A Retrospective Observational Study on Telemedicine in Prescribing Low-Dose Pills for Patients with Dysmenorrhea.

Introduction: In Japan, telemedicine has gradually expanded due to deregulation in response to the COVID-19 pandemic. However, its current status remains unclear, as it is primarily provided by general practitioners. Meanwhile, telemedicine has begun to be utilized for low-dose estrogen-progestin (LEP) prescriptions for dysmenorrhea. Methods: We conducted a retrospective analysis of medical record data from two gynecology clinics and performed an exploratory evaluation between a group that combined telemedicine and in-person visits during the initial 6 months of LEP treatment, and another group with only in-person visits. Results: After propensity score matching, 89 and 83 patients were eligible for the telemedicine and in-person groups, respectively, with 53 patients in both. The characteristics of both groups were similar after matching. There were no significant differences in the probability of abnormal uterine bleeding during the first 6 months of treatment (25% and 43% in each group; p = 0.064), side effects, or treatment efficacy between the two groups. The withdrawal rate at 6 months was significantly higher in the telemedicine group than in the in-person group (13% and 0%, p = 0.013). The average copayment for patients who covered 30% of the total cost was also significantly higher in the telemedicine group after 1 and 3 months of LEP prescription. Conclusion: The appropriate combination of telemedicine and in-person visits is currently employed in hospital visits, which does not differ significantly from in-person visits. Given the retrospective nature of this study and the limited number of cases, further investigation is necessary in the future.

The onset of cerebral infarction may be affected by differences in atmospheric pressure distribution patterns.

Background: Some papers have highlighted a possible causal relationship between the onset of ischemic stroke and weather conditions. This study aimed to elucidate the onset mechanism of cerebral infarction from a meteorological approach. We focused on the atmospheric pressure distribution patterns (APDPs). Methods: The subjects are 221 cases diagnosed as cardiogenic cerebral embolism (Group A) and 612 cases diagnosed as atherosclerotic cerebral thrombosis (Group B). We investigated the APDP on the date closest to the date and time of onset of cerebral infarction in each patient on the website and chose the most similar one from the reported 11 APDPs. Groups A and B were compared for clinical characteristics and the appearance rate of each APDP in each group. Results: The clinical characteristics of Groups A and B were consistent with some previously reported clinical characteristics of cerebral embolism and cerebral thrombosis except for smoking. The appearance rate of the other high-pressure type, which cannot be classified as either the anticyclone belt type or the migratory anticyclone type, in Group B was statistically significantly higher than that in Group A, and the appearance rate of the anticyclone belt type in Group A was statistically significantly higher than that in Group B (p < 0.05, Fisher's exact probability method, respectively). Conclusions: Cerebral embolism and cerebral thrombosis exhibited significant differences in APDPs on the day of onset. Dehydration particularly in the other high-pressure type or in the anticyclone belt type should be prevented. Further investigation should focus on the other meteorological factors.

Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP⁻/⁻ cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP⁻/⁻ Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

OGFOD1, a member of the 2-oxoglutarate and iron dependent dioxygenase family, functions in ischemic signaling.

The 2-oxoglutarate and iron dependent dioxygenase family are crucial for cellular adaptation to changes in oxygen concentration. We found that cells with OGFOD1 gene silencing in this family showed resistance to cell death under ischemia, and cDNA microarray analysis of OGFOD1 knockout human cells revealed downregulation of ATPAF1. Although reintroduction of the OGFOD1 wild-type gene to OGFOD1 KO cells restored ATPAF1 mRNA levels, the catalytically inactive OGFOD1 mutants did not. Furthermore, introduction of ATPAF1 gene to OGFOD1 KO cells induced ischemic cell death. Thus, OGFOD1 plays an important role in ischemic cell survival and an OGFOD1 iron binding residue is required for ATPAF1 gene expression.

FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells.

