DNA cytosine methyltransferases differentially regulate genome-wide hypermutation and interhomolog recombination in Trichoderma reesei meiosis.

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2 and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

Natural products for combating multidrug resistance in cancer.

Cancer cells frequently develop resistance to chemotherapeutic therapies and targeted drugs, which has been a significant challenge in cancer management. With the growing advances in technologies in isolation and identification of natural products, the potential of natural products in combating cancer multidrug resistance has received substantial attention. Importantly, natural products can impact multiple targets, which can be valuable in overcoming drug resistance from different perspectives. In the current review, we will describe the well-established mechanisms underlying multidrug resistance, and introduce natural products that could target these multidrug resistant mechanisms. Specifically, we will discuss natural compounds such as curcumin, resveratrol, baicalein, chrysin and more, and their potential roles in combating multidrug resistance. This review article aims to provide a systematic summary of recent advances of natural products in combating cancer drug resistance, and will provide rationales for novel drug discovery.

Discovery of neuroprotective Agents: Potent, brain Penetrating, lipoic acid derivatives for the potential treatment of ischemic stroke by regulating oxidative stress and inflammation - a Preliminary study.

Stroke poses a serious risk to the physical and mental health of patients. Endogenous compounds are widely used to treat ischemic stroke. Lipoic acid, a naturally occurring (R)-5-(1,2-dithiolan-3-yl)pentanoic acid, has therapeutic potential for the treatment of ischemic stroke. However, the direct application of lipoic acid is limited by its relatively low efficacy and instability. Therefore, there is a need to modify the structure of lipoic acid to improve its pharmaceutical capabilities. Currently, 37 lipoic acid derivatives have been synthesized, and compound AA-9 demonstrated optimal therapeutic potential in an in vitro model of induced oxidative damage using tert-butyl hydroperoxide (t-BHP). In addition, in vitro experiments have shown that compound AA-9 has an excellent safety profile. Subsequently, the therapeutic effect of AA-9 was significant in the rat MCAO ischemic stroke model, which may be attributed to the antioxidant and anti-inflammatory effects of compound AA-9 by activating PGC-1α and inhibiting NLRP3. Notably, compound AA-9 exhibited higher stability and better bioavailability properties than ALA in plasma stability and pharmacokinetic properties. In conclusion, AA-9 may be a promising neuroprotective agent for the treatment of ischemic stroke and warrants further investigation.

Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

Discovery of novel danshensu derivatives bearing pyrazolone moiety as potential anti-ischemic stroke agents with antioxidant activity.

Neuroprotective agents with attenuation of oxidative stress by directly scavenging ROS and indirectly through Keap1-Nrf2 signal pathway activation may be a promising cerebral ischemic stroke therapeutic strategy. In this study, a series of novel danshensu derivatives bearing pyrazolone moieties with dual antioxidant effects were synthesized for the treatment of ischemic stroke. Most compounds exhibited considerable DPPH free radical scavenging ability and neuroprotective activity against H2O2-induced oxidative injury in PC12 neuronal cells, without cytotoxicity. Among these target compounds, Del03 displayed the strongest dose-dependent neuroprotective activity in vitro, directly downregulated intracellular ROS levels, and improved the oxidative stress parameters MDA, SOD, and LDH. Del03 also promoted Nrf2 translocation to the nucleus, subsequently increasing the expression of the Nrf2 downstream target HO-1. Molecular docking analysis revealed that Del03 could anchor to the key site of Keap1. Del03 possessed the ability to penetrate blood-brain barrier and displayed good ability on pharmacokinetic properties in rats Del03 possessed good BBB penetration efficiency, suitable pharmacokinetic properties in vivo. Del03 reduced cerebral infarction volume and promoted neurological function in a middle cerebral artery occlusion (MCAO) mouse model at a dose of 20 mg/kg by intravenous injection. The characteristics of Del03 detailed in this study demonstrate its potential as a therapeutic agent in the treatment of ischemic stroke.

Drosophila Phosphatase of Regenerating Liver Is Critical for Photoreceptor Cell Polarity and Survival during Retinal Development.

