pitfalls of developing EV-based GB therapeutic strategies. The analysis also suggests the main directions to explore to realize the possibility of exploiting EVs for the treatment of GB.

Re-emergence of human leishmaniasis in northern Italy, 2004 to 2022: a retrospective analysis.

BackgroundHuman leishmaniasis is a protozoan disease transmitted by sand flies and endemic in the Mediterranean region. In Italy, leishmaniasis is present in the south and the western coastal regions, with an epidemic peak detected in northern Italy in the early 1970s.AimTo examine temporal trends, and demographic, clinical, geographical and environmental features of human leishmaniasis cases recorded by the local health unit (LHU) of Bologna, northern Italy.MethodsIn this retrospective observational study, we analysed human leishmaniasis cases recorded from 2004 to 2022 within the Bologna LHU. We also conducted serological investigations for canine leishmaniasis in owned dogs living near the place of infection of human cases.ResultsIn total, 173 cases of human leishmaniasis were detected, and 154 cases were considered autochthonous. An increase of human cases was observed since 2004, with incidence peaks above 2 cases/100,000 inhabitants in 2013, 2018 and 2022; epidemic peaks were preceded by dry summers. Most cases lived in the plain and hilly areas less than 400 m above sea level and many resided in isolated housing, in city outskirts, and/or near uncultivated areas, watercourses and railway sections. The incidence of canine leishmaniasis did not increase in the study period.ConclusionAn epidemic of human leishmaniasis with fluctuating annual numbers of cases, probably related to environmental and climatic factors, was identified in the Bologna LHU. Understanding the risk factors and the environmental characteristics related to places of infection is crucial to evaluate the public health implications of leishmaniasis.

G Protein-Coupled Estrogen Receptor (GPER) and ERs Are Modulated in the Testis-Epididymal Complex in the Normal and Cryptorchid Dog.

There is growing evidence by the literature that the unbalance between androgens and estrogens is a relevant condition associated with a common canine reproductive disorder known as cryptorchidism. The role of estrogens in regulating testicular cell function and reproductive events is supposedly due to the wide expression of two nuclear estrogen receptors (ERs), ER-alpha and ER-beta and a trans-membrane G protein-coupled estrogen receptor (GPER) in the testis. In this study, immunohistochemistry, Western blotting and qRT-PCR were used to assess the distribution and expression of GPER in the testis-epididymal complex in the normal and cryptorchid dog. ER-alpha and ER-beta were also evaluated to better characterize the relative abundances of all three receptors. In addition, in these tissues, the expression level of two proteins as SOD1 and Nrf2 normally associated with oxidative stress was investigated to evaluate a possible relationship with ERs. Our data revealed changes in the distribution and expression of the GPER between the normal and cryptorchid dog. In particular, dogs affected by cryptorchidism showed an upregulation of GPER at level of the examined reproductive tract. Also considering the obtained result of a modulation of SOD1 and Nrf2 expression, we could hypothesize the involvement of GPER in the cryptorchid condition. Further studies are, however, necessary to characterize the role of GPER and its specific signaling mechanisms.

Orexins and Prostate Cancer: State of the Art and Potential Experimental and Therapeutic Perspectives.

Prostate cancer (PCa) is the second most common cancer in humans. Peptides have recently been used as targeted therapeutics in cancers, due to their extensive multi-functional applications. Two hypothalamic peptides, orexins A (OXA) and B (OXB) and their specific receptors, orexin receptor 1 (OX1R) and 2 (OX2R), orchestrate several biological processes in the central nervous system and peripheral organs. However, in addition to their role in physiological responses, orexins are involved in numerous inflammatory and/or neoplastic pathologies. The presence and expression of orexins in different cancer models, including prostate cancer, and their role in inducing pro- or anti-apoptotic responses in tumor cell lines, suggest that the orexinergic system might have potential therapeutic action or function as a diagnostic marker in PCa. In addition to the traditional animal models for studying human PCa, the canine model might also serve as an additional tool, due to its clinical similarities with human prostate cancer.

Pathological and Therapeutic Significance of Tumor-Derived Extracellular Vesicles in Cancer Cell Migration and Metastasis.

