Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor.

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

Metabolic and Hormonal Changes Associated with Vitellogenesis in Salvator merianae Lizards.

Vitellogenesis in oviparous vertebrates is a critical marker of the restart of seasonal reproductive activity. During this process of multihormonal regulation, females allocate a considerable amount of organic and mineral reserves to the synthesis of yolk, with changes in their plasma values. In this work, we determined plasma levels of various metabolites and steroid hormones throughout the reproductive cycle in females of Salvator merianae who developed vitellogenic and non-vitellogenic follicular cycles. We worked for two consecutive years with 20 adult females from the Experimental Hatchery of the Facultad de Agronomía y Zootecnia of the Universidad Nacional de Tucumán. Values of metabolites: glucose, triglycerides, cholesterol, calcium, phosphorus, albumin, total proteins, and hormones: estradiol, progesterone, and testosterone, were determined during the following stages of the annual cycle: hibernation, hibernation emergence, courtship-mating, oviposition, and incubation. Vitellogenic females showed significantly higher plasma levels of triglycerides, calcium, phosphorus, and albumin than non-vitellogenic females, mainly in the courtship-mating stage (advanced vitellogenesis). In contrast, annual cholesterol averages were lower in vitellogenic females. Glucose showed changes throughout the annual cycle regardless of the vitellogenic condition. Total proteins plasma levels had very few fluctuations during the cycle. Among the hormones studied, only testosterone showed differences related to vitellogenic condition, with higher levels in non-vitellogenic females during the entire reproductive cycle. The knowledge of these changes associated with vitellogenesis will improve zootechnical management and will allow optimizing the reproductive efficiency of Salvator lizards in captivity.

Current knowledge of the Southern Hemisphere marine microbiome in eukaryotic hosts and the Strait of Magellan surface microbiome project.

Host-microbe interactions are ubiquitous and play important roles in host biology, ecology, and evolution. Yet, host-microbe research has focused on inland species, whereas marine hosts and their associated microbes remain largely unexplored, especially in developing countries in the Southern Hemisphere. Here, we review the current knowledge of marine host microbiomes in the Southern Hemisphere. Our results revealed important biases in marine host species sampling for studies conducted in the Southern Hemisphere, where sponges and marine mammals have received the greatest attention. Sponge-associated microbes vary greatly across geographic regions and species. Nevertheless, besides taxonomic heterogeneity, sponge microbiomes have functional consistency, whereas geography and aging are important drivers of marine mammal microbiomes. Seabird and macroalgal microbiomes in the Southern Hemisphere were also common. Most seabird microbiome has focused on feces, whereas macroalgal microbiome has focused on the epibiotic community. Important drivers of seabird fecal microbiome are aging, sex, and species-specific factors. In contrast, host-derived deterministic factors drive the macroalgal epibiotic microbiome, in a process known as "microbial gardening". In turn, marine invertebrates (especially crustaceans) and fish microbiomes have received less attention in the Southern Hemisphere. In general, the predominant approach to study host marine microbiomes has been the sequencing of the 16S rRNA gene. Interestingly, there are some marine holobiont studies (i.e., studies that simultaneously analyze host (e.g., genomics, transcriptomics) and microbiome (e.g., 16S rRNA gene, metagenome) traits), but only in some marine invertebrates and macroalgae from Africa and Australia. Finally, we introduce an ongoing project on the surface microbiome of key species in the Strait of Magellan. This is an international project that will provide novel microbiome information of several species in the Strait of Magellan. In the short-term, the project will improve our knowledge about microbial diversity in the region, while long-term potential benefits include the use of these data to assess host-microbial responses to the Anthropocene derived climate change.

Body site microbiota of Magellanic and king penguins inhabiting the Strait of Magellan follow species-specific patterns.