Establishing apicobasal polarity, involving intricate interactions among polarity regulators, is key for epithelial cell function. Though phosphatase of regenerating liver (PRL) proteins are implicated in diverse biological processes, including cancer, their developmental role remains unclear. In this study, we explore the role of Drosophila PRL (dPRL) in photoreceptor cell development. We reveal that dPRL, requiring a C-terminal prenylation motif, is highly enriched in the apical membrane of developing photoreceptor cells. Moreover, dPRL knockdown during retinal development results in adult Drosophila retinal degeneration, caused by hid-induced apoptosis. dPRL depletion also mislocalizes cell adhesion and polarity proteins like Armadillo, Crumbs, and DaPKC and relocates the basolateral protein, alpha subunit of Na+/K+-ATPase, to the presumed apical membrane. Importantly, this polarity disruption is not secondary to apoptosis, as suppressing hid expression does not rescue the polarity defect in dPRL-depleted photoreceptor cells. These findings underscore dPRL's crucial role in photoreceptor cell polarity and emphasize PRL's importance in establishing epithelial polarity and maintaining cell survival during retinal development, offering new insights into PRL's role in normal epithelium.

Two Reference-Quality Sea Snake Genomes Reveal Their Divergent Evolution of Adaptive Traits and Venom Systems.

True sea snakes (Hydrophiini) are among the last and most successful clades of vertebrates that show secondary marine adaptation, exhibiting diverse phenotypic traits and lethal venom systems. To better understand their evolution, we generated the first chromosome-level genomes of two representative Hydrophiini snakes, Hydrophis cyanocinctus and H. curtus. Through comparative genomics we identified a great expansion of the underwater olfaction-related V2R gene family, consisting of more than 1,000 copies in both snakes. A series of chromosome rearrangements and genomic structural variations were recognized, including large inversions longer than 30 megabase (Mb) on sex chromosomes which potentially affect key functional genes associated with differentiated phenotypes between the two species. By integrating multiomics we found a significant loss of the major weapon for elapid predation, three-finger toxin genes, which displayed a dosage effect in H. curtus. These genetic changes may imply mechanisms that drove the divergent evolution of adaptive traits including prey preferences between the two closely related snakes. Our reference-quality sea snake genomes also enrich the repositories for addressing important issues on the evolution of marine tetrapods, and provide a resource for discovering marine-derived biological products.

CBCRisk-Black: a personalized contralateral breast cancer risk prediction model for black women.

PURPOSE: Black breast cancer (BC) survivors have a higher risk of developing contralateral breast cancer (CBC) than Whites. Existing CBC risk prediction tools are developed based on mostly White women. To address this racial disparity, it is crucial to develop tools tailored for Black women to help them inform about their actual risk of CBC. METHODS: We propose an absolute risk prediction model, CBCRisk-Black, specifically for Black BC patients. It uses data on Black women from two sources: Breast Cancer Surveillance Consortium (BCSC) and Surveillance, Epidemiology, and End Results (SEER). First, a matched lasso logistic regression model for estimating relative risks (RR) is developed. Then, it is combined with relevant hazard rates and attributable risks to obtain absolute risks. Six-fold cross-validation is used to internally validate CBCRisk-Black. We also compare CBCRisk-Black with CBCRisk, an existing CBC risk prediction model. RESULTS: The RR model uses data from BCSC on 744 Black women (186 cases). CBCRisk-Black has four risk factors (RR compared to baseline): breast density (2.13 for heterogeneous/extremely dense), family history of BC (2.28 for yes), first BC tumor size (2.14 for T3/T4, 1.56 for TIS), and age at first diagnosis of BC (1.41 for < 40). The area under the receiver operating characteristic curve (AUC) for 3- and 5-year predictions are 0.72 and 0.65 for CBCRisk-Black while those are 0.65 and 0.60 for CBCRisk. CONCLUSION: CBCRisk-Black may serve as a useful tool to clinicians in counseling Black BC patients by providing a more accurate and personalized CBC risk estimate.

Molecular imaging on ACE2-dependent transocular infection of coronavirus.

