Effectiveness of Create ML in microscopy image classifications: a simple and inexpensive deep learning pipeline for non-data scientists.

Observing chromosomes is a time-consuming and labor-intensive process, and chromosomes have been analyzed manually for many years. In the last decade, automated acquisition systems for microscopic images have advanced dramatically due to advances in their controlling computer systems, and nowadays, it is possible to automatically acquire sets of tiling-images consisting of large number, more than 1000, of images from large areas of specimens. However, there has been no simple and inexpensive system to efficiently select images containing mitotic cells among these images. In this paper, a classification system of chromosomal images by deep learning artificial intelligence (AI) that can be easily handled by non-data scientists was applied. With this system, models suitable for our own samples could be easily built on a Macintosh computer with Create ML. As examples, models constructed by learning using chromosome images derived from various plant species were able to classify images containing mitotic cells among samples from plant species not used for learning in addition to samples from the species used. The system also worked for cells in tissue sections and tetrads. Since this system is inexpensive and can be easily trained via deep learning using scientists' own samples, it can be used not only for chromosomal image analysis but also for analysis of other biology-related images.

Predictors of increase in pacing threshold after transcatheter pacing system implantation due to micro-dislodgement.

BACKGROUND: Achieving a favorable pacing threshold with a Micra transcatheter pacing system (Micra-TPS) is needed to reduce battery depletion. In some cases, the threshold increases shortly after the device is implanted, and a higher pacing threshold may be required. This study aims to identify the causes and predictors of the increase in pacing threshold observed shortly after Micra-TPS implantation. METHODS: The study included 64 consecutive patients who underwent Micra-TPS implantation between 2017 and 2020. The patients were divided into two groups depending on their pacing threshold: the increased pacing threshold (IPT) group (threshold increased by ≥0.5 V/0.24 ms within 1 month of implantation) and the stable pacing threshold (SPT) group. RESULTS: Excluding four patients who could not be followed up, of the 60 remaining patients, nine (15%) were in the IPT group and 51 (85%) were in the SPT group. The IPT group had significantly lower implant impedance values and higher implant thresholds than the SPT group (582 ± 59 vs 755 ± 167 Ω [P < .001] and 1.29 ± 0.87 vs 0.71 ± 0.40 V/0.24 ms [P = .014]). Implant impedance and threshold may serve as predictors of a threshold increase after implantation (area under the curve: 0.737-0.943 and 0.586-0.926, respectively). CONCLUSIONS: An IPT was noted shortly after Micra-TPS implantation owing to micro-dislodgement because of insufficient anchoring of the device to the myocardium. Impedance >660 Ω and threshold <1.0 V/0.24 ms may predict an increase in pacing threshold.

Minichromosomes and artificial chromosomes in Arabidopsis.

Minichromosomes have been extensively used as tools for revealing the functional structures of eukaryotic chromosomes. However, the definition of a minichromosome is still ambiguous. Based on previous reports on various eukaryotes, minichromosomes are defined here to be chromosomes that are smaller than one third the size of the smallest chromosome in the given species. In Arabidopsis thaliana, therefore, chromosomes <8.5 Mb in length are classified as minichromosomes, although to date only six different minichromosomes have been found or created, probably due to their extremely small sizes that limit detection. Minichromosomes vary from 1.7 to 8.4 Mb in length and are much shorter than authentic chromosomes (25.3 to 38.0 Mb). Linear and circular minichromosomes have been identified, and both types are maintained as experimental lines. Most of the circular, ring-shaped minichromosomes in Arabidopsis are relatively stable at mitosis and transmissible to the next generation, regardless of the centromere form (dicentric or monocentric). Recently, a ring minichromosome was artificially generated by a combination of the Cre/LoxP and Ac/Ds systems. This artificial ring chromosome, AtARC1, has several advantages over the previously reported minichromosomes as a chromosome vector; therefore, this method of generating artificial ring chromosomes is expected to be improved for application to other plant species including important crops.

Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination.

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and 'artificial chromosomes' may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A. thaliana artificial ring chromosome 1), originated from a centromeric edge of the long arm of chromosome 2, but its size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180 bp repeats approximately 250 kb in length, compared with the 3 Mb domain on the original chromosome 2, centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.

Identification and characterization of functional centromeres of the common bean.

In higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution.

Tobacco karyotyping by accurate centromere identification and novel repetitive DNA localization.

