Yeast Ataxin-2 Forms an Intracellular Condensate Required for the Inhibition of TORC1 Signaling during Respiratory Growth.

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.

Intrinsically disordered protein regions are required for cell wall homeostasis in Bacillus subtilis.

Intrinsically disordered protein regions (IDRs) have been implicated in diverse nuclear and cytoplasmic functions in eukaryotes, but their roles in bacteria are less clear. Here, we report that extracytoplasmic IDRs in Bacillus subtilis are required for cell wall homeostasis. The B. subtilis σI transcription factor is activated in response to envelope stress through regulated intramembrane proteolysis (RIP) of its membrane-anchored anti-σ factor, RsgI. Unlike canonical RIP pathways, we show that ectodomain (site-1) cleavage of RsgI is constitutive, but the two cleavage products remain stably associated, preventing intramembrane (site-2) proteolysis. The regulated step in this pathway is their dissociation, which is triggered by impaired cell wall synthesis and requires RsgI's extracytoplasmic IDR. Intriguingly, the major peptidoglycan polymerase PBP1 also contains an extracytoplasmic IDR, and we show that this region is important for its function. Disparate IDRs can replace the native IDRs on both RsgI and PBP1, arguing that these unstructured regions function similarly. Our data support a model in which the RsgI-σI signaling system and PBP1 represent complementary pathways to repair gaps in the PG meshwork. The IDR on RsgI senses these gaps and activates σI, while the IDR on PBP1 directs the synthase to these sites to fortify them.

Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae.

Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.

In Vivo Cross-Linking Sheds Light on the Salmonella Divisome in Which PBP3 and PBP3SAL Compete for Occupancy.

Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen-specific paralogue PBP3SAL. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild-type cells and isogenic mutants lacking PBP3 or PBP3SAL. The complexes were cross-linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3SAL do not extensively cross-link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross-link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain-containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol-Pal proteins. Other cross-linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D-endo- and D,D-carboxypeptidases. PBP3 and PBP3SAL localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic-dead PBP3SAL-S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an 'adjustable' divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities.

Peptidoglycan Endopeptidase PBP7 Facilitates the Recruitment of FtsN to the Divisome and Promotes Peptidoglycan Synthesis in Escherichia coli.

Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C-terminal α-helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D-amino acid HADA, reporting on transpeptidase activity, compared to wild-type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase-mediated PG cleavage may be a rate-limiting step for septal PG synthesis.

Roles of Pbp1, Mkt1, and Dhh1 in the regulation of gene expression in the medium containing non-fermentative carbon sources.

Pbp1, a yeast ortholog of human ataxin-2, is important for cell growth in the medium containing non-fermentable carbon sources. We had reported that Pbp1 regulates expression of genes related to glycogenesis via transcriptional regulation and genes related to mitochondrial function through mRNA stability control. To further analyze the role of Pbp1 in gene expression, we first examined the time course of gene expression after transfer from YPD medium containing glucose to YPGlyLac medium containing glycerol and lactate. At 12 h after transfer to YPGlyLac medium, the pbp1∆ mutant showed decreased expression of genes related to mitochondrial function but no decrease in expression of glycogenesis-related genes. We also examined a role of the Pbp1-binding factor, Mkt1. The mkt1∆ mutant, like the pbp1∆ mutant, showed slow growth on YPGlyLac plate and reduced expression of genes related to mitochondrial function. Furthermore, we found that mutation of DHH1 gene encoding a decapping activator exacerbated the growth of the pbp1∆ mutant on YPGlyLac plate. The dhh1∆ mutant showed reduced expression of genes related to mitochondrial function. These results indicate that Pbp1 and Mkt1 regulate the expression of genes related to mitochondrial function and that the decapping activator Dhh1 also regulates the expression of those genes.

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1.

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used ß-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of ß-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of ß-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing ß-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand-PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum ß-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to ß-lactam/ß-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum ß-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

PBP4 is required for serum-induced cell wall thickening and antibiotic tolerance in Staphylococcus aureus.

