Current Outlook on Autophagy in Human Leukemia: Foe in Cancer Stem Cells and Drug Resistance, Friend in New Therapeutic Interventions.

Autophagy is an evolutionarily conserved cellular recycling process in cell homeostasis and stress adaptation. It confers protection and promotes survival in response to metabolic/environmental stress, and is upregulated in response to nutrient deprivation, hypoxia, and chemotherapies. Autophagy is also known to sustain malignant cell growth and contributes to cancer stem cell survival when challenged by cytotoxic and/or targeted therapies, a potential mechanism of disease persistence and drug resistance that has gathered momentum. However, different types of human leukemia utilize autophagy in complex, context-specific manners, and the molecular and cellular mechanisms underlying this process involve multiple protein networks that will be discussed in this review. There is mounting preclinical evidence that targeting autophagy can enhance the efficacy of cancer therapies. Chloroquine and other lysosomal inhibitors have spurred initiation of clinical trials and demonstrated that inhibition of autophagy restores chemosensitivity of anticancer drugs, but with limited autophagy-dependent effects. Intriguingly, several autophagy-specific inhibitors, with better therapeutic indexes and lower toxicity, have been developed. Promising preclinical studies with novel combination approaches as well as potential challenges to effectively eradicate drug-resistant cells, particularly cancer stem cells, in human leukemia are also detailed in this review.

Computational approach to predict species-specific type III secretion system (T3SS) effectors using single and multiple genomes.

BACKGROUND: Many gram-negative bacteria use type III secretion systems (T3SSs) to translocate effector proteins into host cells. T3SS effectors can give some bacteria a competitive edge over others within the same environment and can help bacteria to invade the host cells and allow them to multiply rapidly within the host. Therefore, developing efficient methods to identify effectors scattered in bacterial genomes can lead to a better understanding of host-pathogen interactions and ultimately to important medical and biotechnological applications. RESULTS: We used 21 genomic and proteomic attributes to create a precise and reliable T3SS effector prediction method called Genome Search for Effectors Tool (GenSET). Five machine learning algorithms were trained on effectors selected from different organisms and a trained (voting) algorithm was then applied to identify other effectors present in the genome testing sets from the same (GenSET Phase 1) or different (GenSET Phase 2) organism. Although a select group of attributes that included the codon adaptation index, probability of expression in inclusion bodies, N-terminal disorder, and G + C content (filtered) were better at discriminating between positive and negative sets, algorithm performance was better when all 21 attributes (unfiltered) were used. Performance scores (sensitivity, specificity and area under the curve) from GenSET Phase 1 were better than those reported for six published methods. More importantly, GenSET Phase 1 ranked more known effectors (70.3%) in the top 40 ranked proteins and predicted 10-80% more effectors than three available programs in three of the four organisms tested. GenSET Phase 2 predicted 43.8% effectors in the top 40 ranked proteins when tested on four related or unrelated organisms. The lower prediction rates from GenSET Phase 2 may be due to the presence of different translocation signals in effectors from different T3SS families. CONCLUSIONS: The species-specific GenSET Phase 1 method offers an alternative approach to T3SS effector prediction that can be used with other published programs to improve effector predictions. Additionally, our approach can be applied to predict effectors of other secretion systems as long as these effectors have translocation signals embedded in their sequences.

Rapid Identification of Bacteria Directly from Positive Blood Cultures by Use of a Serum Separator Tube, Smudge Plate Preparation, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

We analyzed the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of smudge plate growth for bacterial identification from 400 blood cultures. Ninety-seven percent of Gram-negative bacilli and 85% of Gram-positive organisms were correctly identified within 4 h; only eight isolates (2.0%) were misidentified. This method provided rapid and accurate microbial identification from positive blood cultures.

Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB.

The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

Verification, Analytical Sensitivity, Cost-effectiveness, and Comparison of 4 Candida auris Screening Methods.

In this verification study, we compare and contrast the performance characteristics of chromogenic agar culture, direct polymerase chain reaction (PCR), and broth enrichment followed by culture or PCR for the detection of Candida auris colonization. We find that culture and PCR both offer excellent performance, with broth enrichment offering little performance advantage given its cost.

The benefit of a complete reference genome for cancer structural variant analysis.

