Distinct roles of two eIF4E isoforms in the germline of Caenorhabditis elegans.

Germ cells use both positive and negative mRNA translational control to regulate gene expression that drives their differentiation into gametes. mRNA translational control is mediated by RNA-binding proteins, miRNAs and translation initiation factors. We have uncovered the discrete roles of two translation initiation factor eIF4E isoforms (IFE-1, IFE-3) that bind 7-methylguanosine (m7G) mRNA caps during Caenorhabditiselegans germline development. IFE-3 plays important roles in germline sex determination (GSD), where it promotes oocyte cell fate and is dispensable for spermatogenesis. IFE-3 is expressed throughout the germline and localizes to germ granules, but is distinct from IFE-1 and PGL-1, and facilitates oocyte growth and viability. This contrasts with the robust expression in spermatocytes of IFE-1, the isoform that resides within P granules in spermatocytes and oocytes, and promotes late spermatogenesis. Each eIF4E is localized by its cognate eIF4E-binding protein (IFE-1:PGL-1 and IFE-3:IFET-1). IFE-3 and IFET-1 regulate translation of several GSD mRNAs, but not those under control of IFE-1. Distinct mutant phenotypes, in vivo localization and differential mRNA translation suggest independent dormant and active periods for each eIF4E isoform in the germline.

Triclosan induces apoptosis in Burkitt lymphoma-derived BJAB cells through caspase and JNK/MAPK pathways.

Burkitt's lymphoma (BL) is the fastest growing human tumor. Current treatment consists of a multiagent regimen of cytotoxic drugs with serious side effjects including tumor lysis, cardiotoxicity, hepatic impairment, neuropathy, myelosuppression, increased susceptibility to malignancy, and death. Furthermore, therapeutic interventions in areas of BL prevalence are not as feasible as in high-income countries. Therefore, there exists an urgent need to identify new therapies with a safer profile and improved accessibility. Triclosan (TCS), an antimicrobial used in personal care products and surgical scrubs, has gained considerable interest as an antitumor agent due to its interference with fatty acid synthesis. Here, we investigate the antitumor properties and associated molecular mechanisms of TCS in BL-derived BJAB cells. Dose-dependent cell death was observed following treatment with 10-100 µM TCS for 24 h, which was associated with membrane phospholipid scrambling, compromised permeability, and cell shrinkage. TCS-induced cell death was accompanied by elevated intracellular calcium, perturbed redox balance, chromatin condensation, and DNA fragmentation. TCS upregulated Bad expression and downregulated that of Bcl2. Moreover, caspase and JNK MAPK signaling were required for the full apoptotic activity of TCS. In conclusion, this report identifies TCS as an antitumor agent and provides new insights into the molecular mechanisms governing TCS-induced apoptosis in BL cells.

Nucleotide Excision Repair, XPA-1, and the Translesion Synthesis Complex, POLZ-1 and REV-1, Are Critical for Interstrand Cross-Link Repair in Caenorhabditis elegans Germ Cells.

Interstrand cross-links (ICLs) are adducts of covalently linked nucleotides in opposing DNA strands that obstruct replication and prime cells for malignant transformation or premature cell death. ICLs may be caused by alkylating agents or ultraviolet (UV) irradiation. These toxic lesions are removed by diverse repair mechanisms such as the Fanconi anemia (FA) pathway, nucleotide excision repair (NER), translesion synthesis (TLS), and homologous recombination (HR). In mammals, the xeroderma pigmentosum group F (XP-F) protein participates in both the FA pathway and NER, while DNA polymerase ζ (POLZ-1) and REV-1 mediate TLS. Nevertheless, little is known regarding the genetic determinants of these pathways in ICL repair and damage tolerance in germ cells. In this study, we examined the sensitivity of Caenorhabditis elegans germ cells to ICLs generated by trimethylpsoralen/ultraviolet A (TMP/UV-A) combination, and embryonic mortality was employed as a surrogate for DNA damage in germ cells. Our results show that XPA-1, POLZ-1, and REV-1 were more critical than FA pathway mediators in preserving genomic stability in C. elegans germ cells. Notably, mutant worms lacking both XPA-1 and POLZ-1 (or REV-1) were more sensitive to ICLs compared to either single mutant alone. Moreover, knockdown of XPA-1 and REV-1 leads to the retarded disappearance of RPA-1 and RAD-51 foci upon ICL damage. Since DNA repair mechanisms are broadly conserved, our findings may have ramifications for prospective therapeutic interventions in humans.

