SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes.

Although an increased risk of myocarditis has been observed after vaccination with mRNA encoding severe acute respiratory syndrome coronavirus 2 spike protein, its underlying mechanism has not been elucidated. This study investigated the direct effects of spike receptor-binding domain (S-RBD) on human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs). Immunostaining experiments using ACE2 wild-type (WT) and knockout (KO) iPSC-CMs treated with purified S-RBD demonstrated that S-RBD was bound to ACE2 and internalized into the subcellular space in the iPSC-CMs, depending on ACE2. Immunostaining combined with live cell imaging using a recombinant S-RBD fused to the superfolder GFP (S-RBD-sfGFP) demonstrated that S-RBD was bound to the cell membrane, co-localized with RAB5A, and then delivered from the endosomes to the lysosomes in iPSC-CMs. Quantitative PCR array analysis followed by single cell RNA sequence analysis clarified that S-RBD-sfGFP treatment significantly upregulated the NF-kß pathway-related gene (CXCL1) in the differentiated non-cardiomyocytes, while upregulated interferon (IFN)-responsive genes (IFI6, ISG15, and IFITM3) in the matured cardiomyocytes. S-RBD-sfGFP treatment promoted protein ISGylation, an ISG15-mediated post-translational modification in ACE2-WT-iPSC-CMs, which was suppressed in ACE2-KO-iPSC-CMs. Our experimental study demonstrates that S-RBD is internalized through the endolysosomal pathway, which upregulates IFN-responsive genes and promotes ISGylation in the iPSC-CMs.

Molecular detection of Blastocystis sp. subtype 14 in the Yezo sika deer (Cervus nippon yesoensis) in Hokkaido, Japan.

This study describes the first report of Blastocystis sp. colonization in the sika deer (Cervus nippon) in Japan and in other animals in Hokkaido, Japan. Blastocystis sp. is one of the most widespread intestinal protist in a wide range of animals. Blastocystis sp. isolated from mammalian and avian species have been classified into 17 subtypes (STs). Some of the STs are zoonotic. The aim of this study was to evaluate Blastocystis sp. colonization in the Yezo sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. The Yezo sika deer are currently overabundant and they are expanding their habitat to humans and livestock. A total of 132 deer fecal samples were subjected for molecular detection of Blastocystis sp. Of these, 60 (45.5%) samples were positive using PCR, which targets the small subunit ribosomal RNA gene sequence. All Blastocystis sp. DNA sequences from the Yezo sika deer were genotyped into ST14, which were originally reported in cattle. These findings indicate that the current public health risks of Blastocystis sp. from the Yezo sika deer is low, although more detailed future analysis is required.

Usefulness of electron microscopy in the diagnosis of wild-type transthyretin cardiac amyloidosis.

Wild-type transthyretin cardiac amyloidosis (ATTRwt) has been recognized as an important cause of heart failure with preserved ejection fraction; thus, its accurate diagnosis is crucial. Herein, we describe the case of a 76-year-old man who presented with dyspnea and palpitation. On observing the laboratory evaluations and clinical course, we suspected cardiac amyloidosis. However, optical microscopic analysis by Congo-red and direct fast scarlet staining revealed no amyloid deposits in the biopsy samples. Therefore, a more thorough investigation was pursued by examining the myocardial tissue under electron microscopy. We could recognize amyloid deposits between the myocardial fibers using electron microscopy. We submitted all the pathological specimens to a specialized facility for genetic testing to ensure the accurate diagnosis of the amyloidosis disease type. As a result, a biopsy sample from the minor salivary gland was stained with the Congo red stain. Anti-transthyretin antibody detected using immunohistochemical analysis of amyloidosis supported the presence of transthyretin form of amyloid proteins. Genetic testing revealed the absence of TTR gene mutations. The final diagnosis was ATTRwt. We believe that this case suggests the usefulness of electron microscopy in the diagnosis of ATTRwt and other related disorders. Further study is warranted to validate our findings.