The complexities of cancer genomes are becoming more easily interpreted due to advancements in sequencing technologies and improved bioinformatic analysis. Structural variants (SVs) represent an important subset of somatic events in tumors. While detection of SVs has been markedly improved by the development of long-read sequencing, somatic variant identification and annotation remains challenging. We hypothesized that use of a completed human reference genome (CHM13-T2T) would improve somatic SV calling. Our findings in a tumour/normal matched benchmark sample and two patient samples show that the CHM13-T2T improves SV detection and prioritization accuracy compared to GRCh38, with a notable reduction in false positive calls. We also overcame the lack of annotation resources for CHM13-T2T by lifting over CHM13-T2T-aligned reads to the GRCh38 genome, therefore combining both improved alignment and advanced annotations. In this process, we assessed the current SV benchmark set for COLO829/COLO829BL across four replicates sequenced at different centers with different long-read technologies. We discovered instability of this cell line across these replicates; 346 SVs (1.13%) were only discoverable in a single replicate. We identify 49 somatic SVs, which appear to be stable as they are consistently present across the four replicates. As such, we propose this consensus set as an updated benchmark for somatic SV calling and include both GRCh38 and CHM13-T2T coordinates in our benchmark. The benchmark is available at: 10.5281/zenodo.10819636 Our work demonstrates new approaches to optimize somatic SV prioritization in cancer with potential improvements in other genetic diseases.

Methicillin-resistant Staphylococcus aureus colonization among health care workers in a downtown emergency department in Toronto, Ontario.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) acquired in the community, otherwise known as community-acquired MRSA, has emerged rapidly in recent years. Colonization with MRSA has been associated with an increased risk of symptomatic and serious infections and, in some settings, health care workers (HCWs) exhibit a higher prevalence of MRSA colonization. OBJECTIVE: To determine MRSA colonization in emergency department (ED) HCWs in the setting of a moderate prevalence of MRSA in skin and soft tissue infections. METHODS: The present study was conducted at a downtown ED in Toronto, Ontario. ED HCWs completed a brief questionnaire and swabs were taken from one anterior nare, one axilla and any open wounds (if present). Swabs were processed using standard laboratory techniques. RESULTS: None of the 89 staff (registered nurses [n=55], physicians [n=15], other [n=19]) were MRSA positive and 25 (28.1%) were colonized with methicillin-susceptible S aureus. CONCLUSIONS: Contrary to common belief among HCWs and previous studies documenting MRSA colonization of HCWs, MRSA colonization of this particular Canadian ED HCW cohort was very low and similar to that of the local population.

HISTORIQUE: Le Staphylococcus aureus résistant à la méthicilline (SARM) d'origine non nosocomiale, ou SARM d'origine communautaire, a émergé rapidement ces dernières années. La colonisation par le SARM s'associe à une augmentation du risque d'infections graves et symptomatiques. Dans certains milieux, les travailleurs de la santé (TdS) présentent une prévalence plus élevée de colonisation par le SARM. OBJECTIF: Déterminer la colonisation par le SARM desTdS d'un département d'urgence (DU) où l'on observe une prévalence modérée de SARM en cas d'infections de la peau et des tissus mous. MÉTHODOLOGIE: Les chercheurs ont mené la présente étude dans un DU du centre-ville de Toronto, en Ontario. Les TdS du DU ont rempli un bref questionnaire et effectué un prélèvement dans une narine, sous l'aisselle et dans leurs plaies ouvertes (le cas échéant). Les prélèvements ont été traités au moyen de techniques de laboratoire standard. RÉSULTATS: Aucun des 89 employés (infirmières diplômées [n=55], médecins [n=15], autres [n=19]) n'était positif au SARM, mais 25 (28,1 %) étaient colonisés par le S aureus susceptible à la méthicilline. CONCLUSIONS: Contrairement aux idées reçues chez les TdS et aux études antérieures étayant la colonisation des TdS par le SARM, la colonisation par le SARM de cette cohorte de TdS d'un DU canadien était très faible et similaire à celle de la population locale.

Prevalence and incidence of antimicrobial-resistant organisms among hospitalized inflammatory bowel disease patients.