The bacterial pathogen Staphylococcus aureus responds to the host environment by synthesizing a thick peptidoglycan cell wall, which protects the bacterium from membrane-targeting antimicrobials and the immune response. However, the proteins required for this response were previously unknown. Here, we demonstrate by three independent approaches that the penicillin-binding protein PBP4 is crucial for serum-induced cell wall thickening. First, mutants lacking various non-essential cell wall synthesis enzymes were tested, revealing that a mutant lacking pbp4 was unable to generate a thick cell wall in serum. This resulted in reduced serum-induced tolerance of the pbp4 mutant toward the last resort antibiotic daptomycin relative to wild-type cells. Second, we found that serum-induced cell wall thickening occurred in each of a panel of 134 clinical bacteremia isolates, except for one strain with a naturally occurring mutation that results in an S140R substitution in the active site of PBP4. Finally, inhibition of PBP4 with cefoxitin prevented serum-induced cell wall thickening and the resulting antibiotic tolerance in the USA300 strain and clinical MRSA isolates. Together, this provides a rationale for combining daptomycin with cefoxitin, a PBP4 inhibitor, to potentially improve treatment outcomes for patients with invasive MRSA infections.

Fully closed conformation of penicillin-binding protein revealed by structure of PBP2 from Acinetobacter baumannii.

Penicillin-binding protein 2 (PBP2), a vital protein involved in bacterial cell-wall synthesis, serves a target for ß-lactam antibiotics. Acinetobacter baumannii is a pathogen notorious for multidrug resistance; therefore, exploration of PBPs is pivotal in the development of new antimicrobial strategies. In this study, the tertiary structure of PBP2 from A. baumannii (abPBP2) was elucidated using X-ray crystallography. The structural analysis demonstrated notable movement in the head domain, potentially critical for its glycosyltransferase function, suggesting that abPBP2 assumes a fully closed conformation. Our findings offer valuable information for developing novel antimicrobial agents targeting abPBP2 that are applicable in combating multidrug-resistant infections.

Carba PBP: a novel penicillin-binding protein-based lateral flow assay for rapid phenotypic detection of carbapenemase-producing Enterobacterales.

Rapid phenotypic detection assays, including Carba NP and its variants, are widely applied for clinical diagnosis of carbapenemase-producing Enterobacterales (CPE). However, these tests are based on the acidification of the pH indicator during carbapenem hydrolysis, which limits test sensitivity and speed, especially for the detection of CPE producing low-activity carbapenem (e.g., OXA-48 variants). Herein, we developed a novel rapid and sensitive CPE detection method (Carba PBP) that could measure substrate (meropenem) consumption based on penicillin-binding protein (PBP). Meropenem-specific PBP was used to develop a competitive lateral flow assay (LFA) for meropenem identification. For the detection of carbapenemase activity, meropenem concentration was optimized using a checkerboard assay. The performance of Carba PBP was evaluated and compared with that of Carba NP using a panel of 94 clinical strains characterized by whole-genome sequencing and carbapenem susceptibility test. The limit of detection of PBP-based LFA for meropenem identification was 7 ng mL-1. Using 10 ng mL-1 meropenem as the substrate, Carba PBP and Carba NP could detect 10 ng mL-1 carbapenemase within 25 min and 1,280 ng mL-1 CPE in 2 h, respectively. The sensitivity and specificity were 100% (75/75) and 100% (19/19) for Carba PBP and 85.3% (64/75) and 100% (19/19) for Carba NP, respectively. When compared with Carba NP, Carba PBP showed superior performance in detecting all the tested CPE strains (including OXA-48-like variants) within 25 min and presented two orders of magnitude higher analytical sensitivity, demonstrating potential for clinical diagnosis of CPE. IMPORTANCE This study successfully achieved the goal of carbapenemase activity detection with both high sensitivity and convenience, offering a convenient lateral flow assay for clinical diagnosis of carbapenemase-producing Enterobacterales.

