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We present a chip-based extended nano-Coulter counter (XnCC) that can detect nanoparticles affinity-selected from biological samples with low concentration limit-of-detection that surpasses existing resistive pulse sensors by 2–3 orders of magnitude. The XnCC was engineered to contain 5 in-plane pores each with an effective diameter of 350 nm placed in parallel and can provide high detection efficiency for single particles translocating both hydrodynamically and electrokinetically through these pores. The XnCC was fabricated in cyclic olefin polymer (COP) via nanoinjection molding to allow for high-scale production. The concentration limit-of-detection of the XnCC was 5.5 × 103particles/mL, which was a 1,100-fold improvement compared to a single in-plane pore device. The application examples of the XnCC included counting affinity selected SARS-CoV-2 viral particles from saliva samples using an aptamer and pillared microchip; the selection/XnCC assay could distinguish the COVID-19(+) saliva samples from those that were COVID-19(−). In the second example, ovarian cancer extracellular vesicles (EVs) were affinity selected using a pillared chip modified with a MUC16 monoclonal antibody. The affinity selection chip coupled with the XnCC was successful in discriminating between patients with high grade serous ovarian cancer and healthy donors using blood plasma as the input sample.

This study from budding yeast provides mechanistic insights into how accumulation of R-loops at centromeric chromatin perturbs kinetochore integrity and contributes to chromosome instability.

Mothers infected with human T-cell lymphotropic virus type II (HTLV-II) apparently do not pass it on to their children during pregnancy. Researchers measured HTLV-II antibody levels in 185 children between four days and 10 years old. HTLV-II antibody was not found in 62 samples taken from 30 children of intravenous drug user (IVDU) mothers or in those from 117 children of non-IVDU mothers. HTLV-II antibody was detected in the blood of 15 infants under the age of six months whose mothers were IVDUs. Six of these children later tested negative once their maternal antibodies stopped circulating. Children born to HTLV-II-positive mothers may develop antibodies later. Infection with HTLV-II is common among IVDUs.

The structural maintenance of chromosome complexes condensin and cohesin have disparate geometric localizations in chromatin. The different chromatin-tethering modalities of the complexes result in their partitioning in the presence of an extensional force on chromatin. Condensin’s ability to form chromatin loops drives its axial localization.

Mechanisms that drive DNA damage-induced chromosome mobility include relaxation of external tethers to the nuclear envelope and internal chromatin–chromatin tethers. Together with microtubule dynamics, these can mobilize the genome in response to DNA damage.

Cdc5 associates with centromeric chromatin during mitosis. Cdc5 plays a critical role in the differential removal of cohesin from centromeric chromatin compared to chromosome arms.

A novel Pat1-dependent mechanism is identified for the protection of kinetochore-associated Cse4 from ubiquitination in order to ensure faithful chromosome segregation and genomic stability.

Molecular crowding of DNA loops exerts a radial force on the kinetochore. Previous observations of the kinetochore and pericentric cohesin revealed a paradox upon spindle perturbation. Using simulations of the thermodynamics of DNA, we resolve the experimental results and provide evidence for mechanical feedback between chromatin and the kinetochore.

Dynamics of histones under tension in the pericentromere depends on RSC and ISW2 chromatin remodeling. The underlying pericentromeric chromatin forms a platform that is required to maintain kinetochore structure when under spindle-based tension.

ABSTRACTEnteroviruses (EVs) are common seasonal viruses that are associated with a variety of diseases. High-quality monoclonal antibodies (MAbs) are needed to improve the accuracy of EV diagnosis in clinical laboratories. In the present study, the full-length VP1 genes of poliovirus 1 (Polio 1) and coxsackievirus B3 (Cox B3) were cloned, and the encoded proteins were expressed and used as antigens in an attempt to raise broad-spectrum MAbs to EVs. Two pan-EV MAbs were isolated: one raised against Polio 1 VP1 and the other against Cox B3 VP1. The binding sites of both pan-EV MAbs were mapped to an amino acid sequence within a conserved region in the N terminus of Polio 1 VP1 by peptide and competition enzyme-linked immunosorbent assay. Two additional MAbs, an EV70-specific MAb and an EV71/Cox A16-bispecific MAb, developed against EV70 and 71 VP1 proteins, were pooled with the two pan-EV MAbs (pan-EV MAb mix) and tested for their sensitivity and specificity in the staining of various virus-infected cells. The pan-EV MAb mix detected all 40 prototype EVs tested and showed no cross-reactivity to 18 different non-EV human viruses. Compared with two commercially available EV tests, the pan-EV MAb mix exhibited higher specificity than one test and broader spectrum reactivity than the other. Thus, our study demonstrates that full-length Polio 1 VP1 and Cox B3 VP1 can serve as effective antigens for developing a pan-EV MAb and that the pan-EV MAb mix can be used for the laboratory diagnosis of a wide range of EV infections.

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.

ABSTRACTA pan-virus DNA microarray (Virochip) was used to detect a human metapneumovirus (hMPV) strain associated with a critical respiratory tract infection in an elderly adult with chronic lymphocytic leukemia. This infection had previously eluded diagnosis despite extensive microbiological testing for possible etiologic agents. The patient's hMPV strain did not grow in viral culture, and only one of five specific reverse transcription-PCR assays for hMPV was positive.

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being “pushed” by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus “pulling” the nucleus toward the bud neck. Failure of “pulling” is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9,dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.

Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoriais a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein–GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system , the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90–120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 μm-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein–GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology , and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) .