A transocular infection has been proved as one of the main approaches that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the body, and angiotensin-converting enzyme 2 (ACE2) plays a key role in this procedure. Dynamic and quantitative details on virus distribution are lacking for virus prevention and drug design. In this study, a radiotraceable pseudovirus packed with an enhanced green fluorescent protein (EGFP) gene, 125 I-CoV, was prepared and inoculated in the unilateral eye of humanized ACE2 (hACE2) mice or ACE2-knockout (ACE2-KO) mice. Single-photon emission computed tomography/computed tomography images were acquired at multiple time points to exhibit ACE2-dependent procedures from invasion to clearance. Positron emission tomography (PET) and western blot were performed to quantify ACE2 expression and verify the factors affecting transocular infection. For the transocular infection of coronavirus (CoV), the renin-angiotensin-aldosterone system (RAAS), lungs, intestines, and genital glands were the main targeted organs. Due to the specific anchor to ACE2-expressed host cells, virus concentrations in genital glands, liver, and lungs ranked the top three most and stabilized at 3.75 ± 0.55, 3.30 ± 0.25, and 2.10 ± 0.55% inoculated dose (ID)/mL at 48 h post treatment. Meanwhile, ACE2-KO mice had already completed the in vivo clearance. In consideration of organ volumes, lungs (14.50 ± 3.75%ID) and liver (10.94 ± 0.71%ID) were the main in-store reservoirs of CoV. However, the inoculated eye (5.52 ± 1.85%ID for hACE2, 5.24 ± 1.45%ID for ACE2-KO, p > 0.05) and the adjacent brain exhibited ACE2-independent virus infection at the end of 72 h observation, and absolute amount of virus played a key role in host cell infection. These observations on CoV infection were further manifested by infection-driven intracellular EGFP expression. ACE2 PET revealed an infection-related systematic upregulation of ACE2 expression in the organs involved in RAAS (e.g., brain, lung, heart, liver, and kidney) and the organ that was of own local renin-angiotensin system (e.g., eye). Transocular infection of CoV is ACE2-dependent and constitutes the cause of disturbed ACE2 expression in the host. The brain, genital glands, and intestines were of the highest unit uptake, potentially accounting for the sequelae. Lungs and liver were of the highest absolute amount, closely related to the respiratory diffusion and in vivo duplication. ACE2 expression was upregulated in the short term after infection with CoV. These visual and quantitative results are helpful to fully understanding the transocular path of SARS-CoV-2 and other CoVs.

Recent Advances in Targeted Nanotherapies for Ischemic Stroke.

Ischemic stroke (IS) is a severe neurological disease caused by the narrowing or occlusion of cerebral blood vessels and is known for high morbidity, disability, and mortality rates. Clinically available treatments of stroke include the surgical removal of the thrombus and thrombolysis with tissue fibrinogen activator. Pharmaceuticals targeting IS are uncommon, and the development of new therapies is hindered by the low bioavailability and stability of many drugs. Nanomedicine provides new opportunities for the development of novel neuroprotective and thrombolytic strategies for the diagnosis and treatment of IS. Numerous nanotherapeutics with different physicochemical properties are currently being developed to facilitate drug delivery by accumulation and controlled release and to improve their restorative properties. In this review, we discuss recent developments in IS therapy, including assisted drug delivery and targeting, neuroprotection through regulation of the neuron environment, and sources of endogenous biomimetic specific targeting. In addition, we discuss the role and neurotoxic effects of inorganic metal nanoparticles in IS therapy. This study provides a theoretical basis for the utilization of nano-IS therapies that may contribute to the development of new strategies for a range of embolic diseases.

Novel long-acting ropeginterferon alfa-2b: Pharmacokinetics, pharmacodynamics and safety in a phase I clinical trial.