RNA-binding protein PUM2 regulates mesenchymal stem cell fate via repression of JAK2 and RUNX2 mRNAs.

The differentiation of mesenchymal stem cells (MSCs) into unwanted lineages can generate potential problems in clinical trials. Thus, understanding the molecular mechanisms, involved in this process, would help prevent unexpected complications. Regulation of gene expression, at the posttranscriptional level, is a new approach in cell therapies. PUMILIO is a conserved posttranscriptional regulator. However, the underlying mechanisms of PUMILIO, in vertebrate stem cells, remain elusive. Here, we show that depletion of PUMILIO2 (PUM2) blocks MSC adipogenesis and enhances osteogenesis. We also demonstrate that PUM2 works as a negative regulator on the 3'-untranslated regions of JAK2 and RUNX2 via direct binding. CRISPR/Cas9-mediated gene silencing of Pum2 inhibited lipid accumulation and induced excessive bone formation in zebrafish larvae. Our findings reveal novel roles of PUM2 in MSCs and provide potential therapeutic targets for related diseases.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET).

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1-5 mM (0.02%-0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

MPK-1/ERK regulatory network controls the number of sperm by regulating timing of sperm-oocyte switch in C. elegans germline.

The precise regulation of germline sexual fate is crucial for animal fertility. In C. elegans, the production of either type of gamete, sperm or oocyte, becomes mutually exclusive beyond the larval stage. Hermaphrodites initially produce sperm and then switch to produce oocytes. This change of fate during germline development is tightly controlled by several regulators. In C. elegans hermaphrodites, FBF-1 and FBF-2 (>95% identical, members of the Pumilio RNA-binding protein family) proteins function redundantly to promote the sperm-oocyte switch. Here, we demonstrate that loss of LIP-1 (dual specificity phosphatase) in fbf-1(ok91) single mutants leads to excess sperm production due to a delayed sperm-oocyte switch. This phenotype was dramatically rescued by depletion of MPK-1 (an ERK homolog). In contrast, loss of LIP-1 in fbf-2(q738) single mutants leads to a premature sperm-oocyte switch and loss of sperm. Notably, fbf-1 fbf-2; lip-1 triple mutants produce excess sperm. These results suggest that the MPK-1/ERK regulatory network, including FBF-1, FBF-2, and LIP-1, controls the number of sperm by regulating the timing of the sperm-oocyte switch in C. elegans.

Sorbus alnifolia protects dopaminergic neurodegeneration in Caenorhabditis elegans.

CONTEXT: The twigs of Sorbus alnifolia (Sieb. et Zucc.) K. Koch (Rosaceae) have been used to treat neurological disorders as a traditional medicine in Korea. However, there are limited data describing the efficacy of S. alnifolia in Parkinson's disease (PD). OBJECTIVE: This study was conducted to identify the protective effects of the methanol extracts of S. alnifolia (MESA) on the dopaminergic (DA) neurodegeneration in Caenorhabditis elegans. MATERIALS AND METHODS: To test the neuroprotective action of MESA, viability assay was performed after 48 h exposure to 1-methyl-4-phenylpyridine (MMP+) in PC12 cells and C. elegans (400 µM and 2 mM of MMP+, respectively). Fluorescence intensity was quantified using transgenic mutants such as BZ555 (Pdat-1::GFP) and and UA57 (Pdat-1::GFP and Pdat-1::CAT-2) to determine MESA's effects on DA neurodegeneration in C. elegans. Aggregation of α-synuclein was observed using NL5901 strain (unc-54p::α-synuclein::YFP). MESA's protective effects on the DA neuronal functions were examined by food-sensing assay. Lifespan assay was conducted to test the effects of MESA on the longevity. RESULTS: MESA restored MPP+-induced loss of viability in both PC12 cells and C. elegans (85.8% and 54.9%, respectively). In C. elegans, MESA provided protection against chemically and genetically-induced DA neurodegeneration, respectively. Moreover, food-sensing functions were increased 58.4% by MESA in the DA neuron degraded worms. MESA also prolonged the average lifespan by 25.6%. However, MESA failed to alter α-synuclein aggregation. DISCUSSION AND CONCLUSIONS: These results revealed that MESA protects DA neurodegeneration and recovers diminished DA neuronal functions, thereby can be a valuable candidate for the treatment of PD.