Evolution of mutations in the ftsI gene leading to amino acid substitutions in PBP3 in Haemophilus influenzae strains under the selective pressure of ampicillin and cefuroxime.

BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to ß-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

The peptidoglycan synthase PBP interacts with PLASTID DIVISION2 to promote chloroplast division in Physcomitrium patens.

The peptidoglycan (PG) layer, a core component of the bacterial cell wall, has been retained in the Physcomitrium patens chloroplasts. The PG layer entirely encompasses the P. patens chloroplast, including the division site, but how PG biosynthesis cooperates with the constriction of two envelope membranes at the chloroplast division site remains elusive. Here, focusing on the PG synthase penicillin-binding protein (PBP), we performed cytological and molecular analyses to dissect the mechanism of chloroplast division in P. patens. We showed that PBP, acting in the final step of PG biosynthesis, is likely a chloroplast inner envelope protein that can aggregate at mid-chloroplasts during chloroplast division. Physcomitrium patens had five orthologs of PLASTID DIVISION2 (PDV2), an outer envelope component of the chloroplast division complex. Our data indicated that PpPDV2 proteins interact with PpPBP and are responsible for recruiting PpPBP to the chloroplast division site, in addition to PpDRP5B. Furthermore, we found that PBP deletion and carbenicillin application restrain constriction of the chloroplast division complex, rather than its assembly. This work provides direct molecular evidence for a link between chloroplast division of P. patens and PG biosynthesis and indicates that PG biosynthesis is required for the constriction of the chloroplast division apparatus in P. patens.

Corticotropin-releasing factor-dopamine interactions in male and female macaque: Beyond the classic VTA.

Dopamine (DA) is involved in stress and stress-related illnesses, including many psychiatric disorders. Corticotropin-releasing factor (CRF) plays a role in stress responses and targets the ventral midbrain DA system, which is composed of DA and non-DA cells, and divided into specific subregions. Although CRF inputs to the midline A10 nuclei ("classic VTA") are known, in monkeys, CRF-containing terminals are also highly enriched in the expanded A10 parabrachial pigmented nucleus (PBP) and in the A8 retrorubral field subregions. We characterized CRF-labeled synaptic terminals on DA (tyrosine hydroxylase, TH+) and non-DA (TH-) cell types in the PBP and A8 regions using immunoreactive electron microscopy (EM) in male and female macaques. CRF labeling was present mostly in axon terminals, which mainly contacted TH-negative dendrites in both subregions. Most CRF-positive terminals had symmetric profiles. In both PBP and A8, CRF symmetric (putative inhibitory) synapses onto TH-negative dendrites were significantly greater than asymmetric (putative excitatory) profiles. This overall pattern was similar in males and females, despite shifts in the size of these effects between regions depending on sex. Because stress and gonadal hormone shifts can influence CRF expression, we also did hormonal assays over a 6-month time period and found little variability in basal cortisol across similarly housed animals at the same age. Together our findings suggest that at baseline, CRF-positive synaptic terminals in the primate PBP and A8 are poised to regulate DA indirectly through synaptic contacts onto non-DA neurons.

Reduced susceptibility to aztreonam-avibactam conferred by acquired AmpC-type ß-lactamases in PBP3-modified Escherichia coli.