AIMS: Ropeginterferon alfa-2b is a novel, long-acting pegylated interferon alfa-2b. We aimed to evaluate its safety, pharmacokinetics (PK) and pharmacodynamics (PD). METHODS: Thirty-six subjects received single subcutaneous injection of ropeginterferon alfa-2b at doses ranging from 24 to 270 µg, and 12 subjects received pegylated IFN alfa-2a subcutaneously at 180 µg. Primary endpoints were safety/PK profiles of ropeginterferon alfa-2b, while secondary endpoints were to compare PK/PD parameters with pegylated IFN alfa-2a. RESULTS: Adverse events in ropeginterferon alfa-2b and pegylated IFN alfa-2a groups were similar, and most of them were mild or moderate. Mean Cmax increased from 1.78 to 24.84 ng/mL along with the dose escalations in ropeginterferon alfa-2b groups and was 12.95 ng/mL for pegylated IFN alfa-2a. At 180 µg, ropeginterferon alfa-2b showed statistically significant Cmax geometric mean ratio (1.76; P = .0275). Mean Tmax ranged from 74.52 to 115.69 h for ropeginterferon alfa-2b groups, and was 84.25 h for pegylated IFN alfa-2a. Mean AUC0-t increased from 372.3 to 6258 ng•h/mL with the dose escalations in the ropeginterferon alfa-2b groups, while for pegylated IFN alfa-2a it was found to be 2706 ng•h/mL in pegylated IFN alfa-2a. For neopterin and 2',5'-oligoadenylate synthase, mean Emax , Tmax and AUC0-t of ropeginterferon alfa-2b were similar to those of pegylated IFNα-2a at 180 µg. CONCLUSION: Ropeginterferon alfa-2b up to 270 µg was safe and well tolerated. The PK/PD parameters of ropeginterferon alfa-2b showed increase in dose-response. Ropeginterferon alfa-2b had higher drug exposures and showed similar safety profile when compared to pegylated IFN alfa-2a at the same dose level.

Dual roles of yeast Rad51 N-terminal domain in repairing DNA double-strand breaks.

Highly toxic DNA double-strand breaks (DSBs) readily trigger the DNA damage response (DDR) in cells, which delays cell cycle progression to ensure proper DSB repair. In Saccharomyces cerevisiae, mitotic S phase (20-30 min) is lengthened upon DNA damage. During meiosis, Spo11-induced DSB onset and repair lasts up to 5 h. We report that the NH2-terminal domain (NTD; residues 1-66) of Rad51 has dual functions for repairing DSBs during vegetative growth and meiosis. Firstly, Rad51-NTD exhibits autonomous expression-enhancing activity for high-level production of native Rad51 and when fused to exogenous ß-galactosidase in vivo. Secondly, Rad51-NTD is an S/T-Q cluster domain (SCD) harboring three putative Mec1/Tel1 target sites. Mec1/Tel1-dependent phosphorylation antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min. Our results evidence a direct link between homologous recombination and DDR modulated by Rad51 homeostasis.

Drosophila decapping protein 2 modulates the formation of cortical F-actin for germ plasm assembly.

In Drosophila, the deposition of the germ plasm at the posterior pole of the oocyte is essential for the abdomen and germ cell formation during embryogenesis. To assemble the germ plasm, oskar (osk) mRNA, produced by nurse cells, should be localized and anchored on the posterior cortex of the oocyte. Processing bodies (P-bodies) are cytoplasmic RNA granules responsible for the 5'-3' mRNA degradation. Evidence suggests that the components of P-bodies, such as Drosophila decapping protein 1 and Ge-1, are involved in the posterior localization of osk. However, whether the decapping core enzyme, Drosophila decapping protein 2 (dDcp2), is also involved remains unclear. Herein, we generated a dDcp2 null allele and showed that dDcp2 was required for the posterior localization of germ plasm components including osk. dDcp2 was distributed on the oocyte cortex and was localized posterior to the osk. In the posterior pole of dDcp2 mutant oocytes, osk was mislocalized and colocalized with F-actin detached from the cortex; moreover, considerably fewer F-actin projections were observed. Using the F-actin cosedimentation assay, we proved that dDcp2 interacted with F-actin through its middle region. In conclusion, our findings explored a novel function of dDcp2 in assisting osk localization by modulating the formation of F-actin projections on the posterior cortex.