The infiltration of primary tumors and metastasis formation at distant sites strongly impact the prognosis and the quality of life of cancer patients. Current therapies including surgery, radiotherapy, and chemotherapy are limited in targeting the complex cell migration mechanisms responsible for cancer cell invasiveness and metastasis. A better understanding of these mechanisms and the development of new therapies are urgently needed. Extracellular vesicles (EVs) are lipid-enveloped particles involved in inter-tissue and inter-cell communication. This review article focuses on the impact of EVs released by tumor cells, specifically on cancer cell migration and metastasis. We first introduce cell migration processes and EV subtypes, and we give an overview of how tumor-derived EVs (TDEVs) may impact cancer cell migration. Then, we discuss ongoing EV-based cancer therapeutic approaches, including the inhibition of general EV-related mechanisms as well as the use of EVs for anti-cancer drug delivery, focusing on the harnessing of TDEVs. We propose a protein-EV shuttle as a route alternative to secretion or cell membrane binding, influencing downstream signaling and the final effect on target cells, with strong implications in tumorigenesis. Finally, we highlight the pitfalls and limitations of therapeutic EV exploitation that must be overcome to realize the promise of EVs for cancer therapy.

Human-Animal Interaction in Animal-Assisted Interventions (AAI)s: Zoonosis Risks, Benefits, and Future Directions-A One Health Approach.

Animal-assisted interventions (AAI)s represent the expression of integrated medicine, according to the One Health approach. Actually, animal-assisted therapies and animal-assisted activities are implemented in hospitals, rehabilitation centers, etc. The efficacy of AAIs is based on interspecific interactions and would be impacted by different factors, such as the characters of both the animal and the handler, a suitable selection of animal species, an appropriate animal educational protocol, the relationship between the handler and the animal, and mutual relationship among the animal, the patients, and members of the working team. AAIs produce many advantages for the patients but could expose them to zoonotic-pathogens transmission. Therefore, positive animal welfare, as preventative medicine to avoid incidents or transmission of zoonosis, is a relevant aspect with implications for human and animal health and wellbeing. This review aims to summarize the current published knowledge regarding the occurrence of pathogens in AAIs and to discuss their relevance in light of health and safety in AAIs participants. In addition, this review will contribute to defining the state of the art of AAIs through a careful benefits/challenges analysis and offers discussion points on the possible future developments according to the One Health approach.

Neuronal control of the vagina in vertebrates: A review.

BACKGROUND: At present, there is an increased interest in the vaginal microbiome. It is believed that microbes play equally important roles in the vagina, including the modulation of neuronal pathways, as in the gut. However, in man as well as in animals, the vagina is the least well-studied part of the female reproductive system. The vagina, a fibromuscular tract, having two main functions, i.e., childbirth and sexual intercourse, is mainly innervated by the pudendal nerve and the pelvic splanchnic nerves (the uterovaginal nerve plexus) containing sympathetic, parasympathetic and nociceptive nerve fibers. Innervation density in the vaginal wall undergoes significant remodeling due to hormonally mediated physiological activity. Knowledge about expression and function of neuropeptides and neurotransmitters in the vaginal fibers is incomplete or not established. Most research concerning the neuroregulation of the vagina and the function and expression of neuropeptides and neurotransmitters, is performed in several vertebrate species, including large farm animals, rodents, domestic fowl and lizards. METHODS: This review summarizes, on a bibliographic basis, the current knowledge on vaginal innervation and function of neuropeptides and neurotransmitters expressed in vaginal nerve fibers in several vertebrate species, including humans. The presence and role played by the local microbioma is also explored. CONCLUSION: A thorough knowledge of the vaginal innervation is necessary to unravel the putative communication of the vaginal microbiome and vaginal nerve fibers, but also to understand the effects of vaginal pathologies and of administered drugs on the neuroregulation of the vagina.

A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Does Orexin B-Binding Receptor 2 for Orexins Regulate Testicular and Epididymal Functions in Normal and Cryptorchid Dogs?

