Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Multilayered proteomics reveals that JAM-A promotes breast cancer progression via regulation of amino acid transporter LAT1.

Recent studies have shown that transmembrane-type tight junction proteins are upregulated in various cancers compared with their levels in normal tissues and are involved in cancer progression, suggesting that they are potential therapeutic targets. Here, we demonstrated the expression profile and a novel role of junctional adhesion molecule-A (JAM-A) in breast cancer. Immunohistochemistry of surgical specimens showed that JAM-A was highly expressed from carcinoma in situ lesions, as in other adenocarcinomas, with higher expression in invasive carcinomas. High expression of JAM-A contributed to malignant aspects such as lymph node metastasis and lymphatic involvement positivity. In breast cancer cells, JAM-A expression status affects malignant potentials including proliferation and migration. Multilayered proteomics revealed that JAM-A interacts with the amino acid transporter LAT1 in breast cancer cells. JAM-A regulates the expression of LAT1 and interacts with it on the whole cell membrane, leading to enhanced amino acid uptake to promote tumor growth. Double high expression of JAM-A and LAT1 predicts poor prognosis in patients with breast cancer. Of note, an antibody against an extracellular domain of JAM-A suppressed the proliferation of breast cancer cells. Our findings indicate the possibility of JAM-A-targeted therapy ideally combined with LAT1-targeted therapy as a new therapeutic strategy against breast cancer.

Affinity of ß-Lactam Antibiotics for Neisseria gonorrhoeae Penicillin-Binding Protein 2 Having Wild, Cefixime-Reduced-Susceptible, and Cephalosporin (Ceftriaxone)-Resistant penA Alleles.

Multidrug-resistant Neisseria gonorrhoeae is a serious concern worldwide. Resistance to ß-lactam antibiotics occurs through mutations in penicillin-binding proteins (PBPs), acquisition of ß-lactamases, and alteration of antibiotic penetration. Mosaic structures of penA, which encodes PBP2, play a major role in resistance to ß-lactams, especially cephalosporins. Ceftriaxone (CRO) is recognized as the only satisfiable antibiotic for the treatment of gonococcal infections; however, CRO-resistant isolates have emerged in the community. Here, we examined the affinity of ß-lactam antibiotics for recombinant PBP2 in a competition assay using fluorescence-labeled penicillin. We found no or little difference in the affinities of penicillins and meropenem (MEM) for PBP2 from cefixime (CFM)-reduced-susceptible strain and cephalosporin-resistant strain. However, the affinity of cephalosporins, including CRO, for PBP2 from the cephalosporin-resistant strain was markedly lower than that for PBP2 from the CFM-reduced-susceptible-resistant strain. Notably, piperacillin (PIP) showed almost the same affinity for PBP2 from penicillin-susceptible, CFM-reduced-susceptible, and cephalosporin (including CRO)-resistant strains. Thus, PIP/tazobactam and MEM are candidate antibiotics for the treatment of CRO-resistant/multidrug-resistant N. gonorrhoeae.

The clarithromycin-binding proteins NIPSNAP1 and 2 regulate cytokine production through mitochondrial quality control.

The mechanism underlying the anti-inflammatory effect of macrolide antibiotics, such as clarithromycin (CAM), remains to be clarified. The CAM-binding proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25 (SNAP25)-like protein homolog (NIPSNAP) 1 and 2 are involved in the immune response and mitochondrial homeostasis. However, the axis between CAM-NIPSNAP-mitochondria and Toll-like receptor (TLR) and their molecular mechanisms remain unknown. In this study, we sought to elucidate the relationship between mitochondrial homeostasis mediated by NIPSNAP1 and 2 and the immunomodulatory effect of CAM. NIPSNAP1 or 2 knockdown (KD) by RNA interference impaired TLR4-mediated interleukin-8 (IL-8) production. Similar impairment was observed upon treatment with mitochondrial function inhibitors. However, IL-8 secretion was not impaired in NIPSNAP1 and 2 individual knockout (KO) and double KO (DKO) cells. Moreover, the oxygen consumption rate (OCR) in mitochondria measured using a flex analyzer was significantly reduced in NIPSNAP1 or 2 KD cells, but not in DKO cells. CAM also dose-dependently reduced the OCR. These results indicate that CAM suppresses the IL-8 production via the mitochondrial quality control regulated by temporary functional inhibition of NIPSNAP1 and 2. Our findings provide new insight into the mechanisms underlying cytokine production, including the TLR-mitochondria axis, and the immunomodulatory effects of macrolides.

Serotype replacement and an increase in non-encapsulated isolates among community-acquired infections of Streptococcus pneumoniae during post-vaccine era in Japan.