Animal hosts live in continuous interaction with bacterial partners, yet we still lack a clear understanding of the ecological drivers of animal-associated bacteria, particularly in seabirds. Here, we investigated the effect of body site in the structure and diversity of bacterial communities of two seabirds in the Strait of Magellan: the Magellanic penguin (Spheniscus magellanicus) and the king penguin (Aptenodytes patagonicus). We used 16S rRNA gene sequencing to profile bacterial communities associated with body sites (chest, back, foot) of both penguins and the nest soil of Magellanic penguin. Taxonomic composition showed that Moraxellaceae family (specifically Psychrobacter) had the highest relative abundance across body sites in both penguin species, whereas Micrococacceae had the highest relative abundance in nest soil. We were able to detect a bacterial core among 90% of all samples, which consisted of Clostridium sensu stricto and Micrococcacea taxa. Further, the king penguin had its own bacterial core across its body sites, where Psychrobacter and Corynebacterium were the most prevalent taxa. Microbial alpha diversity across penguin body sites was similar in most comparisons, yet we found subtle differences between foot and chest body sites of king penguins. Body site microbiota composition differed across king penguin body sites, whereas it remained similar across Magellanic penguin body sites. Interestingly, all Magellanic penguin body site microbiota composition differed from nest soil microbiota. Finally, bacterial abundance in penguin body sites fit well under a neutral community model, particularly in the king penguin, highlighting the role of stochastic process and ecological drift in microbiota assembly of penguin body sites. Our results represent the first report of body site bacterial communities in seabirds specialized in subaquatic foraging. Thus, we believe it represents useful baseline information that could serve for long-term comparisons that use marine host microbiota to survey ocean health.

The potential of PGPR and Trichoderma-based bioproducts and resistant cultivars as tools to manage clubroot disease in cruciferous crops.

The objective of this research was to determine the potential use of eco-friendly technologies to reduce the clubroot disease caused by Plasmodiophora brassicae, the main constraint of cruciferous crops worldwide. Two commercial bioproducts were evaluated in susceptible broccoli, one based on the PGPR consortium (Bacillus amyloliquefaciens, Bacillus pumilus, and Agrobacterium radiobacter K84) and the other one based on Trichoderma koningiopsis Th003 (Tricotec® WG). Additionally, the resistant broccoli cv. Monclano® was tested under two concentrations of resting spores (RS) of P. brassicae, 1 × 103 and 1 × 105 RS g-1 of soil. The first phase of evaluations with broccoli was carried out under a greenhouse, while susceptible broccoli, cauliflower, and red cabbage were included in a subsequent field phase. Tebuconazole + Trifloxystrobin mixture and Fluazinam were included as positive controls. The effectiveness of the bioproducts depended on the nature of the biocontrol agent, the concentration of P. brassicae, and the dose of treatment. Tricotec® showed consistent plant growth promotion but no biocontrol effect against clubroot, and the rhizobacteria-based bioproduct significantly reduced the disease in both greenhouse and field experiments. Higher disease severity was observed with the higher dose of Tricotec®. Under field conditions, the rhizobacteria reduced the incidence progress by 26%, 39%, and 57% under high, medium, and low pressure of the pathogen, respectively. However, no reduction of clubroot severity under high pressure of the pathogen was observed. Complete inhibition of club formation in roots was achieved via the fungicide, but a phytotoxic effect was observed under greenhouse conditions. Fungicides reduced the incidence progress of clubroot, but not the severity under high inoculum pressure in the field. The fungicides, the bacterial treatment, and the combination of bioproducts tended to delay the progress of the disease compared with the negative control and Tricotec alone. The resistant broccoli showed a low level of disease under high concentrations of P. brassicae (less than 10% incidence and up to 2% severity). These results suggested the overall potential of commercial tools based on the PGPR consortium and plant resistance to control P. brassicae. The integration of control measures, the role of Trichoderma spp. in P. brassicae-cruciferous pathosystems, and the need to recover highly infested soils will be discussed.

Prevalence of CYP2C19 polymorphism in Bogotá, Colombia: The first report of allele *17.

