A novel Golgi mannosidase inhibitor: Molecular design,synthesis, enzyme inhibition,and inhibition of spheroid formation

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell–cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.     

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.     

β-Carotene enhances the expression of inflammation-related genes and histone H3 K9 acetylation,K4 dimethylation,and K36 trimethylation around these genes in juvenile macrophage-like THP-1 cells

β-Carotene is converted into vitamin A in the body and can remove reactive oxygen species. However, it is still unclear whether β-carotene alters the expression levels of inflammation-related genes in macrophages and how this is regulated. In the present study, we investigated whether the administration of β-carotene under hyperglycemic conditions altered the expression level of inflammation-related genes and whether any observed differences were associated with changes in histone modifications in juvenile macrophage-like THP-1 cells. THP-1 cells (from a human monocytic leukemia cell line) were cultured in low glucose (5 mM), high glucose (25 mM), or high glucose (25 mM) + β-carotene (5 μM) media for 1 day, and mRNA expression levels of genes related to oxidative stress and inflammation, and histone modifications were determined by mRNA microarray and qRT-PCR analyses, and chromatin immunoprecipitation assays, respectively. The expression of inflammation-related genes, such as IL31RA, CD38, and NCF1B, and inflammation-associated signaling pathway genes, such as ITGAL, PRAM1, and CSF3R, were upregulated by β-carotene under high-glucose conditions. Under these conditions, histone H3 lysine 4 (K4) demethylation, H3K36 trimethylation, and H3K9 acetylation around the CD38, NCF1B, and ITGAL genes were higher in β-carotene-treated cells than in untreated cells. Treatment of juvenile macrophage-like THP-1 cells with β-carotene under these high glucose conditions induced the expression of inflammation-related genes, K9 acetylation, and K4 di- and K36 trimethylation of histone H3 around these genes.     

Clinical,Hormonal, and Genetic Characteristics of 5α-Reductase Type 2 Deficiency in 103 Chinese Patients

Objective5α-Reductase type 2 (5α-RD2) deficiency causes variable degrees of undervirilization in patients. The correlation between its genotype and phenotype is unclear.MethodsWe retrospectively evaluated 103 patients with 46,XY disorders of sex development who were diagnosed with 5α-RD2 deficiency.ResultsThe prevalence of female sex assignment (P = .008) and the incidences of cryptorchidism (P = .0003) and bifid scrotum (P = .0002) in the non-p.R227Q variant group were higher, but there were no significant differences in the incidences of hypospadias and isolated microphallus. The external masculinization score in the non-p.R227Q variant group was lower than that in the homozygous p.R227Q variant (P = .019) and compound heterozygous p.R227Q variant groups (P = .013). The level of anti-Mullerian hormone in the non-p.R227Q variant group was lower than that in the homozygous p.R227Q variant (P < .001) and compound heterozygous p.R227Q variant groups (P = .006). The testosterone-to-dihydrotestosterone ratio of the homozygous p.R227Q variant group was higher than that of the non-p.R227Q variant (P = .018) and compound heterozygous p.R227Q variant groups (P = .029). Twenty-three reportedly pathogenic variants and 11 novel steroid 5α-reductase 2 (SRD5A2) variants were identified.ConclusionCompared with patients without p.R227Q, patients with p.R227Q exhibited higher external masculinization scores and anti-Mullerian hormone expression, a lower prevalence of female sex assignment, and lower incidences of cryptorchidism and bifid scrotum. We identified 23 reportedly pathogenic SRD5A2 variants and 11 novel SRD5A2 variants that led to 5α-RD2 deficiency. We established a genotype-phenotype correlation, and patients with p.R227Q showed a relatively mild phenotype.     