PURPOSE: Carbapenemase-producing Enterobacterales are a growing threat, and very few therapeutic options remain active against those multidrug resistant bacteria. Aztreonam is the molecule of choice against metallo-beta-lactamases (MBL) producers since it is not hydrolyzed by those enzymes, but the co-production of acquired plasmidic cephalosporinases or extended-spectrum ß-lactamases leading to aztreonam resistance may reduce the efficacy of this molecule. Hence, the development of the aztreonam-avibactam (AZA) combination provides an interesting therapeutic alternative since avibactam inhibits the activity of both cephalosporinases and extended-spectrum ß-lactamases. However, structural modifications of penicillin binding protein PBP3, the target of aztreonam, may lead to reduced susceptibility to aztreonam-avibactam. METHODS: Here the impact of various plasmid-encoded AmpC-type ß-lactamases (ACC-1, ACT-7, ACT-17, CMY-2, CMY-42, DHA-1, FOX-1, and FOX-5) on susceptibility to aztreonam-avibactam was evaluated using isogenic E. coli MG1655 strains harboring insertions in PBP3 (YRIN and YRIK). The inhibitory activity of various ß-lactamase inhibitors (clavulanic acid, tazobactam, avibactam, relebactam, and vaborbactam) were also compared against these enzymes. RESULTS: Hence, we showed that reduced susceptibility to AZA was due to the combined effect of both AmpC production and amino acid insertions in PBP3. The highest resistance level was achieved in strains possessing the insertions in PBP3 in association with the production of ACT-7, ACC-1, or CMY-42. CONCLUSION: Although none of the recombinant strains tested displayed clinical resistance to aztreonam-avibactam, our data emphasize that the occurrence of such profile might be of clinical relevance for MBL-producing strains.

Antibiotic susceptibility and genome analysis of Enterococcus species isolated from inpatients in one hospital with no apparent outbreak of vancomycin-resistant Enterococcus in Japan.

To prevent nosocomial infection, it is important to screen for potential vancomycin-resistant Enterococcus (VRE) among patients. In this study, we analyzed enterococcal isolates from inpatients in one hospital without any apparent outbreak of VRE. Enterococcal isolates were collected from inpatients at Hiroshima University Hospital from April 1 to June 30, 2021 using selective medium for Enterococci. Multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. A total of 164 isolates, including Enterococcus faecium (41 isolates), Enterococcus faecalis (80 isolates), Enterococcus raffinosus (11 isolates), Enterococcus casseliflavus (nine isolates), Enterococcus avium (12 isolates), Enterococcus lactis (eight isolates), Enterococcus gallinarum (two isolates), and Enterococcus malodoratus (one isolate), were analyzed. We found one vanA-positive E. faecium, which was already informed when the patient was transferred to the hospital, nine vanC-positive E. casseliflavus, and two vanC-positive E. gallinarum. E. faecium isolates showed resistance to ampicillin (95.1%), imipenem (95.1%), and levofloxacin (87.8%), and E. faecalis isolates showed resistance to minocycline (49.4%). Ampicillin- and levofloxacin-resistant E. faecium had multiple mutations in penicillin-binding protein 5 (PBP5) (39/39 isolates) and ParC/GyrA (21/36 isolates), respectively. E. raffinosus showed resistance to ampicillin (81.8%), imipenem (45.5%), and levofloxacin (45.5%), and E. lactis showed resistance to ampicillin (37.5%) and imipenem (50.0%). The linezolid resistance genes optrA and cfr(B) were found only in one isolate of E. faecalis and E. raffinosus, respectively. This study, showing the status of enterococci infection in hospitalized patients, is one of the important information when considering nosocomial infection control of VRE.

Berberine hydrochloride reduces staphyloxanthin synthesis by inhibiting fni genes in methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a great health threat to humans. Looking for compounds that could reduce the resistance of S. aureus towards methicillin is an effective way to alleviate the antimicrobial resistance crisis. METHODS AND RESULTS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Time-killing growth curve, staphyloxanthin and penicillin-binding protein 2a (PBP2a) were detected. A quantitative polymerase chain reaction was used to measure the effect of BBH on the gene transcription profiles of MRSA. The MIC of MRSA-ST59-t437 towards oxacillin was 8 µg/ml, and MBC was 128 µg/ml. After adding a sub-inhibitory concentration of BBH, the MIC and MBC of MRSA-ST59-t478 towards oxacillin went down to 0.125 and 32 µg/ml respectively. The amount of PBP2a and staphyloxanthin were reduced after treatment with BBH. Moreover, the transcription levels of sarA, mecA and fni genes were downregulated. CONCLUSIONS: It is for the first time reported that BBH could inhibit staphyloxanthin synthesis by inhibiting fni gene. Moreover, fni might be the target gene of sarA, and there might be another regulatory pathway to inhibit staphyloxanthin biosynthesis. BBH could effectively reduce the methicillin resistance of MRSA-ST59-t437 by downregulating fni, sarA and mecA genes.

