Crucial role of the NSE1 RING domain in Smc5/6 stability and FANCM-independent fork progression.

The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.

A TOPBP1 allele causing male infertility uncouples XY silencing dynamics from sex body formation.

Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis, and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted, including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.

The Practical Application of Bio-Inspired PMA for the Detection of Partial Discharges in High Voltage Equipment.

In this paper, the practical application of a bio-inspired antenna for partial discharge (PD) detection in high voltage equipment was evaluated in order to validate the efficiency of using this technology for PD monitoring purposes. For this, PD measurements using the bio-inspired antenna were performed on operational 69 kV potential transformers (PT) in a real substation. After the field experiment, laboratory measurements using the IEC 60270 standard method and a bio-inspired antenna were performed, simultaneously, over the evaluated PT. The results obtained at the substation indicated suspicious frequencies of partial discharge activity in two out of three evaluated potential transformers, mainly for the frequencies of 461 MHz, 1366 MHz, 1550 MHz and 1960 MHz. During the laboratory tests, the presence of partial discharge activity over the suspicious potential transformers was confirmed with the detection of PD apparent charge levels above 20 pC. Finally, the frequency spectrum obtained from the PD signals detected by the bio-inspired antenna in the laboratory presented similar frequency values to those obtained during the practical application at the substation, making it a promising indicator for future defect classification studies using artificial intelligence.

Acute Kidney Injury and BK Polyomavirus in Urine Sediment Cells.

Polyomaviruses are widespread, with BK viruses being most common in humans who require immunosuppression due to allotransplantation. Infection with BK polyomavirus (BKV) may manifest as BK virus-associated nephropathy and hemorrhagic cystitis. Established diagnostic methods include the detection of polyomavirus in urine and blood by PCR and in tissue biopsies via immunohistochemistry. In this study, 79 patients with pathological renal retention parameters and acute kidney injury (AKI) were screened for BK polyomavirus replication by RNA extraction, reverse transcription, and virus-specific qPCR in urine sediment cells. A short fragment of the VP2 coding region was the target of qPCR amplification; patients with (n = 31) and without (n = 48) a history of renal transplantation were included. Urine sediment cell immunofluorescence staining for VP1 BK polyomavirus protein was performed using confocal microscopy. In 22 patients with acute renal injury, urinary sediment cells from 11 participants with kidney transplantation (KTX) and from 11 non-kidney transplanted patients (nonKTX) were positive for BK virus replication. BK virus copies were found more frequently in patients with AKI stage III (n = 14). Higher copy numbers were detected in KTX patients having experienced BK polyoma-nephropathy (BKPyVAN) in the past or diagnosed recently by histology (5.6 × 109-3.1 × 1010). One patient developed BK viremia following delayed graft function (DGF) with BK virus-positive urine sediment. In nonKTX patients with BK copies, decoy cells were absent; however, positive staining of cells was found with epithelial morphology. Decoy cells were only found in KTX patients with BKPyVAN. In AKI, damage to the tubular epithelium itself may render the epithelial cells more permissive for polyoma replication. This non-invasive diagnostic approach to assess BK polyomavirus replication in urine sediment cells has the potential to identify KTX patients at risk for viremia and BKPyVAN during AKI. This method might serve as a valuable screening tool for close monitoring and tailored immunosuppression decisions.

Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15.

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNß treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNß activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNß on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness.

A TOPBP1 Allele Causing Male Infertility Uncouples XY Silencing Dynamics From Sex Body Formation.

Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1 B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1 B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.

Expression of RAD9B in the mesostriatal system of rats and humans: Overexpression in a 6-OHDA rat model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder that affects primarily the dopaminergic (DAergic) neurons of the mesostriatal system, among other nuclei of the brain. Although it is considered an idiopathic disease, oxidative stress is believed to be involved in DAergic neuron death and therefore plays an important role in the onset and development of the disease. RAD9B is a paralog of the RAD9 checkpoint, sharing some similar functions related to DNA damage resistance and apoptosis, as well as the ability to form 9-1-1 heterotrimers with RAD1 and HUS1. METHODS: In addition to immunohistochemistry, immunofluorescence and Western-blot analysis, we implemented Quantitative RT-PCR and in situ hybridization techniques. RESULTS: We demonstrated RAD9B expression in rat and human mesencephalic DAergic cells using specific markers. Additionally, we observed significant overexpression of RAD9B mRNA (p<0.01) and protein (p<0.01) in the midbrain 48 h after inducing damage with 150 µg of 6-hydroxydopamine (6-OHDA) injected in a rat model of PD. Regarding protein expression, the increased levels were observed in neurons of the mesostriatal system and returned to normal 5 days post-injury. CONCLUSIONS: This response to a neurotoxin, known to produce oxidative stress specifically on DAergic neurons indicates the potential importance of RAD9B in this highly vulnerable population to cell death. In this model, RAD9B function appears to provide neuroprotection, as the induced lesion resulted in only mild degeneration. This observation highlights the potential of RAD9B checkpoint protein as a valuable target for future therapeutic interventions aimed at promoting neuroprotection.

