Fission yeast Cdc14-like phosphatase Flp1/Clp1 modulates the transcriptional response to oxidative stress.

Reactive oxygen species (ROS) are an important source of cellular damage. When ROS intracellular levels increase, oxidative stress takes place affecting DNA stability and metabolic functions. To prevent these effects, stress-activated protein kinases (SAPKs) delay cell cycle progression and induce a transcriptional response that activates antioxidant mechanisms ensuring cell adaptation and survival. Fission yeast Cdc14-like phosphatase Flp1 (also known as Clp1) has a well-established role in cell cycle regulation. Moreover, Flp1 contributes to checkpoint activation during replication stress. Here, we show that Flp1 has a role in fine-tuning the cellular oxidative stress response. Phosphorylation-dependent nucleolar release of Flp1 in response to oxidative stress conditions plays a role in the cellular transcriptional response. Thus, Flp1 ablation increases the transcriptional response to oxidative stress, in both intensity and duration, upregulating both Atf1/Pcr1- and Pap1-dependent stress induced genes. Remarkably, we found that Flp1 interacts with the Atf1/Pcr1 complex with Pcr1 acting as a direct substrate. Our results provide evidence that Flp1 modulates the oxidative stress response by limiting the Atf1/Pcr1-mediated transcription.

Global Emergence of Resistance to Fluconazole and Voriconazole in Candida parapsilosis in Tertiary Hospitals in Spain During the COVID-19 Pandemic.

Background: Candida parapsilosis is a frequent cause of candidemia worldwide. Its incidence is associated with the use of medical implants, such as central venous catheters or parenteral nutrition. This species has reduced susceptibility to echinocandins, and it is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole-nonsusceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last 2 years at the Spanish Mycology Reference Laboratory. Methods: Yeast were identified by molecular biology, and antifungal susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing protocol. The ERG11 gene was sequenced to identify resistance mechanisms, and strain typing was carried out by microsatellite analysis. Results: We examined the susceptibility profile of 1315 C. parapsilosis isolates available at our reference laboratory between 2000 and 2021, noticing an increase in the number of isolates with acquired resistance to fluconazole, and voriconazole has increased in at least 8 different Spanish hospitals in 2020-2021. From 121 recorded clones, 3 were identified as the most prevalent in Spain (clone 10 in Catalonia and clone 96 in Castilla-Leon and Madrid, whereas clone 67 was found in 2 geographically unrelated regions, Cantabria and the Balearic Islands). Conclusions: Our data suggest that concurrently with the coronavirus disease 2019 pandemic, a selection of fluconazole-resistant C. parapsilosis isolates has occurred in Spain, and the expansion of specific clones has been noted across centers. Further research is needed to determine the factors that underlie the successful expansion of these clones and their potential genetic relatedness.

Coinfections among hospitalized patients with covid-19 in the first pandemic wave.

BACKGROUND: COVID19 is the novel respiratory illness caused by SARS-CoV-2. The presence of other potentially pathogenic microorganisms could worsen the prognosis of these patients. AIM: The study aims to describe coinfections in COVID-19 patients and contrast it between standard ward and critical care patients at Hospital Central de la Defensa Gómez Ulla (HCDGU). METHODS: A retrospective study was carried out of patients with COVID-19 confirmed with RTPCR admitted to the HCDGU from March 5, 2020 to May 7 of 2020. FINDINGS: Of a total of 703 patients with COVID-19, 75(10.7%) had other microbiologically confirmed infections: 9% (58/648) in standard ward patients and 31.5%(17/54) in critical care patients. In total 86 samples of the 75 patients presented some microorganism; clinically relevant bacteraemias, 50%, respiratory cultures, 32.6% and pneumococcal positive antigens, 17.4%. CONCLUSIONS: We found a low frequency of microorganism coinfection in COVID-19 patients, however in critical care these coinfections increased considerably.

