Identification and Functional Analysis of the Pre-piRNA 3' Trimmer in Silkworms.

PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3' ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3'-5' exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ∼35-40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.

Iruka Eliminates Dysfunctional Argonaute by Selective Ubiquitination of Its Empty State.

MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. We discovered a RING-type E3 ubiquitin ligase we named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. Our findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function.

The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms.

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.

Zucchini consensus motifs determine the mechanism of pre-piRNA production.

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.

The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells.

PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many "dead" transposon remnants from past invasions and have heterochromatic features. In the genome of silkworm ovary-derived cultured cells called BmN4, a well-established model for piRNA research, torimochi was previously annotated as a unique and specialized genomic region that can capture transgenes and produce new piRNAs bearing a trans-silencing activity. However, the sequence identity of torimochi has remained elusive. Here, we carefully characterized torimochi by utilizing the updated silkworm genome sequence and the long-read sequencer MinION. We found that torimochi is in fact a full-length gypsy-like LTR retrotransposon, which is exceptionally active and has massively expanded its copy number in BmN4 cells. Many copies of torimochi in BmN4 cells have features of open chromatin and the ability to produce piRNAs. Therefore, torimochi may represent a young, growing piRNA cluster, which is still "alive" and active in transposition yet capable of trapping other transposable elements to produce de novo piRNAs.

Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.

The two Gtsf paralogs in silkworms orthogonally activate their partner PIWI proteins for target cleavage.

The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte-specific factor 1 (Gtsf1), an evolutionarily conserved zinc finger protein, promotes catalysis by PIWI proteins. Many animals have multiple Gtsf1 paralogs; however, their respective roles in the piRNA pathway are not fully understood. Here, we dissected the roles of Gtsf1 and its paralog Gtsf1-like (Gtsf1L) in the silkworm piRNA pathway. We found that Gtsf1 and Gtsf1L preferentially bind the two silkworm PIWI paralogs, Siwi and BmAgo3, respectively, and facilitate the endonuclease activity of each PIWI protein. This orthogonal activation effect was further supported by specific reduction of BmAgo3-bound Masculinizer piRNA and Siwi-bound Feminizer piRNA, the unique piRNA pair required for silkworm feminization, upon depletion of Gtsf1 and Gtsf1L, respectively. Our results indicate that the two Gtsf paralogs in silkworms activate their respective PIWI partners, thereby facilitating the amplification of piRNAs.

Impact of Controlling Nutritional Status Score on Comprehensive Clinical Outcomes in Patients With Chronic Limb-Threatening Ischemia in a Local Area of Super-Aged Society.