Specific blood group antibodies inhibit Shigella flexneri interaction with human cells in the absence of spinoculation

The classical models of investigating Shigella flexneri adherence and invasion of tissue culture cells involve either bacterial centrifugation (spinoculation) or the use of AfaE adhesin to overcome the low infection rate observed in vitro. However clinically, S. flexneri clearly adheres and invades the human colon in the absence of ‘spinoculation’. Additionally, certain S. flexneri tissue cell based assays (e.g. plaque assays and infection of T84 epithelial cells on Transwells®), do not require spinoculation. In the absence of spinoculation, we recently showed that glycan-glycan interactions play an important role in S. flexneri interaction with host cells, and that in particular the S. flexneri 2a lipopolysaccharide O antigen glycan has a high affinity for the blood group A glycan. During the investigation of the effect of blood group A antibodies on S. flexneri interaction with cells, we discovered that Panc-1 cells exhibited a high rate of infection in the absence of spinoculation. Select blood group A antibodies inhibited invasion of Panc-1 cells, and adherence to T84 cells. The use of Panc-1 cells represents a simplified model to study S. flexneri pathogenesis and does not require either spinoculation or exogenous adhesins.     

A Novel Monoclonal Antibody Degrades the Thyrotropin Receptor Autoantibodies in Graves' Disease

ObjectiveAutoantibodies against the thyrotropin receptor (TSH-R-Ab) are key mediators for the pathogenesis of Graves' disease (GD). TSH-R-Ab degradation was evaluated using several immunoassays within an exploratory, controlled trial in patients with GD receiving a monoclonal antibody (mAb) targeting the neonatal crystallizable fragment receptor (FcRn).MethodsSerial measurements of TSH-R-Ab serum levels were performed using 3 different binding and cell-based assays in patients with GD either on medication or on placebo.ResultsIn contrast to the placebo group, in which no changes were observed, a 12-week mAb therapy led to an early and significant decrease (>60%) in the serum TSH-R-Ab levels in patients with thyroidal and extrathyroidal GD, as unanimously shown in all 3 assays. These marked changes were noted already at week 7 post baseline (P <.0001 for the binding immunoassay and for the luciferase (readout) bioassay). The 3 TSH-R-Ab binding and bioassays were highly correlated in the samples of both study groups (binding immunoassay vs luciferase bioassay, r =.91, P <.001, binding vs cyclic adenosine monophosphate (cAMP) bioassay, r = 0.86, P <.001, and luciferase vs cAMP bioassay, r = 0.71, P =.006). The serological results correlated with the course of the extrathyroidal clinical parameters of GD, that is, clinical activity score and proptosis.ConclusionTargeting the FcRn markedly reduces the disease-specific TSH-R-Ab in patients with GD. The novel and rapid TSH-R-Ab bioassay improves diagnosis and management of patients with GD.     

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.     

Exposure of Agaricus bisporus to Trichoderma aggressivum f. europaeum leads to growth inhibition and induction of an oxidative stress response

Green mould disease of mushroom, Agaricus bisporus,is caused by Trichodermaspecies and can result in substantial crop losses.Label free proteomic analysis of changes in the abundance of A. bisporusproteins following exposure to T. aggressivumsupernatantin vitroindicated increased abundance of proteins associated with an oxidative stress response (zinc ion binding (+6.6 fold); peroxidase activity (5.3-fold); carboxylic ester hydrolase (+2.4 fold); dipeptidase (+3.2 fold); [2Fe-2S] cluster assembly (+3.3 fold)). Proteins that decreased in relative abundance were associated with growth: structural constituent of ribosome, translation (-12 fold), deadenylation-dependent decapping of nuclear-transcribed mRNA (-3.4 fold), and small GTPase mediated signal transduction (-2.6 fold). In vivoanalysis revealed that 10^-4 T. aggressivuminoculum decreased the mushroom yield by 29% to 56% and 10^-3 T. aggressivuminoculum decreased the mushroom yield by 68% to 100%. Proteins that increased in abundance in A. bisporusin vivofollowing exposure to T. aggressivumindicated an oxidative stress response and included proteins with pyruvate kinase activity (+2.6 fold) and hydrolase activity (+2.1 fold)). The results indicate that exposure of A. bisporusmycelium to T. aggressivum in vitroand in vivoresulted in an oxidative stress response and reduction in growth.     