SARS-COV-2 Infection and Specific Antibody Detection on Health Care Workers from a Military Hospital in Madrid, Spain.

This study aims to assess the COVID-19 seroprevalence in HCW at the Hospital Central de la Defensa Gómez Ulla (HCDGU) (Madrid). From 27 April to 10 June 2020 nasopharyngeal swab and serum samples from employees were processed in order to evaluate their seroprevalence and infective situation. Employees were classified according to their exposure to SARS-CoV-2 infection as high, moderate, and low exposure groups (level 1, level 2, and level 3, respectively). A specific real-time polymerase chain reaction (RT-PCR) was run to diagnose each patient, whereas the qualitative detection of IgG antibodies to SARS-CoV-2 was performed by means of an immunoassay. In total, 2781 HCW were screened. From this sample, 30 employees (1.1%) were infected with SARS-CoV-2 and 450 (16.2%) were positive to SARS-CoV-2-IgG antibodies. The seroprevalence was higher in the high exposure group.The seroprevalence of antibodies against SARS-CoV-2 among employees without any COVID-19 training was higher than in those who received COVID-19 training (14.5% vs 18.6%, P = 0.035). The seroprevalence in military and civilian personnel in level 1 was 18.2% and 20.0%, respectively (P = 0.4616), while in level 2 it was 6.0% and 16.0% (P = 0.0008) and in level 3 it was 16.7% and 10.2% (P = 0.0315). The results from the present study have shown that the high exposure group and HCW not receiving specific training against COVID-19 showed higher seroprevalence. Furthermore, the military employees from this hospital presented low percentage of seroprevalence.

A multiplex antigen microarray for simultaneous IgG and IgM detection against SARS-CoV-2 reveals higher seroprevalence than reported.

The surge of SARS-CoV-2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID-19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 µg ml-1 ) and IgG (~0.017 µg ml-1 ) and is scalable and cost-effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma-based treatments, high-throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.

Comparison of various serological assays for novel SARS-COV-2.

Coronavirus disease-19 (COVID19), the novel respiratory illness caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), is associated with severe morbidity and mortality. The aim of our study was to compare different immunoassays. We evaluated three immunochromatographic test (The StrongStep®SARS-CoV-2 IgG/IgM kit, AllTest COV-19 IgG/IgM kit, and Wondfo® SARS-CoV-2 Antibody) and two chemiluminescence immunoassays (CMIA) (Covid-19 VIRCLIA® IgM+IgA/IgG monotest and the Abbott SARS-CoV-2 IgG assay) in COVID-19 patients. The assays were performed using serum samples of three group patients, i.e., healthy controls, patients with SARS-CoV-2 PCR positive, and patients with SARS-CoV-2 PCR negative clinically diagnosed of COVID-19 infection. The detection percentages of IgG with the StrongStep® SARS-CoV-2 IgG/IgM kit and AllTest COV-19 IgG/IgM kit were similar in both groups (83.3% and 80.6%, respectively in group 2, p = 0.766) and (42.9% and 50.0%, respectively in group 3, p = 0.706). There were some differences on IgM detection between StrongStep® SARS-CoV-2 IgG/IgM kit and AllTest COV-19 IgG/IgM kit (11.1% and 30.6%, respectively in group 2, p = 0.042 and 0.0% and 28.6%, respectively in group 3, p = 0.031). The positive rate of IgG in group 2 is higher compared to group 3 with the two immunoassays tested. We observe the same positive rates of IgG with the two CMIA. Our study shows excellent performance of CMIA compared to immunochromatographic test and confirms its potential use in the diagnosis of the new SARS-CoV-2.

Working on Genomic Stability: From the S-Phase to Mitosis.