Budding yeast Rad51: a paradigm for how phosphorylation and intrinsic structural disorder regulate homologous recombination and protein homeostasis.

The RecA-family recombinase Rad51 is the central player in homologous recombination (HR), the faithful pathway for repairing DNA double-strand breaks (DSBs) during both mitosis and meiosis. The behavior of Rad51 protein in vivo is fine-tuned via posttranslational modifications conducted by multiple protein kinases in response to cell cycle cues and DNA lesions. Unrepaired DSBs and ssDNA also activate Mec1ATR and Tel1ATM family kinases to initiate the DNA damage response (DDR) that safeguards genomic integrity. Defects in HR and DDR trigger genome instability and result in cancer predisposition, infertility, developmental defects, neurological diseases or premature aging. Intriguingly, yeast Mec1ATR- and Tel1ATM-dependent phosphorylation promotes Rad51 protein stability during DDR, revealing how Mec1ATR can alleviate proteotoxic stress. Moreover, Mec1ATR- and Tel1ATM-dependent phosphorylation also occurs on DDR-unrelated proteins, suggesting that Mec1ATR and Tel1ATM have a DDR-independent function in protein homeostasis. In this minireview, we first describe how human and budding yeast Rad51 are phosphorylated by multiple protein kinases at different positions to promote homology-directed DNA repair and recombination (HDRR). Then, we discuss recent findings showing that intrinsic structural disorder and Mec1ATR/Tel1ATM-dependent phosphorylation are coordinated in yeast Rad51 to regulate both HR and protein homeostasis.

Cytoprotective Effects Evaluation of a Novel Danshensu Derivative DEX-018 against Oxidative Stress Injury in HUVECs.

Ischemic heart disease (IHD) is one of the most common cardiovascular diseases with high morbidity and mortality. Danshensu (DSS) is widely used in the treatment of coronary heart disease. In this study, the carboxy group of DSS was esterified with edaravone to synthesize the novel DSS derivative DEX-018 to achieve a synergistic protective effect and overcome the structural deficiency of DSS. The pharmacological effect of DEX-018 against tert-butyl hydrogen peroxide (t-BHP) induced oxidative damage in human umbilical vein endothelial cells (HUVECs) was evaluated. The results demonstrated that pretreatment with DEX-018 significantly increased cell viability and superoxide dismutase (SOD) activity and decreased the lactate dehydrogenase (LDH) leakage rate, malondialdehyde (MDA) level and intracellular reactive oxygen species (ROS) level. In addition, DEX-018 inhibited cell apoptosis and reversed the expression of apoptosis-related proteins (Bcl-2, Bax, and caspase-3) in HUVECs stimulated by t-BHP. Further study on the mechanism of DEX-018 revealed that the expression of p-Akt and p-extracellular signal-regulated kinase 1/2 (ERK1/2) was increased, which suggested that DEX-018 may protect HUVECs against t-BHP induced oxidative injury via the Akt and ERK1/2 signaling pathways. To further validate the correlation, CCK8 was used to detect cell viability after treatment with DEX-018 plus Akt inhibitor (MK2206) and phosphadylinositol 3-kinase (PI3K) inhibitor (LY294002). Compared with DEX-018 alone, MK2206 or LY294002 significantly decreased cell viability of HUVECs, indicating that the protective effect of DEX-018 against t-BHP induced oxidative injury was significantly weakened. It was further verified that the antioxidant and anti-apoptotic effects of DEX-018 were partly related to the PI3K-Akt signaling pathway.

Tlr7 deletion alters expression profiles of genes related to neural function and regulates mouse behaviors and contextual memory.