ATXN3 controls DNA replication and transcription by regulating chromatin structure.

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.

Monopole directional antenna bioinspired in elliptical leaf with golden ratio for WLAN and 4G applications.

In this work, it is proposed the development a new monopole directional antenna, bioinspired in elliptical leaf, with cut by golden ratio, for 4G band application, by the use of the technique of the cut of the radiating element for the increasing of the antenna perimeter, being the first work to use this technique in a bioinspired antenna, promotes resonance frequency turned, and reconfiguring of the antenna parameters as bandwidth, radiation pattern and gain, with the use of the reflector near to the group plane, without the insertion of active devices as the pin diode or change in radiating element. The shape antenna is generated by Gielis formula, built in FR4 substrate, with cuts calculated by golden ratio. To compare the results of the bioinspired monopole on the elliptical sheet, a square-shaped monopole antenna was designed, simulated and measured, the structures were designed in the MATLAB software version 2015(b) and the simulations were performed in the Ansys software version 2016. In the results compared between the square monopole and the bioinspired antenna in the elliptical sheet, it can be seen that the measured bioinspired antenna, compared to the square monopole, presented a bandwidth reduction of 77.27%, a more compact structure, with a reduction of 98%, covering the wireless local area network, and long-time evolution 4G at 2.5 GHz. The proposed technique uses a reflector on the ground plane, to change the parameters of the monopole planar antenna, of omnidirectional radiation pattern to a directional, maintaining the characteristics of the broadband, half-power beamwidth great than 100°, with high current density, and similar gain of a directional antenna. From the results, it has been observed that the elliptical leaf monopole antenna shows broadband characteristics, with a half-power beamwidth of 128°, wideband, the bandwidth of 500 MHz, a gain of 6.28 dBi, a current density of 13.01 A/m2, and circular polarization.

Architecture of an Electrical Equivalence Pyranometer with Temperature Difference Analog Control.

In this paper, an architecture of an electrical equivalence pyranometer with analog control of the temperature difference is presented. The classical electrical equivalence pyranometer employs a Wheatstone bridge with a feedback amplifier to keep the sensor operating at a constant temperature to estimate the incident radiation through the sensor thermal balance employing the electrical equivalence principal. However, this architecture presents limitations under ambient temperature variation, such as sensitivity variation. To overcome those limitations, we propose an architecture that controls the temperature difference between the sensor and ambient via an analog compensating circuit. Analytical results show an improvement near five times in sensitivity over the ambient temperature span and 76.3% increase of useful output voltage. A prototype was developed and validated with a commercial pyranometer, showing a high agreement on the measurement results. It is verified that the use of temperature difference, rather than constant temperature, significantly reduces the effect of ambient temperature variation.

Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.

Regulation of Claspin by the p38 stress-activated protein kinase protects cells from DNA damage.

Stress-activated protein kinases (SAPKs) enhance survival in response to environmental changes. In yeast, the Hog1 SAPK and Mrc1, a protein required for DNA replication, define a safeguard mechanism that allows eukaryotic cells to prevent genomic instability upon stress during S-phase. Here we show that, in mammals, the p38 SAPK and Claspin-the functional homolog of Mrc1-protect cells from DNA damage upon osmostress during S-phase. We demonstrate that p38 phosphorylates Claspin and either the mutation of the p38-phosphorylation sites in Claspin or p38 inhibition suppresses the protective role of Claspin on DNA damage. In addition, wild-type Claspin but not the p38-unphosphorylatable mutant has a protective effect on cell survival in response to cisplatin treatment. These findings reveal a role of Claspin in response to chemotherapeutic drugs. Thus, this pathway protects S-phase integrity from different insults and it is conserved from yeast to mammals.

USP29 Deubiquitinates SETD8 and Regulates DNA Damage-Induced H4K20 Monomethylation and 53BP1 Focus Formation.

SETD8 is a histone methyltransferase that plays pivotal roles in several cellular functions, including transcriptional regulation, cell cycle progression, and genome maintenance. SETD8 regulates the recruitment of 53BP1 to sites of DNA damage by controlling histone H4K20 methylation. Moreover, SETD8 levels are tightly regulated in a cell cycle-dependent manner by ubiquitin-dependent proteasomal degradation. Here, we identified ubiquitin-specific peptidase 29, USP29, as a novel regulator of SETD8. Depletion of USP29 leads to decreased SETD8 protein levels, an effect that is independent of the cell cycle. We demonstrate that SETD8 binds to USP29 in vivo, and this interaction is dependent on the catalytic activity of USP29. Wildtype USP29 can deubiquitinate SETD8 in vivo, indicating that USP29 directly regulates SETD8 protein levels. Importantly, USP29 knockdown inhibits the irradiation-induced increase in H4K20 monomethylation, thereby preventing focus formation of 53BP1 in response to DNA damage. Lastly, depletion of USP29 increases the cellular sensitivity to irradiation. These results demonstrate that USP29 is critical for the DNA damage response and cell survival, likely by controlling protein levels of SETD8.