PURPOSE: This study aimed to evaluate the relationship between nutritional status and hospital outcomes in patients with chronic limb-threatening ischemia (CLTI) in a local area of contemporary super-aged society of Japan. MATERIALS AND METHODS: We analyzed 131 consecutive patients with 179 lower limb diseases admitted to our hospital for the treatment of CLTI between April 2018 and March 2023. These 131 patients were divided into 3 groups according to hospital outcomes: home discharge (HD), out-of-home discharge (OD), and in-hospital death (ID). Patient and lesion backgrounds were compared among the 3 groups, and a multivariable regression analysis was used to analyze the interaction between malnutrition and composite hard endpoints. RESULTS: The median age was 82.8 years, and non-ambulatory patients comprised 61.8% of the study population. The HD group included more ambulatory and fewer patients with higher CONUT score or inflammation than OD or ID group. The Rutherford classification and Wound, Ischemia, and foot Infection stage were significantly more severe in the ID group than in the HD group. Endovascular treatment (EVT) was more often implemented in the HD (94.9%) and OD (81.7%) groups than in the ID group (60.0%). However, all EVT procedures in the ID group were performed until as distally as possible to achieve the target arterial path success contrary to some EVT procedures in the HD or ID group that targeted lesions only above the knee. Multivariate analysis showed that a non-ambulatory state (hazard ratio [HR]=3.65, 95% confidence interval [CI]=1.48-9.02) and a higher controlling nutritional status (CONUT) score (≥5) (HR=7.46, 95% CI=1.66-33.6) were significant predictors for composite endpoints (major amputation or ID). Patients with lower CONUT scores (≤4) showed better outcomes in all indices including overall survival, major amputation-free survival, and wound healing. CONCLUSION: Condition of the CLTI patients represented by higher CONUT score emerged as the most influential predictor of major amputation or ID. Furthermore, non-ambulatory status or condition of higher CONUT score affects the destination after discharge. Implementing multidisciplinary approaches to address patients' nutritional state and physical disability, in addition to revascularization, may enhance comprehensive prognoses in patients with CLTI. CLINICAL IMPACT: In this single-center retrospective study, we analyzed prognoses of 131 consecutive patients with 179 lower limb diseases admitted for the treatment of chronic limb-threatening ischemia (CLTI) between April 2018 and March 2023. Our main finding was that condition of the CLTI patients represented by higher controlling nutritional status (CONUT) score was the most significant predictor of either major amputation or in-hospital death. Furthermore, condition of higher CONUT score or non-ambulatory status affects the destination after discharge. This suggests that multidisciplinary approaches to address patients' nutritional state and physical disability, in addition to revascularization, may enhance the prognosis in patients with CLTI. This is the first report to evaluate nutritional status associated with comprehensive hospital outcomes in addition to previously reported hard endpoints, such as major amputation or overall survival, and will be of great help in future clinical practice.

A latent bleeding complication in ipsilateral common femoral puncture involving delayed diagnosis: a case report.

BACKGROUND: The trans femoral ipsilateral approach is often adopted for endovascular treatment (EVT) for better steerability of guidewires or better device deliverability. However, contrary to the trans femoral contralateral approach, ipsilateral antegrade puncture sometimes causes peculiar bleeding complications. CASE PRESENTATION: A 76-year-old female underwent EVT for chronic occlusion of the left superficial femoral artery (SFA) via the ipsilateral antegrade approach. After guidewire passage, we inflated the drug-coated balloons, but angiography showed blood flow stasis at the mid segment of the SFA. We also ensured prolonged balloon inflation, which resulted in favorable blood flow. While trying to ensure hemostasis, the blood pressure remained decreased, but neither bleeding nor superficial hematoma were observed at the puncture site. After hemostasis was achieved, we removed the surgical drape and noticed a swelling in the mid-portion of the thigh, distant from the puncture point. We then approached the left common femoral artery (CFA) contralaterally. Angiography showed continuous bleeding from a little bit distally to the sheath insertion point that was spreading through an intramuscular space. We stopped the bleeding with balloon tamponade inside the CFA. Angiography after hemostasis demonstrated blood flow stasis at the mid-segment of the SFA, similarly as that seen before. We confirmed compression of the SFA by a large hematoma using both intra- and extra- vascular ultrasound. Therefore, we deployed a self-expandable stent at the compressed SFA position. Finally, we achieved favorable blood flow on angiography. CONCLUSION: We encountered a case that latent bleeding unrecognized in the surgical field persisted while prolonged inflation of DCB was conducted at just proximal SFA. We could have avoided bailout stenting by noticing the bleeding incident in a timely manner. Prediction and prevention are essential for all kinds of procedural complications in EVT.

The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation.

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1-10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5' nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5' adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5' A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.

Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants.

Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA-Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposable elements and safeguard fertility in germ cells. Many protein factors required for piRNA biogenesis localize to perinuclear ribonucleoprotein (RNP) condensates named nuage, where target silencing and piRNA amplification are thought to occur. In mice, some of the piRNA factors are found in discrete cytoplasmic foci called processing bodies (P-bodies). However, the dynamics and biological significance of such compartmentalization of the piRNA pathway remain unclear. Here, by analyzing the subcellular localization of functional mutants of piRNA factors, we show that piRNA factors are actively compartmentalized into nuage and P-bodies in silkworm cells. Proper demixing of nuage and P-bodies requires target cleavage by the PIWI protein Siwi and ATP hydrolysis by the DEAD-box helicase BmVasa, disruption of which leads to promiscuous overproduction of piRNAs deriving from non-transposable elements. Our study highlights a role of dynamic subcellular compartmentalization in ensuring the fidelity of piRNA biogenesis.