A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate.

Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.

Role of PUF-8/PUF protein in stem cell control, sperm-oocyte decision and cell fate reprogramming.

Pumilio and FBF (PUF) proteins are conserved stem cell regulators that maintain germline stem cells (GSCs) in worms and flies. Moreover, they are also present in vertebrate stem cells. The nematode Caenorhabditis elegans has multiple PUF proteins with specialized roles. Among them, PUF-8 protein controls multiple cellular processes, including proliferation, differentiation, sperm-oocyte decision, and cell fate reprogramming, depending on the genetic context in the C. elegans germline. In this review, we describe the possible mechanisms of how PUF-8 protein systematically controls multiple cellular processes in the C. elegans germline. Since PUF proteins are evolutionarily conserved, we suggest that a similar mechanism may be involved in controlling stem cell regulation and differentiation in other organisms, including humans.

Phosphorylation state of a Tob/BTG protein, FOG-3, regulates initiation and maintenance of the Caenorhabditis elegans sperm fate program.

FOG-3, the single Caenorhabditis elegans Tob/BTG protein, directs germ cells to adopt the sperm fate at the expense of oogenesis. Importantly, FOG-3 activity must be maintained for the continued production of sperm that is typical of the male sex. Vertebrate Tob proteins have antiproliferative activity and ERK phosphorylation of Tob proteins has been proposed to abrogate "antiproliferative" activity. Here we investigate FOG-3 phosphorylation and its effect on sperm fate specification. We found both phosphorylated and unphosphorylated forms of FOG-3 in nematodes. We then interrogated the role of FOG-3 phosphorylation in sperm fate specification. Specifically, we assayed FOG-3 transgenes for rescue of a fog-3 null mutant. Wild-type FOG-3 rescued both initiation and maintenance of sperm fate specification. A FOG-3 mutant with its four consensus ERK phosphorylation sites substituted to alanines, called FOG-3(4A), rescued partially: sperm were made transiently but not continuously in both sexes. A different FOG-3 mutant with its sites substituted to glutamates, called FOG-3(4E), had no rescuing activity on its own, but together with FOG-3(4A) rescue was complete. Thus, when FOG-3(4A) and FOG-3(4E) were both introduced into the same animals, sperm fate specification was not only initiated but also maintained, resulting in continuous spermatogenesis in males. Our findings suggest that unphosphorylated FOG-3 initiates the sperm fate program and that phosphorylated FOG-3 maintains that program for continued sperm production typical of males. We discuss implications of our results for Tob/BTG proteins in vertebrates.

C. elegans Germline as Three Distinct Tumor Models.

Tumor cells display abnormal growth and division, avoiding the natural process of cell death. These cells can be benign (non-cancerous growth) or malignant (cancerous growth). Over the past few decades, numerous in vitro or in vivo tumor models have been employed to understand the molecular mechanisms associated with tumorigenesis in diverse regards. However, our comprehension of how non-tumor cells transform into tumor cells at molecular and cellular levels remains incomplete. The nematode C. elegans has emerged as an excellent model organism for exploring various phenomena, including tumorigenesis. Although C. elegans does not naturally develop cancer, it serves as a valuable platform for identifying oncogenes and the underlying mechanisms within a live organism. In this review, we describe three distinct germline tumor models in C. elegans, highlighting their associated mechanisms and related regulators: (1) ectopic proliferation due to aberrant activation of GLP-1/Notch signaling, (2) meiotic entry failure resulting from the loss of GLD-1/STAR RNA-binding protein, (3) spermatogenic dedifferentiation caused by the loss of PUF-8/PUF RNA-binding protein. Each model requires the mutations of specific genes (glp-1, gld-1, and puf-8) and operates through distinct molecular mechanisms. Despite these differences in the origins of tumorigenesis, the internal regulatory networks within each tumor model display shared features. Given the conservation of many of the regulators implicated in C. elegans tumorigenesis, it is proposed that these unique models hold significant potential for enhancing our comprehension of the broader control mechanisms governing tumorigenesis.