Orexins A (OXA) and B (OXB) and the receptors 1 (OX1R) and 2 (OX2R) for orexins are hypothalamic peptides found in several mammalian organs and participated to the control of a wide assortment of physiological and pathological functions. The distribution of OXA and OX1R has been extensively studied in the male gonad of mammals. Here, we examined the expression and localization of OXB and OX2R as well as their possible involvement in the regulation of testicular and epididymal functions, in healthy and cryptorchid dogs, employing some techniques such as immunohistochemistry, Western blotting, and real-time RT-PCR. In vitro tests were also carried out for evaluating the steroidogenic effect of OXB. OXB and OX2R were expressed in spermatocytes, spermatids, and Leydig cells in normal testis. Their localization was restricted to Sertoli and Leydig cells in cryptorchid conditions. OXB was found to be localized in all tracts of both normal and cryptorchid epididymis, whereas OX2R was found only in the caput. Because the small molecular weight of the peptides OXA and OXB, the expression of their precursor prepro-orexin (PPO), OX1R, and OX2R proteins and mRNAs were investigated by means of Western blot and real-time RT-PCR analyses, respectively, in all tested groups of. In particular, the mRNA level expression of all three genes was higher in cryptorchid dogs than in normal ones. In vitro tests demonstrated that OXB-by binding OX2R-is not involved in testicular steroidogenic processes. Therefore, the findings of this study might be the basis for further functional and molecular studies addressing the possible biochemical effects of OXB and OX2R in normal and pathological conditions of the male reproductive system.

A large outbreak of giardiasis in a municipality of the Bologna province, north-eastern Italy, November 2018 to April 2019.

Giardiasis, the disease caused by the flagellate Giardia duodenalis (syn. G.lamblia, G. intestinalis), is the most commonly reported among the five food- and waterborne parasitic diseases under mandatory surveillance in 24 EU countries. From November 2018 to April 2019, an outbreak of giardiasis occurred in a municipality of the Bologna province, in north-eastern Italy. Microscopy and immunochromatography identified cysts and antigens, respectively, of the parasite in stool samples of 228 individuals. Molecular typing of 136 stool samples revealed a vast predominance (95%) of G. duodenalis assemblage B. Investigations into potential sources indicated tap water as the most likely vehicle of infection, although cysts were not detected in water samples. Control measures mostly aimed at preventing secondary transmission by informing citizens about the outbreak, and by treatment of patients with anti-parasitic drugs. This is the first documented human outbreak of giardiasis in Italy; its investigation has highlighted the difficulties in the timely detection and management of this parasite, which is often overlooked as a cause of human gastroenteritis. The long and variable incubation time, absence of specific symptoms and a general lack of awareness about this pathogen contributed to delay in diagnosis.

Design of transfections: Implementation of design of experiments for cell transfection fine tuning.

Transfection is the process by which nucleic acids are introduced into eukaryotic cells. This is fundamental in basic research for studying gene function and modulation of gene expression as well as for many bioprocesses in the manufacturing of clinical-grade recombinant biologics from cells. Transfection efficiency is a critical parameter to increase biologics' productivity; the right protocol has to be identified to ensure high transfection efficiency and therefore high product yield. Design of experiments (DoE) is a mathematical method that has become a key tool in bioprocess development. Based on the DoE method, we developed an operational flow that we called "Design of Transfections" (DoT) for specific transfection modeling and identification of the optimal transfection conditions. As a proof of principle, we applied the DoT workflow to optimize a cell transfection chemical protocol for neural progenitors, using polyethyleneimine (PEI). We simultaneously varied key influencing factors, namely concentration and type of PEI, DNA concentration, and cell density. The transfection efficiency was measured by fluorescence imaging followed by automatic counting of the green fluorescent transfected cells. Taking advantage of the DoT workflow, we developed a new simple, efficient, and economically advantageous PEI transfection protocol through which we were able to obtain a transfection efficiency of 34%.

Aquaporins Are Differentially Regulated in Canine Cryptorchid Efferent Ductules and Epididymis.

The efferent ductules and the epididymis are parts of the male reproductive system where spermatozoa mature. Specialized epithelial cells in these ducts contribute to the transport of fluids produced by spermatozoa's metabolic activity. Aquaporins (AQPs) have been demonstrated to be expressed in the spermatozoan membrane and testis epithelial cells, where they contribute to regulating spermatozoan volume and transit through environments of differing osmolality. Due to the lack of detailed literature regarding AQP expression in the canine male genital tract, the aim of this study was to investigate both the distribution and expression of AQP7, AQP8, and AQP9 in the efferent ductules and epididymal regions (caput, corpus, and cauda) from normal and cryptorchid dogs by using immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction (RT-PCR). Our results show different patterns for the distribution and expression of the examined AQPs, with particular evidence of their upregulation in the caput and downregulation in the cauda region of the canine cryptorchid epididymis. These findings are associated with a modulation of Hsp70 and caspase-3 expression, suggesting the participation of AQPs in the luminal microenvironment modifications that are peculiar characteristics of this pathophysiological condition.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

The Transcription Regulator Patz1 Is Essential for Neural Stem Cell Maintenance and Proliferation.