Objectives: It is feared that the serotype replacement of Streptococcus pneumoniae occurred by the introduction of pneumococcal vaccines as periodical inoculation leads to reduced efficacy of the approved vaccines and altered antimicrobial susceptibility. Methods: We determined serotypes of 351 S. pneumoniae isolates collected at a commercial clinical laboratory in Hokkaido prefecture, Japan, from December 2018 to February 2019 by using the polymerase chain reaction procedure of the US Centers for Disease Control and Prevention. Antimicrobial susceptibility and resistance gene profiles were also examined. Results: Vaccine coverage rates were 7.9% for 13-valent conjugate vaccine, and 32.5% for 23-valent polysaccharide vaccine, respectively. Non-typable strains were 19.7%. cpsA-positive isolates (group I), and null capsule clade (NCC)1, NCC2 and NCC3 (group II) comprised 31.3%, 28.4%, 32.8%, and 7.5% of the 69 non-typable strains, respectively. No penicillin-resistant/intermediate isolates were found; however, serotypes 35B and 15A/F showed low susceptibility to ß-lactams. Only five strains (1.4%) were levofloxacin-resistant, and all were from the older persons, and three strains were serotype 35B. Conclusion: The progression of serotype replacement in non-invasive pneumococcal infections has occurred during the post-vaccine era in Japan, and non-encapsulated isolates, such as NCC, have increased. Antimicrobial susceptibility is not worsened.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.

BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs.

ABBREVIATIONS: AMBRA1 autophagy and beclin 1 regulator 1; ATG14 autophagy related 14; ATG5 autophagy related 5; ATG7 autophagy related 7; BECN1 beclin 1; BECN2 beclin 2; CC coiled-coil; CQ chloroquine CNR1/CB1R cannabinoid receptor 1 DAPI 4',6-diamidino-2-phenylindole; dCCD delete CCD; DRD2/D2R dopamine receptor D2 GPRASP1/GASP1 G protein-coupled receptor associated sorting protein 1 GPCR G-protein coupled receptor; ITC isothermal titration calorimetry; IP immunoprecipitation; KD knockdown; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NRBF2 nuclear receptor binding factor 2; OPRD1/DOR opioid receptor delta 1 PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15 phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; RUBCN rubicon autophagy regulator; SQSTM1/p62 sequestosome 1; UVRAG UV radiation resistance associated; VPS vacuolar protein sorting; WT wild type.

Rapid, simple, and cost-effective plaque assay for murine norovirus using microcrystalline cellulose.

Murine norovirus (MNV) is used widely as a practical alternative to human norovirus (HuNoV). Plaque-forming assays for MNV are important for developing therapeutic agents against HuNoV infections. Although agarose-overlay MNV assays have been reported, recent improvements in cellulose derivatives suggest that they could be optimized further, particularly with respect to improving the overlay material. To determine which overlay material is optimal for the MNV plaque assay, we compared four typical cellulose derivatives [microcrystalline cellulose (MCC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC)] with conventional agarose. We found that 3.5% (w/v) MCC-containing medium provided clear round-shaped plaques on RAW 264.7 cells 1 day after inoculation; the visibility of plaques was comparable with that of the original agarose-overlay assay. Removing residual MCC powder from the MCC-overlay assay before fixing was important for obtaining distinct plaques that are clearly countable. Finally, after calculating the plaque diameter as a percentage of well diameter, we found that 12- and 24-well plates were better than other plates for accurate plaque counting. The MCC-based MNV plaque assay is cost-effective and rapid, and produces plaques that are easy to count. Accurate virus quantification using this optimized plaque assay will enable reliable estimation of norovirus titers.

Invasive pulmonary aspergillosis with candidiasis: usefulness of molecular and ultrastructural morphological analysis on FFPE tissue for invasive fungal infections.

Invasive pulmonary aspergillosis (IPA) is one of the most frequent forms of invasive fungal infections (IFI); however, it is often difficult to identify the pathogenic fungal species and to select appropriate treatments for patients with IFI including IPA. Here, we describe the detailed pathophysiology of an autopsy case of severe respiratory failure due to IPA with candidiasis. The patient developed severe respiratory failure after influenza infection and died, and the autopsy revealed a mixed disease of IPA with candidiasis. In this study, in addition to the routine pathological examination, we further examined formalin-fixed paraffin-embedded (FFPE) tissues by scanning electron microscopy (SEM) and partial genomic DNA sequencing. Although optical microscopy alone was insufficient to identify the pathogenic organisms, SEM clearly depicted the characteristic morphology of Aspergillus sp. and Candida sp. as closely overlapping in a nested fashion, providing evidence of mixed infection of both fungal species in a focal site. The technique using FFPE tissue in combination with ultrastructural observation by SEM, elemental analysis by SEM-EDX, and DNA sequencing is promising for analyzing the pathophysiology of IFI.

A hydroxypropyl methylcellulose plaque assay for human respiratory syncytial virus.