INTRODUCTION: Proton pump inhibitors (PPIs) are a group of drugs that are essential for the treatment of acid-related disorders, such as gastroesophageal reflux (GERD), dyspepsia, gastric ulcers and Helicobacter pylori (H. pylori) infection. PPIs such as omeprazole, esomeprazole, pantoprazole and lansoprazole are metabolized by the CYP2C19 enzyme, which is encoded by a polymorphic gene. Four polymorphisms have an impact on the speed of PPI metabolism: CYP2C19*1/*1 (extensive metabolizers), CYP2C19*2/*2 (intermediate metabolizers), CYP2C19*3/*3 (poor metabolizers) and CYP2C19*17/*17 (ultrarapid metabolizers). Extensive and ultrarapid metabolizers inactivate PPIs quickly, which consequently causes low plasma concentrations of PPIs, while intermediate or poor metabolizers have higher plasma concentrations of PPIs and, therefore, PPIs have greater therapeutic efficacy in individuals with these polymorphisms. OBJECTIVE: To determine the frequency of genetic polymorphisms of the CPY2C19 enzyme in Bogotá, Colombia. METHODS: This observational study was conducted in Bogotá between 2012 and 2015 and was part of a clinical trial (ID: NCT03650543). It included 239 subjects with dyspepsia, H. pylori infection, or GERD symptoms. CYP2C19 genotyping was performed on gastric biopsy samples. Polymorphisms *1, *2, and *3 were analyzed by real-time PCR (Roche®), and PCR-RFLP was used to determine the presence of polymorphism *17. RESULTS: The distribution of different types of PPI metabolizers was as follows: extensive (70.7%), ultrarapid (12.9%), intermediate (8.8%) and poor (0.8%). CONCLUSION: The population studied consisted mainly of extensive and ultrarapid PPI metabolizers. These findings show that it is necessary to increase PPI doses in this group of subjects or to use PPIs that are not metabolized by CYP2C19 (rabeprazole). This is the first Colombian work to identify ultrarapid metabolizers.

Correction: cagA gene EPIYA motif genetic characterization from Colombian Helicobacter pylori isolates: Standardization of a molecular test for rapid clinical laboratory detection.

[This corrects the article DOI: 10.1371/journal.pone.0227275.].

cagA gene EPIYA motif genetic characterization from Colombian Helicobacter pylori isolates: Standardization of a molecular test for rapid clinical laboratory detection.

The aim of this work was to determine current cagA gene EPIYA motifs present in Colombian Helicobacter pylori isolates using a fast and reliable molecular test. DNA from eighty-five Helicobacter pylori-cagA positive strains were analyzed. Strains were obtained from patients diagnosed with functional dyspepsia at Clínica Fundadores in Bogotá. The 3' region of the cagA gene was amplified through conventional Polymerase Chain Reaction (PCR). Obtained amplicons were sequenced using the Sanger method and analyzed with bioinformatics tools. Additionally, a significant Spearman correlation coefficient was determined between the patients' age and the number of EPIYA-C repeats; with p values < 0.05 considered significant. Estimates were obtained using a 95% CI. The 3´ variable region of the cagA gene was amplified and PCR products of the following sizes corresponded to the following EPIYA motifs: 400 bp: EPIYA AB, 500 bp: EPIYA ABC, 600 bp: EPIYA ABCC and 700 bp: ABCCC. A single PCR band was observed for 58 out of 85 Helicobacter pylori isolates, with an EPIYA distribution motif as follows: 7/85 AB (8.2%), 34/85 ABC (40%), 26/85 ABCC (30.6%) and 18/85 ABCCC (21.2%). However, in 27 out of 85 Helicobacter pylori isolates, two or more bands were observed, where the most predominant cagA genotype were ABC-ABCC (26%, 7/27) and ABCC-ABCCC (22.2%, 6/27). A direct proportionality between the number of EPIYA-C repeats and an increase in the patients' age was observed, finding a greater number of EPIYA ABCC and ABCCC repeats in the population over 50 years old. All isolates were of the Western cagA type and 51.8% of them were found to have multiple EPIYA-C repeats. These standardized molecular test allowed to identify the number of EPIYA C motifs based on band size.