From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) subpathway. Yet the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H(2)O(2). This oxidant produces genotoxic lesions that are repaired by BER, suggesting that the cells have a deficiency in BER affecting survival. In experiments with extracts from the isogenic FEN1 null and wild-type cell lines, the LP-BER activity of FEN1 null cells was deficient, whereas repair by the single-nucleotide BER subpathway was normal. Other consequences of the FEN1 deficiency were also evaluated. These results illustrate that FEN1 plays a role in LP-BER in higher eukaryotes, presumably by processing the flap-containing intermediates of BER.

Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line.

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6, a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also inserted the gene for the tetracycline-controlled transactivator under the control of a potent CAG promoter. These modifications enabled us to easily obtain rare clones that express optimal amounts of tetracycline-regulated transgenes. We thereby generated a 'tetracycline-inducible conditional gene knockout' for the proliferation-associated SNF2-like gene (PASG) in a Nalm-6 cell line, in which the expression of PASG can be depleted in a tetracycline-dependent manner on a knockout background. This method is applicable to any human genes, making this gene-targeting system using the Nalm-6 cell line a promising tool for analyzing gene function.

Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting.

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.

KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation.

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.

NK314, a topoisomerase II inhibitor that specifically targets the alpha isoform.

Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2alpha is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2beta is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2alpha-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2alpha and not Top2beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an alpha isoform-specific manner. Heterozygous disruption of the human TOP2alpha gene confers increased NK314 resistance, whereas TOP2beta homozygous knock-out cells display increased NK314 sensitivity, indicating that the alpha isoform is the cellular target. We further show that the absence of Top2beta does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2alpha-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents.

Development of thermotolerance requires interaction between polymerase-beta and heat shock proteins.

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat-induced DNA double-strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat-labile repair proteins such as DNA polymerase-beta (Pol beta). Here the authors show that thermotolerance was partially suppressed in Pol beta(-/-) mouse embryonic fibroblasts (MEF) when compared to the wild-type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat-induced gamma H2AX was suppressed in the thermotolerant cells. These results suggest that Pol beta at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat-induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat-labile proteins besides Pol beta. The present novel findings provide strong evidence that Pol beta functions as a critical element involved in thermotolerance and exerts an important role in heat-induced DSB.

Highly proficient gene targeting by homologous recombination in the human pre-B cell line Nalm-6.

Gene targeting provides a powerful means for studying gene function by a reverse genetic approach. Despite recent rapid progress in gene knockdown technologies, gene knockout studies using human somatic cells will be of greater importance for analyzing the functions of human genes in greater detail. Although the frequency of gene targeting is typically very low in human cultured cells, we have recently shown that a human precursor B cell line, Nalm-6, exceptionally allows for high-efficiency gene targeting by homologous recombination. In addition, we have developed a quick and simplified method to construct gene-targeting vectors, which is applicable to all sequenced organisms as well as embryonic stem cells. The combination of the simplified vector construction technology and the highly efficient gene-knockout system using Nalm-6 cells has enabled us to disrupt virtually any locus of the human genome within one month. Our system will greatly facilitate gene-knockout studies in human cells.

The requirement of Artemis in double-strand break repair depends on the type of DNA damage.

Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.

Human Mus81 and FANCB independently contribute to repair of DNA damage during replication.

Recent studies suggest a crucial role for homologous recombination (HR) in repairing replication-associated DNA lesions. In mammals, the Mus81 endonuclease and the Fanconi anemia (FA) pathway have been implicated in HR repair; however, their functional relationship has remained unexplored. Here, we knockout the genes for Mus81 and FANCB, a component of the FA core complex, in the human Nalm-6 cell line. We show that Mus81 plays an important role in cell proliferation to suppress cell death when FANCB is missing, indicating a functional linkage between Mus81 and the FA pathway. In DNA cross-link repair, roles for Mus81 and the FA pathway appear to have an overlapping function. Intriguingly, Mus81 and FANCB act independently in surviving exposure to camptothecin (CPT). Although CPT-induced FANCD2 and Mus81 foci co-localize with Rad51, loss of Mus81, but not FANCB, results in significantly decreased levels of spontaneous and CPT-induced sister chromatid exchanges (SCEs). In addition, Mus81, unlike FANCB, has no significant role in gene targeting as well as in repairing hydroxyurea (HU)-induced stalls of replication forks. Collectively, our results provide the first evidence for differential functions of Mus81 and the FA pathway in repair of DNA damage during replication in human cells.

Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM.

The Bloom syndrome helicase BLM and the tumor-suppressor protein p53 play important roles in preserving genome integrity. Here, we knock out the genes for BLM and p53 in a human pre-B-cell line, Nalm-6. We show that p53 plays an important role in cell proliferation, but not apoptosis, when BLM is absent. Intriguingly, despite the apoptotic function of p53, BLM(/)TP53(/) cells were more sensitive than either single mutant to etoposide, an anticancer agent that poisons DNA topoisomerase II. Our results suggest a direct, BLM-independent role for p53 in etoposide-induced, topoisomerase II-mediated DNA damage in human cells.

A new system for analyzing LINE retrotransposition in the chicken DT40 cell line widely used for reverse genetics.

Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.

Decreased PARP-1 levels accelerate embryonic lethality but attenuate neuronal apoptosis in DNA polymerase beta-deficient mice.

In mammalian cells, DNA polymerase beta (Polbeta) and poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in base excision repair (BER) and single-strand break repair. Polbeta knockout mice exhibit extensive neuronal apoptosis during neurogenesis and die immediately after birth, while PARP-1 knockout mice are viable and display hypersensitivity to genotoxic agents and genomic instability. Although accumulating biochemical data show functional interactions between Polbeta and PARP-1, such interactions in the whole animal have not yet been explored. To study this, we generate Polbeta(-/-)PARP-1(-/-) double mutant mice. Here, we show that the double mutant mice exhibit a profound developmental delay and embryonic lethality at mid-gestation. Importantly, the degree of the neuronal apoptosis was dramatically reduced in PARP-1 heterozygous mice in a Polbeta null background. The reduction was well correlated with decreased levels of p53 phosphorylation at serine-18, suggesting that the apoptosis depends on the p53-mediated apoptosis pathway that is positively regulated by PARP-1. These results indicate that functional interactions between Polbeta and PARP-1 play important roles in embryonic development and neurogenesis.

Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines.

Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian--particularly human--genes.

Contribution of the myeloperoxidase-dependent oxidative system to host defence against Cryptococcus neoformans.

The in vivo contribution of reactive oxygen species produced by neutrophils against Cryptococcus infection is not widely recognized. Myeloperoxidase (MPO) is a neutrophil-specific enzyme that catalyses the production of hypohalous acids such as HOCl from H2O2. This study investigated the role of MPO in immunological defence against Cryptococcus neoformans in an MPO-deficient (MPO-/-) mouse model. The survival of MPO-/- mice infected either intranasally or intravenously with C. neoformans was lower than that of identically challenged wild-type mice. The MPO-/- mice that received intranasal injection of C. neoformans had significantly larger lung fungal burdens than wild-type mice. On day 7, MPO-/- mice had a significantly higher lung concentration of interleukin (IL)-4 and lower concentrations of IL-2, IL-12p70 and interferon (IFN)-gamma than wild-type mice, suggesting a weak Th1 response in the MPO-/- mice to C. neoformans. Pathologically, the MPO-/- mice with intranasal infection showed more severe pneumonia than wild-type mice, which was associated with an increase in the levels of IL-1alpha/beta in the lungs. In addition, in MPO-/- mice, the pulmonary infection disseminated to the brain with occasional meningitis. The keratinocyte-derived cytokine (KC) level in the brain of infected MPO-/- mice was higher than that of control mice. Both intranasal and intravenous infections resulted in a higher number of fungi in the spleen of MPO-/- mice compared to wild-type, suggesting decreased resistance to C. neoformans not only in the lungs but also in the spleen in the absence of MPO. Taken together, these data suggest a major role of MPO in the response to cryptococcal infection.