A Multicenter Prospective Interventional Trial of Therapeutic Angiogenesis Using Bone Marrow-Derived Mononuclear Cell Implantation for Patients With Critical Limb-Threatening Ischemia Caused by Thromboangiitis Obliterans.

BACKGROUND: Thromboangiitis obliterans (TAO) can lead to the development of critical limb-threatening ischemia (CLTI). Despite conventional treatments, such as smoking cessation or revascularization, young patients (<50 years) still require limb amputation. Therapeutic angiogenesis using bone marrow-derived mononuclear cell (BM-MNC) implantation has been tested and shown to have reasonable efficacy in CLTI. In this multicenter prospective clinical trial, we evaluated the safety and efficacy of BM-MNC implantation in CLTI patients with TAO.Methods and Results: We enrolled 22 CLTI patients with skin perfusion pressure (SPP) <30 mmHg. The primary endpoint of this trial is the recovery of SPP in the treated limb after a 180-day follow-up period. Secondary endpoints include the pain scale score and transcutaneous oxygen pressure (TcPO2). One patient dropped out during follow-up, leaving 21 patients (mean age 48 years, 90.5% male, Fontaine Class IV) for analysis. BM-MNC implantation caused no serious adverse events and increased SPP by 1.5-fold compared with baseline. Surprisingly, this effect was sustained over the longer term at 180 days. Secondary endpoints also supported the efficacy of this novel therapy in relieving pain and increasing TcPO2. Major amputation-free and overall survival probabilities at 3 years among all enrolled patients were high (95.5% and 89.5%, respectively). CONCLUSIONS: BM-MNC implantation showed safety and significant efficacy in CLTI patients with TAO.

Emphysematous changes as red flag signs preceding rapidly progressive infectious aortic disease: two case reports.

BACKGROUND: Infectious aortic disease is a rare and fatal disease, that requires the appropriate intervention. An accurate diagnosis should be promptly established. However, this is difficult because the clinical manifestations of this disease vary and are non-specific. CASE PRESENTATION: (CASE 1) An 87-year-old male, presenting with generalized malaise and weight loss, was admitted for further examination. A chest computed tomography (CT) showed mediastinal emphysema. Empirical intravenous antibiotics were administered to address the non-specific infectious findings in the laboratory data. The treatment was effective, and the patient fully recovered. However, he was in shock due to aortic rupture and marked pseudo aneurysmal formation around the aortic arch day 25 of hospitalization. An emergency total aortic arch replacement was performed, and the patient was discharged. (CASE 2) An 82-year-old male who had undergone Y-graft replacement in the abdominal aorta 15 years previously was admitted due to general malaise and anorexia. Abdominal CT revealed emphysematous changes adjacent to the abdominal aorta. The patient responded favorably to empirical treatment with intravenous antibiotics and was discharged 19 days after admission. Four days after discharge, the patient went into cardiac arrest after an episode of hematemesis. Abdominal CT revealed an enlarged stomach and duodenum, filled with massive high-density contents proximal to the abdominal aorta. He died of hemorrhagic shock despite cardiopulmonary resuscitation. CONCLUSIONS: Although emphysematous changes are rare, they are red flag signs during the early stage of infectious aortic disease. Thus, physicians should remain vigilant for this kind of critical sign.

Tako-Tsubo Syndrome Following Pulmonary Vein Isolation for Atrial Fibrillation in a Patient with Primary Aldosteronism.

