Biosymbiotic haptic feedback - Sustained long term human machine interfaces.

Haptic technology permeates diverse fields and is receiving renewed attention for VR and AR applications. Advances in flexible electronics, facilitate the integration of haptic technologies into soft wearable systems, however, because of small footprint requirements face challenges of operational time requiring either large batteries, wired connections or frequent recharge, restricting the utility of haptic devices to short-duration tasks or low duty cycles, prohibiting continuously assisting applications. Currently many chronic applications are not investigated because of this technological gap. Here, we address wireless power and operation challenges with a biosymbiotic approach enabling continuous operation without user intervention, facilitated by wireless power transfer, eliminating the need for large batteries, and offering long-term haptic feedback without adhesive attachment to the body. These capabilities enable haptic feedback for robotic surgery training and posture correction over weeks of use with neural net computation. The demonstrations showcase that this device class expands use beyond conventional brick and strap or epidermally attached devices enabling new fields of use for imperceptible therapeutic and assistive haptic technologies supporting care and disease management.

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure.

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

Cell-cycle and Age-Related Modulations in Mouse Chromosome Stiffness.

The intricate structure of chromosomes is complex, and many aspects of chromosome configuration/organization remain to be fully understood. Measuring chromosome stiffness can provide valuable insights into their structure. However, the nature of chromosome stiffness, whether static or dynamic, remains elusive. In this study, we analyzed chromosome stiffness in MI and MII oocytes. We revealed that MI oocytes had a ten-fold increase in stiffness compared to mitotic chromosomes, whereas chromosome stiffness in MII oocytes was relatively low chromosome. We then investigated the contribution of meiosis-specific cohesin complexes to chromosome stiffness in MI and MII oocytes. Surprisingly, the Young's modulus of chromosomes from the three meiosis-specific cohesin mutants did not exhibit significant differences compared to the wild type, indicating that these proteins may not play a substantial role in determining chromosome stiffness. Additionally, our findings revealed an age-related increase in chromosome stiffness in MI oocytes. Age correlates with elevated DNA damage levels, so we investigated the impact of etoposide-induced DNA damage on chromosome stiffness, discovering a reduction in stiffness in response to such damage in MI oocytes. Overall, our study underscores the dynamic nature of chromosome stiffness, subject to changes influenced by the cell cycle and age.

SMC5/6 Promotes Replication Fork Stability via Negative Regulation of the COP9 Signalosome.

It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

GRAS-1 is a novel regulator of early meiotic chromosome dynamics in C. elegans.

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.

SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Modeling Human Gonad Development in Organoids.

BACKGROUND: Our learning about human reproductive development is greatly hampered due to the absence of an adequate model. Animal studies cannot truthfully recapitulate human developmental processes, and studies of human fetal tissues are limited by their availability and ethical restrictions. Innovative three-dimensional (3D) organoid technology utilizing human pluripotent stem cells (hPSCs) offered a new approach to study tissue and organ development in vitro. However, a system for modeling human gonad development has not been established, thus, limiting our ability to study causes of infertility. METHODS: In our study we utilized the 3D hPSC organoid culture in mini-spin bioreactors. Relying on intrinsic self-organizing and differentiation capabilities of stem cells, we explored whether organoids could mimic the development of human embryonic and fetal gonad. RESULTS: We have developed a simple, bioreactor-based organoid system for modeling early human gonad development. Male hPSC-derived organoids follow the embryonic gonad developmental trajectory and differentiate into multipotent progenitors, which further specialize into testicular supporting and interstitial cells. We demonstrated functional activity of the generated cell types by analyzing the expression of cell type-specific markers. Furthermore, the specification of gonadal progenitors in organoid culture was accompanied by the characteristic architectural tissue organization. CONCLUSION: This organoid system opens the opportunity for detailed studies of human gonad and germ cell development that can advance our understanding of sex development disorders. Implementation of human gonad organoid technology could be extended to modeling causes of infertility and regenerative medicine applications.

Evaluation of Chemcatcher® passive samplers for pesticide monitoring using high-frequency catchment scale data.