Vaccination with BNT162b2 and ChAdOx1 nCoV-19 Induces Cross-Reactive Anti-RBD IgG against SARS-CoV-2 Variants including Omicron.

SARS-CoV-2 variants of concern (VOCs) have caused a significant increase in infections worldwide. Despite high vaccination rates in industrialized countries, the fourth VOC, Omicron, has outpaced the Delta variant and is causing breakthrough infections in individuals with two booster vaccinations. While the magnitude of morbidity and lethality is lower in Omicron, the infection rate and global spread are rapid. Using a specific IgG multipanel-ELISA with the spike protein's receptor-binding domain (RBD) from recombinant Alpha, Gamma, Delta, and Omicron variants, sera from health-care workers from the Medical University of Vienna were tested pre-pandemic and post-vaccination (BNT162b2; ChAdOx1 nCoV-19). The cohort was continuously monitored by SARS-CoV-2 testing and commercial nucleocapsid IgG ELISA. RBD IgG ELISA showed significantly lower reactivity against the Omicron-RBD compared to the Alpha variant in all individuals (p < 0.001). IgG levels were independent of sex, but were significantly higher in BNT162b2 recipients <45 years of age for Alpha, Gamma, and Delta (p < 0.001; p = 0.040; p = 0.004, respectively). Pre-pandemic cross-reactive anti-Omicron IgG was detected in 31 individuals and was increased 8.78-fold after vaccination, regardless of vaccine type. The low anti-RBD Omicron IgG level could explain the breakthrough infections and their presence could also contribute to a milder COVID-19 course by cross-reactivity and broadening the adaptive immunity.

Diagnostic Classification of Cases of Canine Leishmaniasis Using Machine Learning.

Proposal techniques that reduce financial costs in the diagnosis and treatment of animal diseases are welcome. This work uses some machine learning techniques to classify whether or not cases of canine visceral leishmaniasis are present by physical examinations. For validation of the method, four machine learning models were chosen: K-nearest neighbor, Naïve Bayes, support vector machine and logistic regression models. The tests were performed on three hundred and forty dogs, using eighteen characteristics of the animal and the ELISA (enzyme-linked immunosorbent assay) serological test as validation. Logistic regression achieved the best metrics: Accuracy of 75%, sensitivity of 84%, specificity of 67%, a positive likelihood ratio of 2.53 and a negative likelihood ratio of 0.23, showing a positive relationship in the evaluation between the true positives and rejecting the cases of false negatives.

Preclinical Establishment of a Divalent Vaccine against SARS-CoV-2.

First-generation vaccines against SARS-CoV-2 do not provide adequate immune protection. Therefore, we engineered a divalent gene construct combining the receptor-binding domain (RBD) of the spike protein and the immunodominant region of the viral nucleocapsid. This fusion protein was produced in either E. coli or a recombinant baculovirus system. Subsequently, the fusion protein was mixed with adjuvant and administered to mice in a prime-booster mode. Mice (72%) produced an IgG response against both proteins (titer: 10-4-10-5) 14 days after the first booster injection, which was increased to 100% by a second booster. Comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed IgGs beyond 90 days. In addition, cytolytic effector cell molecules were increased in lymphocytes isolated from peripheral blood. Ex vivo stimulation of T cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 among the CD4+ and CD8+ T cells of vaccinated mice. No side effect was documented in the central nervous system. Cumulatively, these data represent a proof-of-principle approach alternative to existing mRNA vaccination strategies.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Phosphoproteomics of ATR signaling in mouse testes.

The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

The CDK1-TOPBP1-PLK1 axis regulates the Bloom's syndrome helicase BLM to suppress crossover recombination in somatic cells.

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability.

Autofocus Entropy Repositioning Method Bioinspired in the Magnetic Field Memory of the Bees Applied to Pollination.

In this paper, a bioinspired method in the magnetic field memory of the bees, applied in a rover of precision pollination, is presented. The method calculates sharpness features by entropy and variance of the Laplacian of images segmented by color in the HSV system in real-time. A complementary positioning method based on area feature extraction between active markers was developed, analyzing color characteristics, noise, and vibrations of the probe in time and frequency, through the lateral image of the probe. From the observed results, it can be seen that the unsupervised method does not require previous calibration of target dimensions, histogram, and distances involved in positioning. The algorithm showed less sensitivity in the extraction of sharpness characteristics regarding the number of edges and greater sensitivity to the gradient, allowing unforeseen operation scenarios, even in small sharpness variations, and robust response to variance local, temporal, and geophysical of the magnetic declination, not needing luminosity after scanning, with the two freedom of degrees of the rotation.