FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis.

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Network Rewiring of Homologous Recombination Enzymes during Mitotic Proliferation and Meiosis.

Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.

Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis.

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.

Regulation of the MLH1-MLH3 endonuclease in meiosis.

During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.

CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

Variation in monitoring: Glucose measurement in the ICU as a case study to preempt spurious correlations.

OBJECTIVE: Health inequities can be influenced by demographic factors such as race and ethnicity, proficiency in English, and biological sex. Disparities may manifest as differential likelihood of testing which correlates directly with the likelihood of an intervention to address an abnormal finding. Our retrospective observational study evaluated the presence of variation in glucose measurements in the Intensive Care Unit (ICU). METHODS: Using the MIMIC-IV database (2008-2019), a single-center, academic referral hospital in Boston (USA), we identified adult patients meeting sepsis-3 criteria. Exclusion criteria were diabetic ketoacidosis, ICU length of stay under 1 day, and unknown race or ethnicity. We performed a logistic regression analysis to assess differential likelihoods of glucose measurements on day 1. A negative binomial regression was fitted to assess the frequency of subsequent glucose readings. Analyses were adjusted for relevant clinical confounders, and performed across three disparity proxy axes: race and ethnicity, sex, and English proficiency. RESULTS: We studied 24,927 patients, of which 19.5% represented racial and ethnic minority groups, 42.4% were female, and 9.8% had limited English proficiency. No significant differences were found for glucose measurement on day 1 in the ICU. This pattern was consistent irrespective of the axis of analysis, i.e. race and ethnicity, sex, or English proficiency. Conversely, subsequent measurement frequency revealed potential disparities. Specifically, males (incidence rate ratio (IRR) 1.06, 95% confidence interval (CI) 1.01 - 1.21), patients who identify themselves as Hispanic (IRR 1.11, 95% CI 1.01 - 1.21), or Black (IRR 1.06, 95% CI 1.01 - 1.12), and patients being English proficient (IRR 1.08, 95% CI 1.01 - 1.15) had higher chances of subsequent glucose readings. CONCLUSION: We found disparities in ICU glucose measurements among patients with sepsis, albeit the magnitude was small. Variation in disease monitoring is a source of data bias that may lead to spurious correlations when modeling health data.

Participant flow diagrams for health equity in AI.

Selection bias can arise through many aspects of a study, including recruitment, inclusion/exclusion criteria, input-level exclusion and outcome-level exclusion, and often reflects the underrepresentation of populations historically disadvantaged in medical research. The effects of selection bias can be further amplified when non-representative samples are used in artificial intelligence (AI) and machine learning (ML) applications to construct clinical algorithms. Building on the "Data Cards" initiative for transparency in AI research, we advocate for the addition of a participant flow diagram for AI studies detailing relevant sociodemographic and/or clinical characteristics of excluded participants across study phases, with the goal of identifying potential algorithmic biases before their clinical implementation. We include both a model for this flow diagram as well as a brief case study explaining how it could be implemented in practice. Through standardized reporting of participant flow diagrams, we aim to better identify potential inequities embedded in AI applications, facilitating more reliable and equitable clinical algorithms.

Concept and Design of a Multi-Polarization Reconfigurable Microstrip Antenna with Symmetrical Biasing Control.

Wireless communication systems have grown rapidly, moving towards being highly compact, intelligent, and flexible to adapt to changing operating requirements. Multifunctional and highly versatile antennas are key in this development to ensure system quality. Reconfigurable antennas, particularly regarding polarization, allow frequency reuse and enable the mitigation of fading effects. This work presents a square microstrip patch antenna operating in the ISM 5.8 GHz band with reconfigurable polarization by controlling its feeding. This antenna has four different states through the application of a symmetrical DC voltage that controls an RF circuit with PIN diodes. As a result, the microstrip patch can operate with three different polarizations: linear polarization and both circular polarizations (right-handed and left-handed). The antenna was fabricated to validate the proposed concept. The good agreement between the measurement and the simulation results was possible to observe regarding its polarization behaviour, impedance adaptation and radiation pattern.

Imaging Acute and Chronic Cardiac Complications of COVID-19 and after COVID-19 Vaccination.

COVID-19 is associated with acute and longer-term cardiovascular manifestations including myocardial injury, myopericarditis, stress-induced cardiomyopathy, myocardial infarction, and thromboembolic disease. Although the morbidity and mortality related to acute COVID-19 have decreased substantially, there is growing concern about the longer-term cardiovascular effects of the disease and postacute sequelae. Myocarditis has also been reported after messenger ribonucleic acid (mRNA)-based COVID-19 vaccination, with the highest risk among adolescent boys and young adult men. Noninvasive imaging including cardiac MRI has a key role in identifying the presence of cardiovascular disease, evaluating for potential mechanisms of injury, stratifying risk of future adverse cardiovascular events, and potentially guiding treatment in patients with suspected cardiovascular injury after COVID-19 and vaccination. Patterns of injury identified at cardiac MRI after COVID-19 include myocarditis and pericarditis, myocardial ischemia, and infarction. Myocardial edema and late gadolinium enhancement have been described months after the initial infection in a minority of patients with persistent cardiac symptoms after COVID-19. In patients with myocarditis after receiving a COVID-19 vaccination, the most common pattern of late gadolinium enhancement is subepicardial at the basal inferolateral wall, and patients tend to have milder imaging abnormalities compared with those from other causes of myocarditis. This article describes the role of multimodality cardiac imaging and imaging findings in patients with acute and longer-term cardiovascular manifestations of COVID-19 and in patients with myocarditis after receiving an mRNA-based COVID-19 vaccination. ©RSNA, 2023 Online supplemental material is available for this article. Quiz questions for this article are available through the Online Learning Center.

Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I.

Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.

Racial Disparities in Invasive ICU Treatments Among Septic Patients: High Resolution Electronic Health Records Analysis from MIMIC-IV.

Background: Low-resolution administrative databases can give biased results, whereas high-resolution, time-stamped variables from clinical databases like MIMIC-IV might provide nuanced insights. We evaluated racial-ethnic disparities in life-sustaining ICU-treatments (Invasive Mechanical Ventilation (IMV), Renal Replacement Therapy (RRT), and Vasopressors (VP)) among patients with sepsis. Methods: In this observational retrospective cohort study, patients fulfilling sepsis-3 criteria were categorized by treatment assignment within the first 4 days. The outcomes were treatment allocations. The likelihood of receiving treatment was calculated by race-ethnicity (Racial-ethnic group (REG) or White group (WG)) using 5-fold sub-sampling nested logistic regression and XGBoost. Results: In 23,914 admissions, 82% were White, 42% were women. REG were less likely to receive IMV across all eligibility days (day 1 odds ratio (OR) 0.87, 95% confidence interval (CI) 0.83-0.94, day 4 OR 0.80, 95% CI 0.72 - 0.87). There were no differences in RRT (day 1 OR 1.00, 95% CI 0.96-1.09, day 4 OR 1.00, 95% CI 0.94-1.06). REG were also less likely to be treated with VP at days 1 to 3 (day 1 OR 0.87, 95% CI 0.76-0.94), but not at day 4 (OR 0.95, 95% CI 0.87-1.01). These findings remained robust when relaxing eligibility criteria for treatment allocation. Conclusion: Our findings reveal significant disparities in the use of invasive life-saving ICU treatments among septic patients from racial and ethnic minority backgrounds, particularly with respect to IMV and VP use. These disparities underscore not only the need to address inequality in critical care settings, but also highlight the importance of high-resolution data.

Dual control of Yen1 nuclease activity and cellular localization by Cdk and Cdc14 prevents genome instability.

The careful orchestration of cellular events such as DNA replication, repair, and segregation is essential for equal distribution of the duplicated genome into two daughter cells. To ensure that persistent recombination intermediates are resolved prior to cell division, the Yen1 Holliday junction resolvase is activated at anaphase. Here, we show that the master cell-cycle regulators, cyclin-dependent kinase (Cdk) and Cdc14 phosphatase, control the actions of Yen1. During S phase, Cdk-mediated phosphorylation of Yen1 promotes its nuclear exclusion and inhibits catalytic activity by reducing the efficiency of DNA binding. Later in the cell cycle, at anaphase, Cdc14 drives Yen1 dephosphorylation, leading to its nuclear relocalization and enzymatic activation. Using a constitutively activated form of Yen1, we show that uncontrolled Yen1 activity is detrimental to the cell: spatial and temporal restriction of Yen1 protects against genotoxic stress and, by avoiding competition with the noncrossover-promoting repair pathways, prevents loss of heterozygosity.

A cell cycle-regulated Slx4-Dpb11 complex promotes the resolution of DNA repair intermediates linked to stalled replication.

A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81-Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11-Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81-Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81-Mms4 binding to the Dpb11-Slx4 complex. Thus, Dpb11-Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81.

Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution.

Coordinated actions of SLX1-SLX4 and MUS81-EME1 for Holliday junction resolution in human cells.

Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted in vitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.

The Use of 3D Printing Technology for Manufacturing Metal Antennas in the 5G/IoT Context.

With the rise of 5G, Internet of Things (IoT), and networks operating in the mmWave frequencies, a huge growth of connected sensors will be a reality, and high gain antennas will be desired to compensate for the propagation issues, and with low cost, characteristics inherent to metallic radiating structures. 3D printing technology is a possible solution in this way, as it can print an object with high precision at a reduced cost. This paper presents different methods to fabricate typical metal antennas using 3D printing technology. These techniques were applied as an example to pyramidal horn antennas designed for a central frequency of 28 GHz. Two techniques were used to metallize a structure that was printed with polylactic acid (PLA), one with copper tape and other with a conductive spray-paint. A third method consists of printing an antenna completely using a conductive filament. All prototypes combine good results with low production cost. The antenna printed with the conductive filament achieved a better gain than the other structures and showed a larger bandwidth. The analysis recognizes the vast potential of these 3D-printed structures for IoT applications, as an alternative to producing conventional commercial antennas.

The Cdc14 Phosphatase Controls Resolution of Recombination Intermediates and Crossover Formation during Meiosis.

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination.

Study of Potentially Toxic Elements Uptake into Organs of Quercus spp. from Copper Deposits in Slovakia, Italy and Portugal.

The article is focused on the application of Energy dispersive micro X-ray fluorescence spectroscopy as a specific method to determine the contents of potentially toxic elements and its spread in plant tissues. As a model species, Quercus spp. were selected. In order to compare the obtained results with previous research, four well-described abandoned Cu-deposits were selected for sampling: Lubietová (Slovakia), Libiola and Caporciano (Italy), and São Domingos (Portugal). The results of micro X-ray fluorescence spectrometry confirm the irregular contamination of Quercus spp. by potentially toxic elements. The level of contamination is the highest predominantly in the root cortex, where is also the highest Ca contents (with exception of São Domingos). At Lubietová and Caporciano, high Ni content was described in branches cortex, in branches mesoderm also Fe, Cu and Zn. At the same time, the inhibition influence of Ca was also confirmed regarding the input of these elements into plants.