Tako-tsubo syndrome (TTS) can be triggered by emotional or physical stress and is characterized by transient left ventricular dysfunction with apical ballooning. Some neurologic disorders and pheochromocytoma serve as triggers for TTS, however, its association with primary aldosteronism (PA) is not well known. Pulmonary vein isolation (PVI) with catheter ablation for atrial fibrillation (AF) has been performed worldwide, and TTS following PVI has been reported as a rare complication. Sympathetic stimulation can play an important role in TTS development, however, its mechanism and risk factors are not yet understood.We describe a 72-year-old woman with PA who developed TTS after PVI with radiofrequency catheter ablation (RFCA) for symptomatic paroxysmal AF. Complete isolation of the pulmonary vein was carried out without any complications, however, she complained of epigastric discomfort 7 hours after the procedure. An electrocardiogram showed recurrent AF with a new negative-T wave and prolonged QT interval. Transthoracic echocardiography revealed apical ballooning and basal hypercontraction, characteristic of TTS, and coronary angiography showed no significant stenosis. She was diagnosed with TTS following RFCA for AF and managed well with conservative therapy.The present case suggests that TTS should be recognized as a complication associated with AF ablation. Moreover, PA may be involved in TTS development by increasing sympathetic activity. Further studies on the mechanism and characteristics of TTS are required.

Impact of Door-to-Balloon Time Reduction Depending on the Killip Classification in Patients with ST-Segment Elevation Myocardial Infarction Transported by Emergency Medical Services.

The coronavirus disease 2019 pandemic occurred in several countries, making the conventional medical system difficult to maintain. Recent recommendations aim to prevent nosocomial infections and infections among health care workers. Therefore, establishing a cardiovascular medical system under an emergency for patients with ST-segment elevation myocardial infarction (STEMI) is desired. This study aimed to determine the relationship between prognosis and door-to-balloon time (DBT) shortening based on the severity on arrival.This retrospective, multi-center, observational study included 1,127 consecutive patients with STEMI. These patients were transported by emergency medical services and underwent primary percutaneous coronary intervention. Patients were stratified according to the Killip classification: Killip 1 (n = 738) and Killip ≥ 2 (n = 389) groups.Patients in the Killip ≥ 2 group were older, with more females, and more severity on arrival than those in the Killip 1 group. The 30-day mortality rate in the Killip 1 and Killip ≥ 2 groups was 2.2% and 18.0%, respectively. The Killip ≥ 2 group had a significant difference in the 30-day mortality between patients with DBT ≤ 90 minutes and those with DBT > 90 minutes; however, this did not occur in the Killip 1 group. Furthermore, multivariate analysis revealed that DBT ≤ 90 minutes was not a significant predictive factor in the Killip 1 group; however, it was an independent predictive factor in the Killip ≥ 2 group.DBT shortening affected the 30-day mortality in STEMI patients with Killip ≥ 2, although not those with Killip 1.

H3K4me3 histone modification in baculovirus-infected silkworm cells.

Baculovirus infection modulates the chromatin states and gene expression of host insect cells. Here we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of H3 trimethylated at Lys4 (H3K4me3) histone modification in Bombyx mori nucleopolyhedrovirus-infected Bombyx mori cells. The ChIP-seq data revealed the changes of the genome-wide distribution and accumulation of euchromatic histone marks in host insect cells during the progression of baculovirus infection.

Early stent thrombosis confirmed in a cancer patient receiving regorafenib, despite triple antithrombotic therapy: a case report.