Quantifying proliferative virus particles is one of the most important experimental procedures in virology. Compared with classical overlay materials, newly developed cellulose derivatives enable a plaque-forming assay to produce countable clear plaques easily. HEp-2 cells are widely used in plaque assays for human respiratory syncytial virus (RSV). It is crucial to use an overlay material to keep HEp-2 cell proliferation and prevent RSV particles from spreading over the fluid. Among four cellulose derivatives, carboxymethyl cellulose sodium salt (CMC), hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), and hydroxyethyl cellulose (HEC), we found that HPMC was the optimal overlay material because HPMC maintained HEp-2 cell proliferation and RSV infectivity. Although MCC was unsuitable for RSV, it assisted the plaque-forming by human metapneumovirus in TMPRSS2-expressing cells. Therefore, depending on the cells and viruses, it is necessary to use different overlay materials at varying concentrations.

Reduction of susceptibility to azoles and 5-fluorocytosine and growth acceleration in Candida albicans in glucosuria.

Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia and glucosuria, and is a risk factor for Candida infections. To reveal the potential effects of glucosuria on Candida spp., we investigated their growth and antifungal susceptibilities in normal human urine to which glucose was added. The viable cell numbers of Candida spp. were more than 10 fold higher in the urine added 3000 mg/dL glucose than in plain urine. In antifungal susceptibility, more than 80% of Candida albicans clinical isolates increased minimum inhibitory concentrations of azoles and 5-fluorocytosine with the addition of glucose, and exceeded their breakpoints. In most of the C. albicans clinical isolates, the mRNA expression of the azole resistance genes ERG11, CDR1, CDR2, and MDR1 in the presence of glucose in urine. These observations provide valuable information about the clinical course and therapeutic effects of azoles against C. albicans infections in patients with diabetes mellitus and hyperglucosuria.

An autophagy-related protein Becn2 regulates cocaine reward behaviors in the dopaminergic system.

Drug abuse is a foremost public health problem. Cocaine is a widely abused drug worldwide that produces various reward-related behaviors. The mechanisms that underlie cocaine-induced disorders are unresolved, and effective treatments are lacking. Here, we found that an autophagy-related protein Becn2 is a previously unidentified regulator of cocaine reward behaviors. Becn2 deletion protects mice from cocaine-stimulated locomotion and reward behaviors, as well as cocaine-induced dopamine accumulation and signaling, by increasing presynaptic dopamine receptor 2 (D2R) autoreceptors in dopamine neurons. Becn2 regulates D2R endolysosomal trafficking, degradation, and cocaine-induced behaviors via interacting with a D2R-bound adaptor GASP1. Inactivating Becn2 by upstream autophagy inhibitors stabilizes striatal presynaptic D2R, reduces dopamine release and signaling, and prevents cocaine reward in normal mice. Thus, the autophagy protein Becn2 is essential for cocaine psychomotor stimulation and reward through regulating dopamine neurotransmission, and targeting Becn2 by autophagy inhibitors is a potential strategy to prevent cocaine-induced behaviors.

Aberrant expression of junctional adhesion molecule-A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155.

Recent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule-A (JAM-A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM-A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM-A significantly suppressed cell proliferation and colony-forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM-A reduced cell proliferation ability and that loss of JAM-A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM-A and formed a physical interaction with JAM-A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155-positive cases expressed a high level of JAM-A, and patients with the expression pattern of PVR/CD155 positive/JAM-A high had significantly shorter periods of relapse-free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM-A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM-A is therefore a potential therapeutic target for this malignancy.

Sitafloxacin has a potent activity for eradication of extended spectrum ß-lactamase-producing fluoroquinolone-resistant Escherichia coli forming intracellular bacterial communities in uroepithelial cells.

INTRODUCTION: Eradication of asymptomatic bacteriuria (ASB) before urological procedures is important to reduce the risk for infectious complications after surgery. However, the appropriate regimen for antimicrobial treatment has not been fully determined. We experienced continuous (over 10 months) isolation of extended spectrum ß-lactamase (ESBL)-producing fluoroquinolone-resistant Escherichia coli from urine of an asymptomatic patient. The four isolates obtained (SMESC1 to 4) were international high-risk clones of O25b:H4-ST131-H30R, and originated from one strain, as revealed by the whole genome sequences. Although the patient received meropenem (MEPM) and fosfomycin (FOM), to which the strains were susceptible before the urological procedures, they could not be eradicated. METHODS: To explore the reason for the continuous isolation even after MEPM and FOM administration, antimicrobial killing of adherent and/or intracellular bacterial communities (IBC) formed by coculture of the E. coli cells and T24 bladder epithelial cells were examined. RESULTS: FOM and levofloxacin did not decrease viable E. coli cells compared with gentamicin. MEPM partly decreased them, and sitafloxacin (STFX) decreased them most potently. These observations indicate that E. coli can survive in the urinary tract under antimicrobial administration, and some antimicrobials such as FOM and MEPM cannot eradicate E. coli in uroepithelial cells. Adhesion on urinary epithelial cells and/or IBC formation might result in continuous isolation from the urinary tract and recurrence of ASB and urinary tract infections. CONCLUSIONS: The present study suggests that STFX is a promising optional agent for the eradication of ESBL-producing fluoroquinolone-resistant E. coli in the urinary tract before urological procedures.