Perioperative management of a patient with unexpectedly detected early-stage ovarian mucinous carcinoma combined with progressive bulbar paralysis: a case report and literature review.

BACKGROUND: Giant ovarian cysts (GOCs)complicated with progressive bulbar paralysis (PBP) are very rare, and no such literature about these cases have been reported. Through the diagnosis and treatment of this case, the perioperative related treatment of such patients was analyzed in detail, and early-stage ovarian mucinous carcinoma was unexpectedly found during the treatment, which provided reference for clinical diagnosis and treatment of this kind of diseases. CASE PRESENTATION: In this article, we reported a 38-year-old female patient. The patient was diagnosed with PBP 2 years ago. Examination revealed a large fluid-dominated cystic solid mass in the pelvis measuring approximately 28.6×14.2×8.0 cm. Carbohydrate antigen19-9(CA19-9) 29.20 IU/mL and no other significant abnormalities were observed. The patient eventually underwent transabdominal right adnexal resection under regional anesthesia, epidural block. Postoperative pathology showed mucinous carcinoma in some areas of the right ovary. The patient was staged as stage IA, and surveillance was chosen. With postoperative follow-up 1 month later, her CA19-9 decreased to 14.50 IU/ml. CONCLUSIONS: GOCs combined with PBP patients require a multi-disciplinary treatment. Preoperative evaluation of the patient's PBP progression, selection of the surgical approach in relation to the patient's fertility requirements, the nature of the ovarian cyst and systemic condition are required. Early mucinous ovarian cancer accidentally discovered after operation and needs individualized treatment according to the guidelines and the patient's situation. The patient's dysphagia and respiratory function should be closely monitored during the perioperative period. In addition, moral support from the family is also very important.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Expression of Wild-Type and Mutant Human TDP-43 in Yeast Inhibits TOROID (TORC1 Organized in Inhibited Domain) Formation and Autophagy Proportionally to the Levels of TDP-43 Toxicity.

TDP-43 forms aggregates in the neurons of patients with several neurodegenerative diseases. Human TDP-43 also aggregates and is toxic in yeast. Here, we used a yeast model to investigate (1) the nature of TDP-43 aggregates and (2) the mechanism of TDP-43 toxicity. Thioflavin T, which stains amyloid but not wild-type TDP-43 aggregates, also did not stain mutant TDP-43 aggregates made from TDP-43 with intragenic mutations that increase or decrease its toxicity. However, 1,6-hexanediol, which dissolves liquid droplets, dissolved wild-type or mutant TDP-43 aggregates. To investigate the mechanism of TDP-43 toxicity, the effects of TDP-43 mutations on the autophagy of the GFP-ATG8 reporter were examined. Mutations in TDP-43 that enhance its toxicity, but not mutations that reduce its toxicity, caused a larger reduction in autophagy. TOROID formation, which enhances autophagy, was scored as GFP-TOR1 aggregation. TDP-43 inhibited TOROID formation. TORC1 bound to both toxic and non-toxic TDP-43, and to TDP-43, with reduced toxicity due to pbp1Δ. However, extragenic modifiers and TDP-43 mutants that reduced TDP-43 toxicity, but not TDP-43 mutants that enhanced toxicity, restored TOROID formation. This is consistent with the hypothesis that TDP-43 is toxic in yeast because it reduces TOROID formation, causing the inhibition of autophagy. Whether TDP-43 exerts a similar effect in higher cells remains to be determined.