Proper regulation of neurogenesis, the process by which new neurons are generated from neural stem and progenitor cells (NS/PCs), is essential for embryonic brain development and adult brain function. The transcription regulator Patz1 is ubiquitously expressed in early mouse embryos and has a key role in embryonic stem cell maintenance. At later stages, the detection of Patz1 expression mainly in the developing brain suggests a specific involvement of Patz1 in neurogenesis. To address this point, we first got insights in Patz1 expression profile in different brain territories at both embryonic and postnatal stages, evidencing a general decreasing trend with respect to time. Then, we performed in vivo and ex vivo analysis of Patz1-knockout mice, focusing on the ventricular and subventricular zone, where we confirmed Patz1 enrichment through the analysis of public RNA-seq datasets. Both embryos and adults showed a significant reduction in the number of Patz1-null NS/PCs, as well as of their self-renewal capability, compared to controls. Consistently, molecular analysis revealed the downregulation of stemness markers in NS/PCs derived from Patz1-null mice. Overall, these data demonstrate the requirement of Patz1 for NS/PC maintenance and proliferation, suggesting new roles for this key transcription factor specifically in brain development and plasticity, with possible implications for neurodegenerative disorders and glial brain tumors.

Differential abundances of AQP3 and AQP5 in reproductive tissues from dogs with and without cryptorchidism.

Aquaporins (AQPs) are integral transmembrane proteins facilitating transport of water and small solutes, such as glycerol and urea, between cells. In male reproductive tracts, AQPs maintain a milieu conducive for sperm formation, maturation, and storage. The aim of this study was to clarify effects of testicular and epidydimal function on male fertility by investigating localisation and abundances of AQP3 and AQP5 in testes and epididymal segments from dogs with and without unilateral cryptorchidism. Immunohistochemistry results indicated AQP3 and AQP5 have different distribution patterns in reproductive tissues of dogs with and without unilateral cryptorchidism. The AQP3, an aquaglyceroprotein, is present in different germ and Sertoli cells in testis of dogs without cryptorchidism. The AQP5 protein was not detected in germ cells but was present in Sertoli and Leydig cells and in endothelia of blood vessels. In cryptorchid dogs, AQP3 was detected in early-developing germ and Sertoli cells, and AQP5 had a distribution pattern similar to testes of dogs without cryptorchidism. In the epididymis, AQP3 and AQP5 were localised in epithelial cells of dogs with and without cryptorchidism in a cell-specific manner. The AQP3 and AQP5 protein was in larger abundance in the gonads from dogs with and without cryptorchidism. In contrast, AQP3 and AQP5 abundance increased in each segment of the cryptorchid epididymis, likely as a compensatory mechanism associated with the pathologic condition. These results indicate involvement of AQP3 and AQP5 in spermatogenesis and sperm maturation. Results from the present study indicate dogs are a useful for comparative reproductive biology studies.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue.

Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Vaccination with (1-11)E2 in alum efficiently induces an antibody response to ß-amyloid without affecting brain ß-amyloid load and microglia activation in 3xTg mice.

Immunization against ß-amyloid (Aß) is pursued as a possible strategy for the prevention of Alzheimer's disease (AD). In clinical trials, Aß 1-42 proved poorly immunogenic and caused severe adverse effects; therefore, safer and more immunogenic candidate vaccines are needed. Multimeric protein (1-11)E2 is able to induce an antibody response to Aß, immunological memory, and IL-4 production, with no concomitant anti-Aß T cell response. Antisera recognize Aß oligomers, protofibrils, and fibrils. In this study, we evaluated the effect of prophylactic immunization with three doses of (1-11)E2 in alum in the 3xTg mouse model of AD. Immunization with (1-11)E2 efficiently induced anti-Aß antibodies, but afforded no protection against Aß accumulation and neuroinflammation. The identification of the features of the anti-Aß immune response that correlate with the ability to prevent Aß accumulation remains an open problem that deserves further investigation.