Structural analysis of the lipoteichoic acid anchor glycolipid: Comparison of methods for degradation of the glycerophosphate backbone polymer.

Acetic acid treatment [98% (v/v), 100 °C, 3 h] was proposed as a new method for degrading the glycerophosphate polymer moiety of Gram-positive bacterial lipoteichoic acid. We demonstrated that this method resulted in partial O-acetylation on the carbohydrate residues of the anchor glycolipid. Hence, the acetic acid treatment is not suitable for the chemical structural analysis of lipoteichoic acid.

Comparison of measurements of anti-PLA2R antibodies in Japanese patients with membranous nephropathy using in-house and commercial ELISA.

BACKGROUND: The prevalence of antibodies against M-type anti-phospholipase A2 receptor (PLA2R) was reported to be ~ 70-80% in early studies on idiopathic membranous nephropathy (iMN) cohorts from Western countries, China, and Korea, and ~ 50% in recent studies on two Japanese iMN cohorts. METHODS: We developed an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of anti-PLA2R antibodies, and examined sera from 217 patients with iMN, 22 patients with secondary MN (sMN), and 50 healthy individuals. All patients and healthy individuals were Japanese. The relationships between levels of anti-PLA2R antibodies and clinical parameters were analyzed. Serum samples were also tested using a standardized commercial ELISA (Euroimmun, Germany). RESULTS: In our ELISA, OD values greater than the mean + 3 standard deviation of healthy subjects were considered to be positive for anti-PLA2R antibodies. Of the patients with iMN, 33.6% (73/217) were positive, but all sMN patients were negative. Our ELISA and the Euroimmun ELISA had a high concordance (93.5%). The proportion of patients with nephrotic syndrome was significantly higher in anti-PLA2R antibody-positive patients than in antibody-negative patients (65.8 vs. 37.5%, P < 0.001). Levels of anti-PLA2R antibodies were significantly correlated with levels of urinary protein and serum albumin (P = 0.004 and P < 0.001, respectively). CONCLUSIONS: The prevalence of anti-PLA2R antibodies in our Japanese iMN cohort was lower than that in the previous studies from other countries and other Japanese institutes. The low prevalence of antibodies may be related with the characteristics of enrolled patients with mild proteinuria and undetectable antibody levels.

Autophagy Differentially Regulates Insulin Production and Insulin Sensitivity.

Autophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing ß cells versus insulin-responsive cells, utilizing knockin (Becn1F121A) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in ß cells, a process we term "vesicophagy." The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, ß cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development.

Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin.

Pseudomonas aeruginosa is an important etiological agent of opportunistic infections. Injectable colistin is available as a last-line treatment option for multidrug-resistant P. aeruginosa infections. When cells were inoculated at a high number, colistin-susceptible P. aeruginosa grew on agar medium containing colistin at a concentration 10-fold higher than the minimum inhibitory concentration without acquiring colistin resistance. This study examined the responsible mechanism for growth in the presence of a high concentration of colistin. Cell wash fluid derived from P. aeruginosa efficiently reduced colistin antimicrobial activity. This reduction was mediated by lipopolysaccharide (LPS) in the wash fluid. Extracellular LPS inhibited colistin activity more effectively than cell-bound LPS in fixed cells. Cell wash fluids from Escherichia coli and Acinetobacter baumannii also reduced colistin activity; however, they were less potent than those from P. aeruginosa. The amount of LPS in cell wash fluid from P. aeruginosa was approximately 10-fold higher than that in fluid from E. coli or A. baumannii. In conclusion, cell-free LPS derived from bacterial cells inhibited the antimicrobial activity of colistin, and this effect was greatest for P. aeruginosa. Thus, large amounts of broken and dead cells of P. aeruginosa at infection foci will reduce the effectiveness of colistin, even against cells that have not yet acquired resistance.

Evaluation of consistency in quantification of gene copy number by real-time reverse transcription quantitative polymerase chain reaction and virus titer by plaque-forming assay for human respiratory syncytial virus.

The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease.

Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid ß (Aß) peptides. Yet the role of autophagy in Aß production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aß clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/Becn1 in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. Becn1F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aß oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by Becn1 gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar Becn1-dependent protective effects on Aß removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of Becn1-mediated autophagy hyperactivation in the prevention of AD.