BACKGROUND: While developments in oncology have lengthened survival in patients with cancer, such patients often develop cardiovascular diseases. Thus, percutaneous coronary intervention (PCI) is frequently undertaken in them. Although stent thrombosis remains a fatal complication in stent-based PCI, worldwide consensus panels tend to recommend shorter duration of dual-antiplatelet therapy. This is based on its clinical efficacy that has resulted from technological innovation. However, there is insufficient discussion on the risk of stent thrombosis in cancer patients with coronary artery disease, especially in those undergoing chemotherapeutic regimens that have a risk for thrombosis, such as regimens with the anti-vascular endothelial growth factor. Presented here is a case of early stent thrombosis that occurred in a cancer patient on regorafenib, despite the administration of triple antithrombotic therapy. Case presentation A 66-year-old Japanese male patient received regorafenib for metastatic colorectal carcinoma and apixaban for deep vein thrombosis. Coronary angiography revealed severe stenosis in the proximal left anterior descending artery. A sirolimus-eluting stent was implanted, without malapposition and under-expansion, under intravascular ultrasound guidance while administering a triple antithrombotic therapy (aspirin: 100 mg/day, prasugrel: 3.75 mg/day, and apixaban: 5 mg/day). However, he was admitted to the hospital for exacerbation of heart failure 1 month after PCI. Coronary angiography revealed contrastive defects in the previous stent. Optical frequency domain imaging confirmed stent thrombosis. PCI was successfully performed with perfusion balloon long-inflation. Antithrombotic therapy was enhanced (aspirin: 100 mg/day, ticagrelor: 120 mg/day, and apixaban: 10 mg/day) and regorafenib was discontinued permanently. While ischemic events did not occur thereafter, the patient died due to metastatic carcinoma progression. CONCLUSIONS: This case suggests that anti-vascular endothelial growth factor might contribute to early stent thrombosis, despite triple antithrombotic therapy. Further discussion is needed on the surveillance and management of cancer patients with coronary artery disease receiving chemotherapy, which carries a risk of thrombosis.

Potential for small RNA production against Bombyx mori latent virus in Bombyx mori ovaries.

Bombyx mori latent virus (BmLV) is a positive, single-stranded insect RNA virus closely related to plant maculaviruses. BmLV was first isolated from Bombyx mori ovary-derived cell line BmN-4, and this virus has already infected most B. mori-derived cultured cell lines. We previously reported that small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways function cooperatively to maintain the amount of BmLV RNA for normal BmN-4 cell growth. On the other hand, BmLV does not propagate in B. mori larvae. Here we conducted BmLV injection into the larval body cavities of B. mori, and examined BmLV accumulation in larval ovaries where siRNA and piRNA pathways are both active, to investigate whether this in vivo resistance is governed by small RNA pathways. Expression levels of RNA-dependent RNA polymerase, coat protein, and p15 genes in BmLV-injected larval ovaries were extremely low compared with those in B. mori cultured cells, indicating that B. mori larval ovaries are more resistant to BmLV than B. mori cultured cells. We also sequenced small RNAs prepared from BmLV-injected larval ovaries and mapped them onto the BmLV genome. Although their amounts were very small, we were able to detect BmLV-derived small RNAs in the ovaries. According to their length distribution and nucleotide bias, they were likely to be siRNAs and piRNAs. These results suggest that B. mori ovaries can potentially produce small RNAs against BmLV, but the resistance of larval ovaries against BmLV is not dependent on RNA silencing pathways.

Whole-genome sequencing and comparative transcriptome analysis of Bombyx mori nucleopolyhedrovirus La strain.

The Bombyx mori nucleopolyhedrovirus (BmNPV) La is a variant BmNPV strain isolated in Laos. La has different features from BmNPV type strain T3 in virulence, production of the polyhedrin protein, and the formation of multicapsid occlusion-derived viruses. Here, the whole-genome sequence of La was compared to the sequences of nine BmNPV and two Bombyx mandarina nucleopolyhedrovirus strains. The complete La genome consisted of 127,618 base pairs with a G + C content of 40.3% and contained putative 136 open reading frames encoding more than 60 amino acids. The La genome lacked the bro-b gene and had the highest identity with that of the T3 strain. A comparison of the transcriptomes of La- and T3-infected cells showed that the expression levels of the polyhedrin and cathepsin genes were greater in cells infected with La as compared to those infected with T3. Interestingly, the virus genes with different RNA levels between the two BmNPV strains were assembled into five clusters in the genome of La. Also, the RNA levels of host ribosomal protein genes were significantly decreased in cells infected with La as compared to those infected with T3.