Tobacco (Nicotiana tabacum) is an amphidiploid species (2n = 4x = 48, genome constitution SSTT) derived from a natural hybrid between Nicotiana sylvestris (2n = 2x = 24, SS) and Nicotiana tomentosiformis (2n = 2x = 24, TT). Genomic in situ hybridization (GISH), using the genomic DNA from these ancestral species as probes, revealed the chromosomal origins (S or T) and the occurrence of intergenomic translocations in N. tabacum. Fluorescence in situ hybridization (FISH) was also used to distinguish between chromosomes. However, the use of repetitive DNA sequences as probes for FISH analysis is limited by an inability to identify all chromosomes. In addition to this limitation, the occurrence of chromosomal tertiary constrictions can easily lead to the misclassification of chromosomes. To overcome these issues, immunostaining with anti-N. tabacum centromere-specific histone H3 antibody was carried out to determine the centromere position of each chromosome, followed by FISH analysis with ten distinct repetitive DNA probes. This approach allowed us to identify 22 of the 24 chromosome pairs in N. tabacum and revealed novel intergenomic chromosome rearrangements and B-chromosome-like minichromosomes. Hence, the combination of immunostaining with FISH and GISH is critical to accurately karyotype tobacco.

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

[Multiple mitral valve aneurysms, mycotic arterial embolism and aneurysms with infective endocarditis].

A 30-year-old woman with a more than 6-month history of fever, weight loss, general fatigue and dysesthesia of lower extremities was admitted to our hospital with a diagnosis of infective endocarditis. Blood cultures revealed Staphylococcus oralis. Echocardiography revealed severe mitral and moderate tricuspid regurgitation, as well as massive vegetations and aneurysms on the mitral valve. Computed tomography revealed an abdominal aortic aneurysm, left common and external iliac arterial aneurysms, and occlusion of the left common iliac, the deep femoral arteries and the bilateral tibioperoneal trunk. The ankle brachial pressure indices (ABI) were 0.94 (right) and 0.61 (left). She initially underwent mitral valve replacement and tricuspid annuloplasty. On postoperative day 24, the affected segments of the arteries were replaced with a woven Dacron bifurcated graft after resection of the mycotic abdominal and the iliac arterial aneurysms. We could not obtain a sufficient amount of omental pedicle to wrap the prosthesis. Her postoperative course was uneventful and mycotic arterial embolism and aneurysm did not recur.

Functional centromeres in Astragalus sinicus include a compact centromere-specific histone H3 and a 20-bp tandem repeat.

The centromere plays an essential role for proper chromosome segregation during cell division and usually harbors long arrays of tandem repeated satellite DNA sequences. Although this function is conserved among eukaryotes, the sequences of centromeric DNA repeats are variable. Most of our understanding of functional centromeres, which are defined by localization of a centromere-specific histone H3 (CENH3) protein, comes from model organisms. The components of the functional centromere in legumes are poorly known. The genus Astragalus is a member of the legumes and bears the largest numbers of species among angiosperms. Therefore, we studied the components of centromeres in Astragalus sinicus. We identified the CenH3 homolog of A. sinicus, AsCenH3 that is the most compact in size among higher eukaryotes. A CENH3-based assay revealed the functional centromeric DNA sequences from A. sinicus, called CentAs. The CentAs repeat is localized in A. sinicus centromeres, and comprises an AT-rich tandem repeat with a monomer size of 20 nucleotides.

Stability of monocentric and dicentric ring minichromosomes in Arabidopsis.

A dicentric ring minichromosome (miniδ) was identified in transgenic Arabidopsis thaliana and added to a wild type as a supernumerary chromosome. This line is relatively stable and has been maintained for generations, notwithstanding its ring and dicentric structure. To determine the mechanism for stable transmission of miniδ, the structure and behavior of two new types of ring minichromosomes (miniδ1 and miniδ1-1) derived from miniδ were investigated. Fluorescence in situ hybridization analysis revealed that miniδ1 is dicentric just like miniδ, whereas miniδ1-1 is monocentric. The estimated sizes of miniδ1 and miniδ1-1 were 3.8~5.0 and 1.7 Mb, respectively. The sizes of the two centromeres on miniδ1 were identical (ca. 270 kb) and similar to that of miniδ1-1 (ca. 250 kb). Miniδ1 was relatively stable during mitosis and meiosis, as is miniδ, whereas miniδ1-1 was unstable during mitosis, and the number of minichromosomes per cell varied. This possibly resulted from misdivision caused by a short centromere on monocentric miniδ1-1. Transmission through the female was quite limited for all three ring minichromosomes (0-3.2%), whereas that through the male was relatively high (15.4-27.3%) compared with that of other supernumerary chromosomes in Arabidopsis. Ring structure without telomeres itself seems not to limit the female transmission.

Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres.

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco.

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.

A New Total Knee Arthroplasty Technique for Valgus Knees that preserves the deep layer of the medial collateral ligament.

OBJECTIVE: The aim of this study was to assess the outcomes and complications, such as tibiofemoral instability and recurrence of valgus deformity, of total knee arthroplasty for valgus knees with a new technique preserving the deep layer of the medial collateral ligament. METHODS: In this study 33 (4 male and 29 female) patients, and a total of 36 (26 knees with osteoarthritis and 10 with rheumatoid arthritis) knees with a standing femorotibial angle (FTA) of <170° were included. Posterior Stabilized (PS) implants were used in 34 knees, rotating hinged knee implants were used in 2 knees. The procedures were carried out by a single surgeon protecting the deep layer of the medial collateral ligament. The patients' average age at the time of the operation was 67.6 ± 12 years, and the average follow-up period was 9.0 ± 3 years (range, 4-15 years). The Japanese Orthopaedic Association (JOA) knee score, range of motion (ROM) (extension/flexion; measured in degrees), FTA (measured in degrees) and complications were investigated. RESULTS: The Japanese Orthopaedic Association knee score significantly improved from an average of 51 ± 12 points before the operation to 86 ± 9 points after the operation (P <0.001). The extension ROM and flexion ROM improved from, -13 ± 13° to a postoperative average of -2 ± 4°, and 115 ± 25° to a postoperative average of 125 ± 18° respectively (P <0.001). The standing FTA significantly improved from 158 ± 9° to an average of 173 ± 2° after the operation (P <0.001). Thirty-four knees with severe valgus deformity were operated on using pos- terior stabilised implants, while only two knees required constrained implants. During follow-up, no complications, such as tibiofemoral instability, recurrence of valgus deformity, patellar necrosis, deep infection, wound problems, or peroneal nerve paralysis were observed. CONCLUSION: This study has shown us that after performing TKA while preserving the d-MCL for valgus knee deformity good clinical results were obtained and no complications were observed. LEVEL OF EVIDENCE: Level IV, Therapeutic Study.

Minichromosome stability induced by partial genome duplication in Arabidopsis thaliana.

Two partially reconstructed karyotypes (RK1 and RK2) of Arabidopsis thaliana have been established from a transformant, in which four structurally changed chromosomes (alpha, beta, gamma, and delta) were involved. Both karyotypes are composed of 12 chromosomes, 2n = 1" + 3" + 4" + 5" + alpha" + gamma" = 12 for RK1 and 2n = 3" + 4" + 5" + alpha" + beta" + gamma" = 12 for RK2, and these chromosome constitutions were relatively stable at least for three generations. Pairing at meiosis was limited to the homologues (1, 3, 4, 5, alpha, beta, or gamma), and no pairing occurred among non-homologous chromosomes in both karyotypes. For minichromosome alpha (mini alpha), precocious separation at metaphase I was frequently observed in RK2, as found for other minichromosomes, but was rare in RK1. This stable paring of mini alpha was possibly caused by duplication of the terminal tip of chromosome 1 that is characteristic of RK1.

Centromere targeting of alien CENH3s in Arabidopsis and tobacco cells.

The centromere is a region utilized for spindle attachment on a eukaryotic chromosome and essential for accurate chromatid segregation. In most eukaryotes, centromeres have specific DNA sequences and are capable of assembling specific proteins to form a complex called the kinetochore. Among these proteins, centromeric histone H3 (CENH3) is one of the most fundamental, since CENH3s have been found in all investigated functional centromeres and recruits other centromeric proteins. In this study, the localization of alien CENH3s were analyzed in Arabidopsis and tobacco-cultured cells to determine the interaction between species-specific centromeric DNA and CENH3. Results showed that CENH3 of Arabidopsis and tobacco were localized on centromeres in the tobacco-cultured cells, unlike the case with CENH3 of rice and Luzula. In addition to these CENH3s, CENH3 of Luzula was partially localized in the Arabidopsis cultured cells. These data suggest that only evolutionally close CENH3s are able to target centromeres in alien species. Furthermore, the ability to target alien centromeres of histone fold domains was investigated using amino-terminal deleted CENH3s.

Functional centromeres in soybean include two distinct tandem repeats and a retrotransposon.