The Sun is less active than other solar-like stars.

The magnetic activity of the Sun and other stars causes their brightness to vary. We investigated how typical the Sun's variability is compared with other solar-like stars, i.e., those with near-solar effective temperatures and rotation periods. By combining 4 years of photometric observations from the Kepler space telescope with astrometric data from the Gaia spacecraft, we were able to measure photometric variabilities of 369 solar-like stars. Most of those with well-determined rotation periods showed higher variability than the Sun and are therefore considerably more active. These stars appear nearly identical to the Sun except for their higher variability. Therefore, we speculate that the Sun could potentially also go through epochs of such high variability.

RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies.

INTRODUCTION: Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described. METHODS: Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays. RESULTS: After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs. CONCLUSIONS: RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.

Schizosaccharomyces pombe histone acetyltransferase Mst1 (KAT5) is an essential protein required for damage response and chromosome segregation.

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.

Schizosaccharomyces pombe mst2+ encodes a MYST family histone acetyltransferase that negatively regulates telomere silencing.

Histone acetylation and deacetylation are associated with transcriptional activity and the formation of constitutively silent heterochromatin. Increasingly, histone acetylation is also implicated in other chromosome transactions, including replication and segregation. We have cloned the only Schizosaccharomyces pombe MYST family histone acetyltransferase genes, mst1(+) and mst2(+). Mst1p, but not Mst2p, is essential for viability. Both proteins are localized to the nucleus and bound to chromatin throughout the cell cycle. Deltamst2 genetically interacts with mutants that affect heterochromatin, cohesion, and telomere structure. Mst2p is a negative regulator of silencing at the telomere but does not affect silencing in the centromere or mating type region. We generated a census of proteins and histone modifications at wild-type telomeres. A histone acetylation gradient at the telomeres is lost in Deltamst2 cells without affecting the distribution of Taz1p, Swi6p, Rad21p, or Sir2p. We propose that the increased telomeric silencing is caused by histone hypoacetylation and/or an increase in the ratio of methylated to acetylated histones. Although telomere length is normal, meiosis is aberrant in Deltamst2 diploid homozygote mutants, suggesting that telomeric histone acetylation contributes to normal meiotic progression.

Suppressors of Bir1p (Survivin) identify roles for the chromosomal passenger protein Pic1p (INCENP) and the replication initiation factor Psf2p in chromosome segregation.

Fission yeast Bir1p/Cut17p/Pbh1p, the homolog of human Survivin, is a conserved chromosomal passenger protein that is required for cell division and cytokinesis. To study how Bir1p promotes accurate segregation of chromosomes, we generated and analyzed a temperature-sensitive allele, bir1-46, and carried out genetic screens to find genes that interact with bir1(+). We identified Psf2p, a component of the GINS complex required for DNA replication initiation, as a high-copy-number suppressor of the bir1-46 growth defect. Loss of Psf2p function by depletion or deletion or by use of a temperature-sensitive allele, psf2-209, resulted in chromosome missegregation that was associated with mislocalization of Bir1p. We also found that the human homolog of Psf2p, PSF2, was required for proper chromosome segregation. In addition, we observed that high-copy-number expression of Pic1p, the fission yeast homolog of INCENP (inner centromere protein), suppressed bir1-46. Pic1p exhibited a localization pattern typical of chromosomal passenger proteins. Deletion of pic1(+) caused chromosome missegregation phenotypes similar to those of bir1-46. Our data suggest that Bir1p and Pic1p act as part of a conserved chromosomal passenger complex and that Psf2p/GINS indirectly affects the localization and function of this complex in chromosome segregation, perhaps through an S-phase role in centromere replication.

Schizosaccharomyces pombe Rad4/Cut5 protein modification and chromatin binding changes in DNA damage.