Direct Proteomic Detection and Prioritization of 19 Onchocerciasis Biomarker Candidates in Humans

Onchocerca volvulus, the causative agent of onchocerciasis, infects over 20 million people and can cause severe dermatitis and ocular conditions including blindness. Current treatments employed in mass drug administration programs do not kill adult female worms, and common diagnostic tests cannot reliably assess viability of adult worms. There is an urgent need for better diagnostic tests to facilitate monitoring the efficacy of new treatments and disease elimination efforts. Here, eight plasma samples collected from individuals infected with O. volvulus and seven from uninfected individuals were analyzed by MS/MS spectrometry to directly identify O. volvulus proteins present in infected but absent in uninfected control samples. This direct proteomic approach for biomarker discovery had not been previously employed for onchocerciasis. Among all detected proteins, 19 biomarker candidates were supported by two or more unique peptides, identified in the plasma of at least three O. volvulus-infected human samples and absent in all control samples. Comprehensive analysis and ranking of these candidates included detailed functional annotation and a review of RNA-seq gene expression profiles. Isotope-labeled standard peptides were run in parallel and validated MS/MS peptide identifications for 15 peptides from 11 of the 19 proteins, and two infected urine and one uninfected urine sample was used for additional validation. A major antigen/OVOC11613 was identified as the most promising candidate with eight unique peptides across five plasma samples and one urine sample. Additional strong candidates included OVOC1523/ATP synthase, OVOC247/laminin and OVOC11626/PLK5, and along with OVOC11613, and were also detected in urine samples from onchocerciasis patients. This study has identified a promising novel set of proteins that will be carried forward to develop assays that can be used for diagnosis of O. volvulus infections and for monitoring treatment efficacy.     

Effects of gingerols-rich extract of ginger on growth performance,serum metabolites,meat quality and antioxidant activity of heat-stressed broilers

In order to investigate the effects of dietary ginger extract (GE) enriched in gingerols on broilers under heat stress (HS) from 21 to 42 days of age, a total of 144 Ross 308 male broilers were randomly allocated to three groups with six replicates of eight broilers per replicate. Broilers in the control group were raised at 22 °C and fed a basal diet, and broilers in the other two groups were raised under cyclic HS (34 °C from 9:00 to 17:00 and at 22 °C for the rest of the time) and fed the basal diet with or without 1000 mg/kg GE. Supplementation of GE improved (P < 0.05) final body weight, average daily gain and feed conversion ratio of broilers under HS, and tended (P < 0.1) to increase breast muscle yield. The alterations of serum total protein, albumin, total cholesterol levels and aspartate aminotransferase activity under HS were reversed (P < 0.05) by GE, which also decreased (P < 0.05) serum triglyceride level and alanine aminotransferase activity. The decreased redness (a* value) and increased drip loss of breast muscle induced by HS were restored (P < 0.05) by GE. Moreover, GE supplementation increased (P < 0.05) total antioxidant capacity and decreased (P < 0.05) malondialdehyde content in liver and breast muscle, and increased (P < 0.05) glutathione peroxidase activity in serum and breast muscle. In conclusion, dietary GE supplementation restored growth performance, serum metabolites and meat quality of broilers under HS possibly by improving antioxidant activity.     

Proteomic Analysis of Trichomonas vaginalis Phagolysosome,Lysosomal Targeting,and Unconventional Secretion of Cysteine Peptidases

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.     

Cryopreservation of three Saprolegnia species (Oomycota): Preliminary evidence for the long-term archiving of water mould species

Saprolegnia spp. water moulds are opportunistic pathogens that can cause economic losses to aquaculture. The diseases caused by them are difficult to control since use of the effective drug, malachite green oxalate, is no longer permitted in several regions (including the European Union and USA). To develop an effective control strategy, Saprolegnia isolates must be maintained in the laboratory. Cryopreservation is a useful solution for long-term maintenance; however, at present, there is no developed protocol for the cryopreservation of Saprolegnia spp. Here, we isolated and identified three Saprolegnia species, S. parasitica, S. australis and S. ferax, and developed a deep-freezing protocol that enables the long-term archiving of these species. The survival and growth rates of isolates kept at −80 °C for 3, 6, 9 and 12 months, were tested and compared among the species examined. Although the growth rates of frozen isolates were significantly lower than those of the control (i.e. non-frozen) isolates, the overall survival rate (>90%) indicated the effectiveness of the technique developed. Thus, the protocol developed appears to be a promising method for the long-term preservation of Saprolegnia isolates and may facilitate the creation of stock collections.     