Mitochondrial stress in GABAergic neurons non-cell autonomously regulates organismal health and aging.

Mitochondrial stress within the nervous system can trigger non-cell autonomous responses in peripheral tissues. However, the specific neurons involved and their impact on organismal aging and health have remained incompletely understood. Here, we demonstrate that mitochondrial stress in γ-aminobutyric acid-producing (GABAergic) neurons in Caenorhabditis elegans ( C. elegans ) is sufficient to significantly alter organismal lifespan, stress tolerance, and reproductive capabilities. This mitochondrial stress also leads to significant changes in mitochondrial mass, energy production, and levels of reactive oxygen species (ROS). DAF-16/FoxO activity is enhanced by GABAergic neuronal mitochondrial stress and mediates the induction of these non-cell-autonomous effects. Moreover, our findings indicate that GABA signaling operates within the same pathway as mitochondrial stress in GABAergic neurons, resulting in non-cell-autonomous alterations in organismal stress tolerance and longevity. In summary, these data suggest the crucial role of GABAergic neurons in detecting mitochondrial stress and orchestrating non-cell-autonomous changes throughout the organism.

The Ras-ERK MAPK regulatory network controls dedifferentiation in Caenorhabditis elegans germline.

How a committed cell can be reverted to an undifferentiated state is a central question in stem cell biology. This process, called dedifferentiation, is likely to be important for replacing stem cells as they age or get damaged. Tremendous progress has been made in understanding this fundamental process, but its mechanisms are poorly understood. Here we demonstrate that the aberrant activation of Ras-ERK MAPK signaling promotes cellular dedifferentiation in the Caenorhabditis elegans germline. To activate signaling, we removed two negative regulators, the PUF-8 RNA-binding protein and LIP-1 dual specificity phosphatase. The removal of both of these two regulators caused secondary spermatocytes to dedifferentiate and begin mitotic divisions. Interestingly, reduction of Ras-ERK MAPK signaling, either by mutation or chemical inhibition, blocked the initiation of dedifferentiation. By RNAi screening, we identified RSKN-1/P90(RSK) as a downstream effector of MPK-1/ERK that is critical for dedifferentiation: rskn-1 RNAi suppressed spermatocyte dedifferentiation and instead induced meiotic divisions. These regulators are broadly conserved, suggesting that similar molecular circuitry may control cellular dedifferentiation in other organisms, including humans.

Genetic and Chemical Controls of Sperm Fate and Spermatocyte Dedifferentiation via PUF-8 and MPK-1 in Caenorhabditis elegans.

Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) repress sperm fate at 20 °C and the dedifferentiation of spermatocytes into mitotic cells (termed "spermatocyte dedifferentiation") at 25 °C. Thus, double mutants lacking both PUF-8 and LIP-1 produce excess sperm at 20 °C, and their spermatocytes return to mitotically dividing cells via dedifferentiation at 25 °C, resulting in germline tumors. To gain insight into the molecular competence for spermatocyte dedifferentiation, we compared the germline phenotypes of three mutant strains that produce excess sperm-fem-3(q20gf), puf-8(q725); fem-3(q20gf), and puf-8(q725); lip-1(zh15). Spermatocyte dedifferentiation was not observed in fem-3(q20gf) mutants, but it was more severe in puf-8(q725); lip-1(zh15) than in puf-8(q725); fem-3(q20gf) mutants. These results suggest that MPK-1 (the C. elegans ERK1/2 MAPK ortholog) activation in the absence of PUF-8 is required to promote spermatocyte dedifferentiation. This idea was confirmed using Resveratrol (RSV), a potential activator of MPK-1 and ERK1/2 in C. elegans and human cells, respectively. Notably, spermatocyte dedifferentiation was significantly enhanced by RSV treatment in the absence of PUF-8, and its effect was blocked by mpk-1 RNAi. We, therefore, conclude that PUF-8 and MPK-1 are essential regulators for spermatocyte dedifferentiation and tumorigenesis. Since these regulators are broadly conserved, we suggest that similar regulatory circuitry may control cellular dedifferentiation and tumorigenesis in other organisms, including humans.