A cavotricuspid isthmus pouch revealed to be a breakout site for gap conduction of recurrent common atrial flutter.

A cavotricuspid isthmus pouch can be a breakout site for gap conduction of cavotricuspid isthmus block line. If the previous block line is electrically silent, high-density 3-D mapping and pouchgraphy are useful to find the pouch and ablate within it.

Pseudoblock of cavotricuspid isthmus via detouring gap conduction.

To detect detouring gap conduction, as demonstrated in this case, 3-D mapping is useful in addition to conventional methods.

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer.

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.

Disappearance of an anticoagulation-resistant left atrial appendage thrombus after administration of low-dose pimobendan.

Anticoagulation therapy is essential for preventing thrombus formation in the left atrial appendage (LAA) and subsequent ischemic strokes in patients with atrial fibrillation (AF). The complete disappearance of any existing LAA thrombi is crucial before AF ablation. Currently, warfarin and direct oral anticoagulants are widely used for this purpose. However, treatment strategies for anticoagulation-resistant LAA thrombi are not well established. Here, we present a case of an 85-year-old male who was scheduled to undergo AF ablation. He developed an LAA thrombus that was resistant to 300 mg/day of dabigatran. Low-dose pimobendan was prescribed in addition to dabigatran; three months later, the thrombus was dissolved successfully. This case demonstrates the potential efficacy of a low-dose oral inotrope for treating an anticoagulation-resistant LAA thrombus. .

Prevalence and phylogenetic analysis of Cryptosporidium infections in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido, Japan.

The Yezo sika deer (Cervus nippon yesoensis) on the island of Hokkaido, Japan are currently recognized as overabundant. Hunting is used to control the deer population, and this has increased the supply of game meat, which is associated with a high risk of various food-borne infections. Additionally, the sub-prefecture Tokachi has a dense population of livestock, which are potentially at risk of cross-species infections from the deer. In this study, we undertook the first analysis of the incidence of Cryptosporidium infection in the Yezo sika deer in the Tokachi area using polymerase chain reaction testing and phylogenetic analysis. Polymerase chain reaction analysis showed Cryptosporidium species present in 7.5% of fecal samples (13/173) collected from deer hunted between 2016 and 2017. However, the zoonotic Cryptosporidium paruvm parasite was not detected in the phylogenetic analysis; when sequenced, all species in the positive samples matched the Cryptosporidium deer genotype. However, deer may act as a reservoir of the zoonotic Cryptosporidium parvum parasite, which affects both humans and livestock. Therefore, we recommend the continuation of surveys of the incidence of Cryptosporidium infections in Yezo sika deer.

Incidence of deep vein thrombosis from screening by venous ultrasonography in Japanese patients.

The aim of the present study was to investigate the incidence of deep vein thrombosis (DVT) patients in a Japanese population by screening them with venous ultrasonography. This retrospective, single-center analysis examined 963 patients who underwent venous ultrasonography in 2015. The primary outcome was the incidence of DVT, and secondary outcomes were predictive factors of DVT in patient characteristics, treatment strategy for DVT patients, and proportion of post thrombotic syndrome (PTS). The overall incidence rate of DVT was 10.3% (100/963). The location of thrombus was the iliac vein in 3.6% (n = 35), the femoral vein in 4.4% (n = 43), and the calf vein in 8.2% (n = 79) of the patients. The main complaint or purpose of examination was perioperative screening in 37% of the patients, leg edema and/or limb swelling in 27% of the patients, and skin disease in 8% of the patients. In a multivariate analysis, the incidence of DVT was significantly higher in the hospitalization group and the "having symptoms" group. Fifty percent of DVTs received treatment, and almost all therapies were medical treatment with oral anticoagulants (OAC). Within a three-year follow-up period, the proportion of PTS was 27% with no significant difference between anticoagulation or non-anticoagulation patients; the risk of PTS was in concomitant varicose veins. In conclusion, of the patients, 10.3% were diagnosed with DVT, and only half received treatment with only anticoagulation.