The centromere as a kinetochore assembly site is fundamental to the partitioning of genetic material during cell division. In order to determine the functional centromeres of soybean, we characterized the soybean centromere-specific histone H3 (GmCENH3) protein and developed an antibody against the N-terminal end. Using this antibody, we cloned centromere-associated DNA sequences by chromatin immunoprecipitation. Our analyses indicate that soybean centromeres are composed of two distinct satellite repeats (GmCent-1 and GmCent-4) and retrotransposon-related sequences (GmCR). The possible allopolyploid origin of the soybean genome is discussed in view of the centromeric satellite sequences present.

Functional analysis of the Arabidopsis centromere by T-DNA insertion-induced centromere breakage.

Two minichromosomes (alpha and delta) in addition to two other aberrant chromosomes (beta and gamma) were found in a transgenic Arabidopsis plant produced by an in planta vacuum infiltration technique. The minichromosomes were successfully separated by successive crossing and selfing and added to wild-type Columbia (Col-0) as a supernumerary chromosome. FISH indicated that both of the two minichromosomes originated from the short arm of chromosome 2. The mini alpha chromosome contained the whole short-arm 2S and a truncated centromere (180-bp repeat cluster), whereas mini delta lacked the terminal region including telomere repeats. Pachytene FISH clearly revealed that mini delta comprised a ring chromosome carrying two copies of the region from the 180-bp repeat cluster to BAC-F3C11. Both of the 180-bp clusters (each approximately 500 kb in length) were thought to possess normal centromere functions because the centromere-specific histone H3 variant (HTR12) was detected on both clusters. Notwithstanding this dicentric and ring form, mini delta was stably transmitted to the next generations, perhaps because of its compact size (<4 Mb). Chromosome beta also comprised a dicentric-like structure, with one of the two 180-bp repeat sites derived from chromosome 1 and the other from chromosome 2. However, the latter was quite small and failed to bind HTR12. The data obtained in this study indicated that 500 kb of the 180-bp array of the chromosome 2 centromere, from the edge of the 180-bp array on the short-arm side, is sufficient to form a functional domain.

A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco.

Centromeres play an important role in segregating chromosomes into daughter cells, and centromeric DNA assembles specific proteins to form a complex referred to as the kinetochore. Among these proteins, centromere-specific histone H3 (CENH3) is one of the most characterized and found to be located only on active centromeres. We isolated four different CENH3-coding complementary DNAs (cDNAs), two from Nicotiana tabaccum and one each from the ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis and raised an antibody against N-terminal amino acid sequences deduced from the cDNAs. Immunostaining with the antibody revealed the preferential centromere localization, indicating that the cDNAs cloned in this study encode authentic tobacco CENH3. A tobacco centromeric DNA sequence (Nt2-7) was also identified by chromatin immunoprecipitation cloning using the antibody.

Characterization of the two centromeric proteins CENP-C and MIS12 in Nicotiana species.

Centromeres play an important role in chromosome transmission in eukaryotes and comprise specific DNA and proteins that form complexes called kinetochores. In tobacco, although a centromere-specific histone H3 (NtCENH3) and centromeric DNA sequence (Nt2-7) have been identified, no other kinetochore components have been determined. In this study, we isolated and characterized cDNAs encoding two centromeric proteins CENP-C and MIS12 from Nicotiana tabaccum. Two CENP-C homologues, NtCENP-C-1 and -2, isolated from N. tabaccum were similar to CENP-C from N. sylvestris and N. tomentosiformis, respectively. Similarly, two Mis12 homologues, NtMIS12-1 and -2, in N. tabaccum were shown to originate from N. sylvestris and N. tomentosiformis, respectively. Both respective homologues for CENP-C and Mis12 were expressed at the same level. This indicates that in a tetraploid species, N. tabaccum, two ancestral genes encoding the centromeric proteins participate equally in the functioning of centromeres.

Primary cardiac B-cell lymphoma presented as heart tamponade and atrioventricular block: a case report.

BACKGROUND: Primay cardiac lymphoma is rare, and its diagnosis is not determined until autopsy. CASE: A 49-year-old man presented with heart tamponade and atrioventricular block. Bloody pericardiac effusion showed a monotonous proliferation of atypical large mononuclear cells, which demonstrated a lambda light-chain monoclonality by the fluorescence-activated cell-sorter method and clonal rearrangement bands by Southern blot analysis of the IgH gene. Transvenous biopsy excised from the right atrial tumor was diagnosed as diffuse large B-cell lymphoma. He underwent chemotherapy and permanent pacemaker implantation and is alive and well. CONCLUSION: Liquid cytology of cardiac effusion was very useful for rapid diagnosis, leading to a better prognosis.