Passive samplers (PS) have been proposed as an enhanced water quality monitoring solution in rivers, but their performance against high-frequency data over the longer term has not been widely explored. This study compared the performance of Chemcatcher® passive sampling (PS) devices with high-frequency sampling (HFS: 7-hourly to daily) in two dynamic rivers over 16 months. The evaluation was based on the acid herbicides MCPA (2-methyl-4-chlorophenoxyacetic acid), mecoprop-P, fluroxypyr and triclopyr. The impact of river discharge parameters on Chemcatcher® device performance was also explored. Mixed effects modelling showed that time-weighted mean concentration (TWMC) and flow-weighted mean concentration (FWMC) values obtained by the HFS approach were both significantly higher (p < 0.001) than TWMC values determined from PS regardless of river or pesticide. Modelling also showed that TWMCPS values were more similar to TWMCHFS than FWMCHFS values. However, further testing revealed that MCPA TWMC values from HFS and PS were not significantly different (p > 0.05). There was little indication that river flow parameters altered PS performance-some minor effects were not significant or consistent. Despite this, the PS recovery of very low concentrations indicated that Chemcatcher® devices may be used to evaluate the presence/absence and magnitude of acid herbicides in hydrologically dynamic rivers in synoptic type surveys where space and time coverage is required. However, a period of calibration of the devices in each river would be necessary if they were intended to provide a quantitative review of pesticide concentration as compared with HFS approaches.

Upstream open reading frames control PLK4 translation and centriole duplication in primordial germ cells.

Centrosomes are microtubule-organizing centers comprised of a pair of centrioles and the surrounding pericentriolar material. Abnormalities in centriole number are associated with cell division errors and can contribute to diseases such as cancer. Centriole duplication is limited to once per cell cycle and is controlled by the dosage-sensitive Polo-like kinase 4 (PLK4). Here, we show that PLK4 abundance is translationally controlled through conserved upstream open reading frames (uORFs) in the 5' UTR of the mRNA. Plk4 uORFs suppress Plk4 translation and prevent excess protein synthesis. Mice with homozygous knockout of Plk4 uORFs (Plk4 Δu/Δu ) are viable but display dramatically reduced fertility because of a significant depletion of primordial germ cells (PGCs). The remaining PGCs in Plk4 Δu/Δu mice contain extra centrioles and display evidence of increased mitotic errors. PGCs undergo hypertranscription and have substantially more Plk4 mRNA than somatic cells. Reducing Plk4 mRNA levels in mice lacking Plk4 uORFs restored PGC numbers and fully rescued fertility. Together, our data uncover a specific requirement for uORF-dependent control of PLK4 translation in counterbalancing the increased Plk4 transcription in PGCs. Thus, uORF-mediated translational suppression of PLK4 has a critical role in preventing centriole amplification and preserving the genomic integrity of future gametes.

Deletion of NFIX results in defective progression through meiosis within the mouse testis†.

Members of the nuclear factor I (NFI) family are key regulators of stem cell biology during development, with well-documented roles for NFIA, NFIB, and NFIX in a variety of developing tissues, including brain, muscle, and lung. Given the central role these factors play in stem cell biology, we posited that they may be pivotal for spermatogonial stem cells or further developing spermatogonia during testicular development. Surprisingly, in stark contrast to other developing organ systems where NFI members are co-expressed, these NFI family members show discrete patterns of expression within the seminiferous tubules. Sertoli cells (spermatogenic supporting cells) express NFIA, spermatocytes express NFIX, round spermatids express NFIB, and peritubular myoid cells express each of these three family members. Further analysis of NFIX expression during the cycle of the seminiferous epithelium revealed expression not in spermatogonia, as we anticipated, but in spermatocytes. These data suggested a potential role for NFIX in spermatogenesis. To investigate, we analyzed mice with constitutive deletion of Nfix (Nfix-null). Assessment of germ cells in the postnatal day 20 (P20) testes of Nfix-null mice revealed that spermatocytes initiate meiosis, but zygotene stage spermatocytes display structural defects in the synaptonemal complex, and increased instances of unrepaired DNA double-strand breaks. Many developing spermatocytes in the Nfix-null testis exhibited multinucleation. As a result of these defects, spermatogenesis is blocked at early diplotene and very few round spermatids are produced. Collectively, these novel data establish the global requirement for NFIX in correct meiotic progression during the first wave of spermatogenesis.

PLK1 depletion alters homologous recombination and synaptonemal complex disassembly events during mammalian spermatogenesis.

Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation. Recombination is facilitated by the formation and repair of programmed DNA double-strand breaks. These DNA breaks are repaired via recombination between maternal and paternal homologous chromosomes and a subset result in the formation of crossovers. HR and crossover formation is facilitated by synapsis of homologous chromosomes by a proteinaceous scaffold structure known as the synaptonemal complex (SC). Recent studies in yeast and worms have indicated that polo-like kinases (PLKs) regulate several events during meiosis, including DNA recombination and SC dynamics. Mammals express four active PLKs (PLK1-4), and our previous work assessing localization and kinase function in mouse spermatocytes suggested that PLK1 coordinates nuclear events during meiotic prophase. Therefore, we conditionally mutated Plk1 in early prophase spermatocytes and assessed stages of HR, crossover formation, and SC processes. Plk1 mutation resulted in increased RPA foci and reduced RAD51/DMC1 foci during zygonema, and an increase of both class I and class II crossover events. Furthermore, the disassembly of SC lateral elements was aberrant. Our results highlight the importance of PLK1 in regulating HR and SC disassembly during spermatogenesis.

SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

Meiotic recombination mirrors patterns of germline replication in mice and humans.

Genetic recombination generates novel trait combinations, and understanding how recombination is distributed across the genome is key to modern genetics. The PRDM9 protein defines recombination hotspots; however, megabase-scale recombination patterning is independent of PRDM9. The single round of DNA replication, which precedes recombination in meiosis, may establish these patterns; therefore, we devised an approach to study meiotic replication that includes robust and sensitive mapping of replication origins. We find that meiotic DNA replication is distinct; reduced origin firing slows replication in meiosis, and a distinctive replication pattern in human males underlies the subtelomeric increase in recombination. We detected a robust correlation between replication and both contemporary and historical recombination and found that replication origin density coupled with chromosome size determines the recombination potential of individual chromosomes. Our findings and methods have implications for understanding the mechanisms underlying DNA replication, genetic recombination, and the landscape of mammalian germline variation.

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes.

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell-specific conditional knockout strategy, we show that Polo-like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.

Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway.

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53-mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53-mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non-centrosomal protein SMC5 is also TP53-dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.

Assessing the impact of fine sediment on high status river sites.

The European Union (EU) Water Framework Directive (WFD) designates as "high status" rivers, lakes, transitional and coastal waters that are close to natural status and relatively un-impacted by anthropogenic activities. These high status water-bodies (HSWs) are sensitive areas that require special attention. Ireland had a globally important distribution of HSWs (10.5% of rivers and 16.2% of lakes classified as high ecological status in Europe occurred in Ireland), but there have been declines of almost 50% between 1987 and 2018, with excessive sediment implicated as a pressure. In this study, an extensive assessment of macro-invertebrate sediment metrics were used to assess sediment as a pressure in sixty-five high or formerly high status river sites in Ireland that were determined to have either: "Lost" their high status (e.g. gone from high to good, moderate, poor or bad; 20 sites); consistently "Maintained" high status (24 sites); or "Gained" in status (e.g. from good to high; 21 sites). Macro-invertebrate taxa occurring in the sixty-five sites were pre-dominantly sediment sensitive taxa. However, for two specific sediment metrics, the Proportion of Sediment-sensitive Index (PSI) and the Empirically-weighted PSI (E-PSI), significant differences were observed between sites that Lost status and those that Maintained status, implying that at some sites, sediment is impacting on macro-invertebrates. However, no significant difference between Lost and Gained sites was observed, leaving an important caveat. While weak to moderate relationships were observed between the macro-invertebrate sediment metrics and the physical sediment variables, no difference between status categories for any of the physical sediment variables was observed. Further research priorities should consider the sampling resolution of these physical variables (e.g. patch vs reach scale), the properties of sediment (e.g. chemical composition) in addition to concentration, the potential interaction of multiple-stressors, and the life cycle characteristics of invertebrate taxa.

Approaches to herbicide (MCPA) pollution mitigation in drinking water source catchments using enhanced space and time monitoring.

Freshwater occurrences of the selective acid herbicide 2-methyl-4-chloro-phenoxyacetic acid (MCPA) are an ongoing regulatory and financial issue for water utility industries as the number and magnitude of detections increase, particularly in surface water catchments. Assessments for mitigating pesticide pollution in catchments used as drinking water sources require a combination of catchment-based and water treatment solutions, but approaches are limited by a lack of empirical data. In this study, an enhanced spatial (11 locations) and temporal (7-hourly to daily sampling) monitoring approach was employed to address these issues in an exemplar surface water source catchment (384 km2). The spatial sampling revealed that MCPA was widespread, with occurrences above the 0.1 µg L-1 threshold for a single pesticide being highly positively correlated to sub-catchments with higher proportions of 'Improved Grassland' land use (r = 0.84). These data provide a strong foundation for targeting catchment-based mitigation solutions and also add to the debate on the ecosystems services provided by such catchments. Additionally, of the 999 temporal samples taken over 12 months from the catchment outlet, 25% were above the drinking water threshold of 0.1 µg L-1. This prevalence of high concentrations presents costly problems for source water treatment. Using these data, abstraction shutdowns were simulated for five scenarios using hydrometeorological data to explore the potential to avoid intake of high MCPA concentrations. The scenarios stopped abstraction for 4.2-9.3% of the April-October period and reduced intake of water containing over 0.1 µg L-1 of MCPA by 16-31%. This represents an important development for real-time proxy assessments for water abstraction in the absence of more direct pesticide monitoring data.

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis.

Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.