The neuronal innate immune system recognizes endogenous danger signals and regulates neuronal development and function. Toll-like receptor 7 (TLR7), one of the TLRs that trigger innate immune responses in neurons, controls neuronal morphology. To further assess the function of TLR7 in the brain, we applied next generation sequencing to investigate the effect of Tlr7 deletion on gene expression in hippocampal and cortical mixed cultures and on mouse behaviors. Since previous in vivo study suggested that TLR7 is more critical for neuronal morphology at earlier developmental stages, we analyzed two time-points (4 and 18 DIV) to represent young and mature neurons, respectively. At 4 DIV, Tlr7 KO neurons exhibited reduced expression of genes involved in neuronal development, synaptic organization and activity and behaviors. Some of these Tlr7-regulated genes are also associated with multiple neurological and neuropsychiatric diseases. TLR7-regulated transcriptomic profiles differed at 18 DIV. Apart from neuronal genes, genes related to glial cell development and differentiation became sensitive to Tlr7 deletion at 18 DIV. Moreover, Tlr7 KO mice exhibited altered behaviors in terms of anxiety, aggression, olfaction and contextual fear memory. Electrophysiological analysis further showed an impairment of long-term potentiation in Tlr7 KO hippocampus. Taken together, these results indicate that TLR7 regulates neural development and brain function, even in the absence of infectious or pathogenic molecules. Our findings strengthen evidence for the role of the neuronal innate immune system in fine-tuning neuronal morphology and activity and implicate it in neuropsychiatric disorders.

Repeat-induced point (RIP) mutation in the industrial workhorse fungus Trichoderma reesei.

Trichoderma reesei (syn. Hypocrea jecorina) is a filamentous ascomycete. Due to its capability of producing large amounts of lignocellulolytic enzymes and various heterologous proteins, this fungus has been widely used for industrial applications for over 70 years. It is also a model organism for lignocellulosic biomass degradation and metabolic engineering. Recently, we experimentally and computationally demonstrated that Trichoderma reesei exhibits high homology pairing and repeat-induced point (RIP) mutation activities at a premeiotic stage, i.e., between fertilization and karyogamy or premeiotic DNA replication. The discovery of RIP in Trichoderma reesei not only reveals significant impacts of sexual reproduction on evolution and chromosome architecture but also provides intriguing perspectives for industrial strain improvement. This review emphasizes two major points about RIP and RIP-like processes in Pezizomycotina fungi. First, the molecular mechanisms of RIP and RIP-like processes in Trichoderma reesei and other Pezizomycotina fungi are apparently distinct from those originally described in the model fungus Neurospora crassa. Second, orthologs of the rid1 (deficient in RIP-1) DNA methyltransferase gene were shown to be essential for sexual development in at least four Pezizomycotina fungi, including Trichoderma reesei. In contrast, rid1 is dispensable for Neurospora crassa sexual development. We suggest that the rid1-like gene products and/or their DNA methyltransferase activities play critical roles in promoting fungal sexual development. The Neurospora crassa rid1 gene might have lost this evolutionarily conserved function.

S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex.

Double-strand breaks (DSBs) in chromosomes are the most challenging type of DNA damage. The yeast and mammalian Mre11-Rad50-Xrs2/Nbs1 (MRX/N)-Sae2/Ctp1 complex catalyzes the resection of DSBs induced by secondary structures, chemical adducts or covalently-attached proteins. MRX/N also initiates two parallel DNA damage responses-checkpoint phosphorylation and global SUMOylation-to boost a cell's ability to repair DSBs. However, the molecular mechanism of this SUMO-mediated response is not completely known. In this study, we report that Saccharomyces cerevisiae Mre11 can non-covalently recruit the conjugated SUMO moieties, particularly the poly-SUMO chain. Mre11 has two evolutionarily-conserved SUMO-interacting motifs, Mre11(SIM1) and Mre11(SIM2), which reside on the outermost surface of Mre11. Mre11(SIM1) is indispensable for MRX assembly. Mre11(SIM2) non-covalently links MRX with the SUMO enzymes (E2/Ubc9 and E3/Siz2) to promote global SUMOylation of DNA repair proteins. Mre11(SIM2) acts independently of checkpoint phosphorylation. During meiosis, the mre11(SIM2) mutant, as for mre11S, rad50S and sae2Δ, allows initiation but not processing of Spo11-induced DSBs. Using MRX and DSB repair as a model, our work reveals a general principle in which the conjugated SUMO moieties non-covalently facilitate the assembly and functions of multi-subunit protein complexes.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels.

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.IMPORTANCETrichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development.

Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over $100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.