Turbinaria ornata and its associated epiphytic Bacillus sp. A promising molecule supplier to discover new natural product approaches

Marine ecosystems are highly dependent on macroalgea in providing food and shelter for aquatic organisms, interacting with many bacteria and mostly producing secondary metabolites of potent therapeutic antibacterial property. Screening of marine microbial secondary metabolites of valuable biotechnological and therapeutical applications are now extensively studied. In this study, Bacillus spp. identified by DNA sequencing and found associated with Turbinaria ornata, was screened and characterized for its cell free supernatant (CFS) possible antimicrobial and antibiofilm applications. Among the 7 microbial isolates tested, CFS greatly affected Bacillus subitilis (12 mm) and inhibited equally the yeast isolates Candida albicans, Candida tropicalis and Candida glabrata (10 mm) and had no or negligible effect on S.aureus, E.coli, P. aeruginosa. As for the CFS antibiofilm activity, no difference was revealed from the positive control. Algal crude extracts (methanol, acetone and aqueous), on the other hand, were similarly tested for their antimicrobial activity against the seven microbial isolates, where highest activity was observed with the aqueous crude extract against Staphylococcus aureus(10 mm) and Pseudomonas aeruginosa (9 mm) compared to the negligible effects of methanol and acetone crude extracts. Chemical analysis was performed to reveal the major constituents of both crude algal extracts and Bacillus spp. CFS. FTIR spectrum of the bacterial CFS indicated the presence of bacteriocin as the major lipopeptide responsible for its biological activity. Whereas, methanol and water crude algal extract GC–MS spectra revealed different chemical groups of various combined therapeutical activity mainly Naphthalene, amino ethane-sulfonic acid, pyrlene, Biotin and mercury chloromethyl correspondingly. Thus, the present study, demonstrated the moderate activity of both crude algal extract and the bacterial CFS, however, further investigations are needed for a better biological activity.     

Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.     

Magnetospirillum sulfuroxidans sp. nov., capable of sulfur-dependent lithoautotrophy and a taxonomic reevaluation of the order Rhodospirillales

A spiral-shaped, highly motile bacterium was isolated from freshwater sulfidic sediment. Strain J10^T is a facultative autotroph utilizing sulfide, thiosulfate, and sulfur as the electron donors in microoxic conditions. Despite high 16S rRNA gene sequence sequence identity to Magnetospirillum gryphiswaldense MSR-1 ^T (99.6 %), digital DNA-DNA hybridisation homology and average nucleotide identity between the two strains was of the different species level (25 % and 83 %, respectively). Strain J10^T is not magnetotactic. The DNA G + C content of strain J10^T is 61.9 %. The predominant phospholipid ester-linked fatty acids are C18:1ω7, C16:1ω7, and C16:0. Strain J10^T (=DSM 23205 ^T = VKM B-3486 ^T) is the first strain of the genus Magnetospirillum showing lithoautotrophic growth and is proposed here as a novel species, Magnetospirillum sulfuroxidans sp. nov. In addition, we propose to establish a framework for distinguishing genera and families within the order Rhodospirillales based on phylogenomic analysis using the threshold values for average amino acid identity at ̴ 72 % for genera and ̴ 60 % for families. According to this, we propose to divide the existing genus Magnetospirillum into three genera: Magnetospirillum, Paramagnetospirillum, and Phaeospirillum, constituting a separate family Magnetospirillaceae fam. nov. in the order Rhodospirillales. Furthermore, phylogenomic data suggest that this order should accomodate six more new family level groups including Magnetospiraceae fam. nov., Magnetovibrionaceae fam. nov., Dongiaceae fam. nov., Niveispirillaceae fam. nov., Fodinicurvataceae fam. nov., and Oceanibaculaceae fam. nov.     