The Schizosaccharomyces pombe Rad4/Cut5 protein is essential for DNA replication and checkpoint control. We have analyzed the behavior of the protein during unperturbed DNA replication, in different replication and checkpoint mutant backgrounds and in response to DNA-damaging agents. In an unperturbed cell cycle, Rad4 is chromatin bound and the mobility of the protein is not altered. Rad4 protein level and thus chromatin binding are dependent on a functional DNA polymerase epsilon. In response to replication arrest and DNA damage, the protein is modified in a Rad3-dependent manner. These data indicate that Rad4 undergoes diverse forms of regulation that are distinct in both DNA replication and checkpoint response.

Breathing of the Nevado del Ruiz volcano reservoir, Colombia, inferred from repeated seismic tomography.

Nevado del Ruiz volcano (NRV), Columbia, is one of the most dangerous volcanoes in the world and caused the death of 25,000 people in 1985. Using a new algorithm for repeated tomography, we have found a prominent seismic anomaly with high values of the Vp/Vs ratio at depths of 2-5 km below the surface, which is associated with a shallow magma reservoir. The amplitude and shape of this anomaly changed during the current phase of unrest which began in 2010. We interpret these changes as due to the ascent of gas bubbles through magma and to degassing of the reservoir. In 2011-2014, most of this gas escaped through permeable roof rocks, feeding surface fumarole activity and leading to a gradual decrease of the Vp/Vs ratio in the reservoir. This trend was reversed in 2015-2016 due to replenishment of the reservoir by a new batch of volatile-rich magma likely to sustain further volcanic activity. It is argued that the recurring "breathing" of the shallow reservoir is the main cause of current eruptions at NRV.

Trypanosoma cruzi Tcp12CKS1 interacts with parasite CRKs and rescues the p13SUC1 fission yeast mutant.

The complex mechanism of cell division in trypanosomatids is not completely fully understood. CRKs (cdc2-related kinases), Cyclins and CKSs (cdc2-kinase subunit) are involved in the progression through the cell cycle. The CKS proteins were first described as components of the cell cycle machinery in yeast and their action has been implicated in the regulation of CDK function. In the present work we identified Tcp12CKS1 a member of the CKS family in the parasite Trypanosoma cruzi. TcCKS1 is expressed in the three forms of T. cruzi. By using anti-Tcp12CKS1 antiserum, protein kinase (PK) activities were immunoprecipitated. The PK activity level varies depending on the stage analyzed, being lower in trypomastigotes and thus suggesting that different stages have different CKS-CRK complexes. Moreover, these PK activities were inhibited by using Flavopiridol, a known CDKs inhibitor. Western blot analyses demonstrated that in the epimastigote stage, p12CKS1 stably interacts with TcCRK1 and TcCRK3. In addition, Tcp12CKS1 was able to rescue the p13SUC1 null mutant of S. pombe. The functional complementation between the CKS proteins of two evolutionary distant organisms supports the role of Tcp12CKS1 as a key regulator in T. cruzi cell cycle.

A screen for Schizosaccharomyces pombe mutants defective in rereplication identifies new alleles of rad4+, cut9+ and psf2+.

Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes. We designed a screen for temperature-sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to rereplicate their DNA in the absence of cyclin B (Cdc13). Of the 42 rereplication-defective mutants analyzed, we were able to clone complementing plasmids for 10. This screen identified new alleles of the APC subunit cut9(+), the initiation/checkpoint factor rad4(+)/cut5(+), and the first mutant allele of psf2(+), a subunit of the novel GINS replication complex. Other genes identified are likely to play general roles in gene expression and protein localization.

Conserved locus-specific silencing functions of Schizosaccharomyces pombe sir2+.

In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+), and hst4(+), encode members of the Sir2 family of conserved NAD(+)-dependent protein deacetylases. The S. pombe sir2(+) gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2(+), suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.

Trisomy 21 consistently activates the interferon response.

Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits.

Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins.

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.