Fidelity in chromosome duplication and segregation is indispensable for maintaining genomic stability and the perpetuation of life. Challenges to genome integrity jeopardize cell survival and are at the root of different types of pathologies, such as cancer. The following three main sources of genomic instability exist: DNA damage, replicative stress, and chromosome segregation defects. In response to these challenges, eukaryotic cells have evolved control mechanisms, also known as checkpoint systems, which sense under-replicated or damaged DNA and activate specialized DNA repair machineries. Cells make use of these checkpoints throughout interphase to shield genome integrity before mitosis. Later on, when the cells enter into mitosis, the spindle assembly checkpoint (SAC) is activated and remains active until the chromosomes are properly attached to the spindle apparatus to ensure an equal segregation among daughter cells. All of these processes are tightly interconnected and under strict regulation in the context of the cell division cycle. The chromosomal instability underlying cancer pathogenesis has recently emerged as a major source for understanding the mitotic processes that helps to safeguard genome integrity. Here, we review the special interconnection between the S-phase and mitosis in the presence of under-replicated DNA regions. Furthermore, we discuss what is known about the DNA damage response activated in mitosis that preserves chromosomal integrity.

PCNA Deubiquitylases Control DNA Damage Bypass at Replication Forks.

DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNA-linked ubiquitin chains. Ubp10 and Ubp12 associate with replication forks in a fashion determined by Ubp10 dependency on lagging-strand PCNA residence, and they downregulate translesion polymerase recruitment and template switch events engaging nascent strands. These findings reveal PCNAK164 deubiquitylation as a key mechanism for the modulation of lesion bypass during replication, which might set a framework for establishing strand-differential pathway choices. We propose that damage tolerance is tempered at replication forks to limit the extension of bypass events and sustain chromosome replication rates.

Disruption of the Caenorhabditis elegans Integrator complex triggers a non-conventional transcriptional mechanism beyond snRNA genes.

Gene expression is generally regulated by recruitment of transcription factors and RNA polymerase II (RNAP II) to specific sequences in the gene promoter region. The Integrator complex mediates processing of small nuclear RNAs (snRNAs) as well as the initiation and release of paused RNAP II at specific genes in response to growth factors. Here we show that in C. elegans, disruption of the Integrator complex leads to transcription of genes located downstream of the snRNA loci via a non-conventional transcription mechanism based on the lack of processing of the snRNAs. RNAP II read-through generates long chimeric RNAs containing snRNA, the intergenic region and the mature mRNA of the downstream gene located in sense. These chimeric sn-mRNAs remain as untranslated long non-coding RNAs, in the case of U1- and U2-derived sn-mRNAs, but can be translated to proteins in the case of SL-derived sn-mRNAs. The transcriptional effect caused by disruption of the Integrator complex is not restricted to genes located downstream of the snRNA loci but also affects key regulators of signal transduction such as kinases and phosphatases. Our findings highlight that these transcriptional alterations may be behind the correlation between mutations in the Integrator complex and tumor transformation.

ENSA and ARPP19 differentially control cell cycle progression and development.

Greatwall (GWL) is an essential kinase that indirectly controls PP2A-B55, the phosphatase counterbalancing cyclin B/CDK1 activity during mitosis. In Xenopus laevis egg extracts, GWL-mediated phosphorylation of overexpressed ARPP19 and ENSA turns them into potent PP2A-B55 inhibitors. It has been shown that the GWL/ENSA/PP2A-B55 axis contributes to the control of DNA replication, but little is known about the role of ARPP19 in cell division. By using conditional knockout mouse models, we investigated the specific roles of ARPP19 and ENSA in cell division. We found that Arpp19, but not Ensa, is essential for mouse embryogenesis. Moreover, Arpp19 ablation dramatically decreased mouse embryonic fibroblast (MEF) viability by perturbing the temporal pattern of protein dephosphorylation during mitotic progression, possibly by a drop of PP2A-B55 activity inhibition. We show that these alterations are not prevented by ENSA, which is still expressed in Arpp19 Δ/Δ MEFs, suggesting that ARPP19 is essential for mitotic division. Strikingly, we demonstrate that unlike ARPP19, ENSA is not required for early embryonic development. Arpp19 knockout did not perturb the S phase, unlike Ensa gene ablation. We conclude that, during mouse embryogenesis, the Arpp19 and Ensa paralog genes display specific functions by differentially controlling cell cycle progression.