Etiology-, Sex-, and Tumor Size-Based Differences in Adrenocorticotropin-Dependent Cushing Syndrome

ObjectiveTo examine demographic, clinical, and biochemical differences in patients with adrenocorticotropin (ACTH)-dependent Cushing syndrome (CS) based on etiology, sex, and tumor size.MethodsThis was a single-center study of 211 patients with ACTH-dependent CS followed for 35 years. Patients were stratified into 3 groups based on etiology: Cushing disease (CD)/transsphenoidal surgery, Cushing disease/total bilateral adrenalectomy (CD/TBA), and ectopic ACTH secretion (EAS). Patients were also stratified based on sex and tumor size (nonvisualized, microadenoma, and macroadenoma).ResultsCD was the commonest cause of ACTH-dependent CS (190; 90%). Most patients presented in the third decade (median age, 29 years). Clinical features, cortisol, and ACTH were significantly greater in the EAS group. The CD/TBA group had more nonvisualized tumors (22% vs 8%; P = .000) and smaller tumor size (4 vs 6 mm; P = .001) compared with the CD/transsphenoidal surgery group. There was female predominance in CD (2.06:1) and male predominance in EAS (2:1). Men had shorter duration of symptoms (2 years; P = .014), were younger (23 years; P = .001), had lower body mass index (25.1 kg/m²; P = .000), and had more severe disease (low bone mineral density, hypokalemia). Macroadenomas were frequent (46; 24.2%), and ACTH correlated with tumor size in CD (r = 0.226; P = .005).ConclusionOur cohort presented at an earlier age than the Western population with a distinct, but slightly lower, female predilection. Patients with CD undergoing TBA had frequent negative imaging. Men had a clinical profile suggesting aggressive disease. Microadenoma and macroadenoma were difficult to distinguish on a clinicobiochemical basis.     

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ～10 nt, but it was required when flaps were ～500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ～20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ～20 nt and is completely required at ～50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ～20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.     

CNS anti-depressant,anxiolytic and analgesic effects of Ganoderma applanatum (mushroom) along with ligand-receptor binding screening provide new insights: Multi-disciplinary approaches

This research was designed to evaluate the CNS depressant, anxiolytic, and analgesic action of aqueous and ethanol extract of Ganoderma applanatum, a valuable medicinal fungus used in multiple disorders belongs to Ganodermataceae family. Two extracts of G. applanatum were prepared using distilled water and ethanol as solvents and named AEGA and EEGA. Open field method, rotarod method, tail suspension method, and hole cross method were utilized for the CNS depressant action. In contrast, elevated plus-maze test and hole board method were utilized for the anxiolytic action. For determining the analgesic potential, acetic acid-induced writhing test, hot plate method, and tail immersion test were used. Besides, molecular docking has been implemented by using Discovery studio 2020, UCSF Chimera and PyRx autodock vina. At both doses (200 and 400 mg/kg) of AEGA and EEGA showed significant CNS depressant effect (p < 0.05 to 0.001) against all four tests used for CNS depressant activity. Both doses of AEGA and EEGA exhibited important anxiolytic activity effect (p < 0.05 to 0.001)against the EPM and hole board test. Both doses of AEGA and EEGA also exhibited a potential analgesic effect (p < 0.05 to 0.001) against all three tests used for analgesic action. In addition, in the molecular docking the compounds obtained the scores of ?5.2 to ?12.8 kcal/mol. Ganoapplanin, sphaeropsidin D and cytosporone C showed the best binding affinity to the selected recptors. It can be concluded that AEGA and EEGA have potential CNS depressant, anxiolytic, and analgesic action, which can be used as a natural antidepressant, anxiolytic, and analgesic source.