Post-transcriptional regulation of the Ras-ERK/MAPK signaling pathway.

The Ras-ERK/MAP (Mitogen-Activated Protein) kinase signaling pathway governs many cellular processes such as proliferation, differentiation, cell fate, homeostasis, and survival in all eukaryotes. Constitutive activation of the Ras-ERK/MAPK signaling pathway often leads to promotion of abnormal cell growth and tumorigenesis. Although the regulation of the Ras-ERK/MAPK signaling pathway by post-translational modification has been well elucidated, post-transcriptional regulations of this pathway are beginning to emerge in invertebrates and this work is extended to humans. In this review, we describe the conserved regulation of Ras-ERK/MAPK signaling by RNA-binding proteins (PUF, KH-domain, HuR, and LARP) and microRNAs (let-7 family miRNAs) and important implications for human diseases including cancers.

Chemical reprogramming of Caenorhabditis elegans germ cell fate.

Small molecules can control cell fate in vivo and may allow directed induction of desired cell types, providing an attractive alternative to transplant-based approaches in regenerative medicine. We have chemically induced functional oocytes in Caenorhabditis elegans adults that otherwise produced only sperm. These findings suggest that chemical approaches to therapeutic cell reprogramming may be feasible and provide a powerful platform for analyzing molecular mechanisms of in vivo cell reprogramming.

Non-invasive cancer detection in canine urine through Caenorhabditis elegans chemotaxis.

Cancer is the leading cause of death in companion animals, and successful early treatment has been a challenge in the veterinary field. We have developed the Non-Invasive Cancer Screening (N.C.S.) Study to perform cancer detection through the analysis of canine urine samples. The test makes use of the strong olfactory system of the nematode Caenorhabditis elegans, which was previously shown to positively respond to urine samples from human cancer patients. We performed a proof-of-concept study to optimize the detection capability in urine samples obtained from dogs with naturally occurring cancers. In this study, we established a scale for identifying the cancer risk based on the magnitude of the chemotaxis index of C. elegans toward a canine urine sample. Through validation, the N.C.S. Study achieved a sensitivity of 85%, showing that it is highly sensitive to indicate the presence of cancer across multiple types of common canine cancers. The test also showed a 90% specificity to cancer samples, indicating a low rate of over-identifying cancer risk. From these results, we have demonstrated the ability to perform low-cost, non-invasive cancer detection in companion animals-a method that can increase the ability to perform cancer diagnosis and treatment.

Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining genome stability, morphogenesis, and life span.

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.

Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death.

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.

The soma-germline communication: implications for somatic and reproductive aging.

Aging is characterized by a functional decline in most physiological processes, including alterations in cellular metabolism and defense mechanisms. Increasing evidence suggests that caloric restriction extends longevity and retards age-related diseases at least in part by reducing metabolic rate and oxidative stress in a variety of species, including yeast, worms, flies, and mice. Moreover, recent studies in invertebrates - worms and flies, highlight the intricate interrelation between reproductive longevity and somatic aging (known as disposable soma theory of aging), which appears to be conserved in vertebrates. This review is specifically focused on how the reproductive system modulates somatic aging and vice versa in genetic model systems. Since many signaling pathways governing the aging process are evolutionarily conserved, similar mechanisms may be involved in controlling soma and reproductive aging in vertebrates. [BMB Reports 2021; 54(5): 253-259].