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1.

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.

The T300A Crohn's disease risk polymorphism impairs function of the WD40 domain of ATG16L1.

A coding polymorphism of human ATG16L1 (rs2241880; T300A) increases the risk of Crohn's disease and it has been shown to enhance susceptibility of ATG16L1 to caspase cleavage. Here we show that T300A also alters the ability of the C-terminal WD40-repeat domain of ATG16L1 to interact with an amino acid motif that recognizes this region. Such alteration impairs the unconventional autophagic activity of TMEM59, a transmembrane protein that contains the WD40 domain-binding motif, and disrupts its normal intracellular trafficking and its ability to engage ATG16L1 in response to bacterial infection. TMEM59-induced autophagy is blunted in cells expressing the fragments generated by caspase processing of the ATG16L1-T300A risk allele, whereas canonical autophagy remains unaffected. These results suggest that the T300A polymorphism alters the function of motif-containing molecules that engage ATG16L1 through the WD40 domain, either by influencing this interaction under non-stressful conditions or by inhibiting their downstream autophagic signalling after caspase-mediated cleavage.

Orderly progression through S-phase requires dynamic ubiquitylation and deubiquitylation of PCNA.

Proliferating-cell nuclear antigen (PCNA) is a DNA sliding clamp with an essential function in DNA replication and a key role in tolerance to DNA damage by ensuring the bypass of lesions. In eukaryotes, DNA damage tolerance is regulated by ubiquitylation of lysine 164 of PCNA through a well-known control mechanism; however, the regulation of PCNA deubiquitylation remains poorly understood. Our work is a systematic and functional study on PCNA deubiquitylating enzymes (DUBs) in Schizosaccharomyces pombe. Our study reveals that the deubiquitylation of PCNA in fission yeast cells is a complex process that requires several ubiquitin proteases dedicated to the deubiquitylation of a specific subnuclear fraction of mono- and di-ubiquitylated PCNA or a particular type of poly-ubiquitylated PCNA and that there is little redundancy among these enzymes. To understand how DUB activity regulates the oscillatory pattern of ubiquitylated PCNA in fission yeast, we assembled multiple DUB mutants and found that a quadruple mutation of ubp2(+), ubp12(+), ubp15(+), and ubp16(+) leads to the stable accumulation of mono-, di-, and poly-ubiquitylated forms of PCNA, increases S-phase duration, and sensitizes cells to DNA damage. Our data suggest that the dynamic ubiquitylation and deubiquitylation of PCNA occurs during S-phase to ensure processive DNA replication.

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation.

The activity of Cdk1-cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1-cyclin B1 activity through Wee1 dephosphorylation.

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription.

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit(2,3) and for segregation of repetitive regions(4). Cdc14 is also a subunit of the silencing complex RENT (refs 5,6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y' repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants(4) correlate with the presence of subtelomeric Y' elements and can be rescued by transcriptional inhibition of RNA polymerase II.

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition.

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) -dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.

Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription.

Cdc14 is an essential phosphatase in yeast but its role in the mammalian cell cycle remains obscure. We report here that Cdc14b-knockout cells display unscheduled induction of multiple cell cycle regulators resulting in early entry into DNA replication and mitosis from quiescence. Cdc14b dephosphorylates Ser5 at the C-terminal domain (CTD) of RNA polymerase II, a major substrate of cyclin-dependent kinases. Lack of Cdc14b results in increased CTD-Ser5 phosphorylation, epigenetic modifications that mark active chromatin, and transcriptional induction of cell cycle regulators. These data suggest a function for mammalian Cdc14 phosphatases in the control of transcription during the cell cycle.

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B.

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.