BACKGROUND: Patients with inflammatory bowel disease (IBD) experience frequent hospitalizations and use of immunosuppressive medications, which may predispose them to colonization with antimicrobial-resistant organisms (ARO). OBJECTIVE: To determine the prevalence of ARO colonization on admission to hospital and the incidence of infection during hospitalization among hospitalized IBD patients. METHODS: A chart review comparing the prevalence of colonization and incidence of infection with methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL) in hospitalized IBD patients with those of non-IBD controls was performed. RESULTS: On admission, there were no significant differences between IBD inpatients and controls in the prevalence of colonization of methicillin-resistant S aureus (1.0% versus 1.2%; P=0.74), vancomycin-resistant enterococci (0.2% versus 0%; P=1.0) or ESBL (4.1% versus 5.5%; P=0.33). Pooling data from historical clinic-based cohorts, IBD patients were more likely than controls to have ESBL colonization (19% versus 6.6%; P<0.05). Antibiotic use on admission was associated with ESBL colonization among IBD inpatients (OR 4.2 [95% CI 1.4 to 12.6]). The incidence of ARO infections during hospitalization was not significantly different between IBD patients and controls. Among IBD patients who acquired ARO infections during hospitalizations, the mean time interval from admission to infection was shorter for those who were already colonized with ARO on admission. CONCLUSIONS: This particular population of hospitalized IBD patients was not shown to have a higher prevalence or incidence of ARO colonization or infection compared with non-IBD inpatients.

HISTORIQUE: Les patients atteints d'une maladie inflammatoire de l'intestin (MII) sont souvent hospitalisés et prennent souvent des immunosuppresseurs, ce qui peut les prédisposer à des organismes résistants aux antimicrobiens (ORA). OBJECTIF: Déterminer la prévalence de colonisation par des ORA à l'admission à l'hôpital ainsi que l'incidence d'infection pendant l'hospitalisation de patients atteints d'une MII. MÉTHODOLOGIE: Les chercheurs ont procédé à l'analyse des dossiers comparant la prévalence de colonisation et d'incidence d'infection par le Staphylococcus aureus résistant à la méthicilline (SARM), les entérocoques résistant à la vancomycine (EVM) et les entérobactériacées productrices de bêta-lactamase à spectre étendu (BLSE) des patients hospitalisés atteints d'une MII à celle de sujets témoins n'ayant pas de MII. RÉSULTATS: À l'admission, les chercheurs n'ont pas constaté de dif-férences significatives entre les patients hospitalisés atteints d'une MII et les sujets témoins pour ce qui est de la prévalence de colonisation par le SARM (1,0 % par rapport à 1,2 %; P=0,74), les EVM (0,2 % par rapport à 0 %; P=1,0) ou les BLSE (4,1 % par rapport à 5,5 %; P=0,33). Selon les données regroupées de cohortes cliniques rétrospectives, les patients atteints d'une MII étaient plus susceptibles que les sujets témoins d'être colonisés par des BLSE (19 % par rapport à 6,6 %; P<0,05). L'utilisation d'antibiotiques à l'admission s'associait à une colonisation par les BLSE chez les patients atteints d'une MII (RRR 4,2 [95 % IC1,4 à 12,6]). L'incidence d'infections par des ORA pendant l'hospitalisation n'était pas significativement différente entre les patients atteints d'une MII et les sujets témoins. Chez les patients atteints d'une MII qui avaient contracté une infection par des ORA pendant l'hospitalisation, l'intervalle moyen entre l'admission et l'infection était plus court pour ceux qui étaient déjà colonisés par des ORA à l'admission. CONCLUSIONS: Cette population de patients hospitalisés atteints d'une MII ne présentait pas de prévalence ou d'incidence plus élevée de colonisation par des ORA que les patients n'ayant pas de MII.

Genomic structures and regulation patterns at HPV integration sites in cervical cancer.

Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.

The Drivers, Mechanisms, and Consequences of Genome Instability in HPV-Driven Cancers.

Human papillomavirus (HPV) is the causative driver of cervical cancer and a contributing risk factor of head and neck cancer and several anogenital cancers. HPV's ability to induce genome instability contributes to its oncogenicity. HPV genes can induce genome instability in several ways, including modulating the cell cycle to favour proliferation, interacting with DNA damage repair pathways to bring high-fidelity repair pathways to viral episomes and away from the host genome, inducing DNA-damaging oxidative stress, and altering the length of telomeres. In addition, the presence of a chronic viral infection can lead to immune responses that also cause genome instability of the infected tissue. The HPV genome can become integrated into the host genome during HPV-induced tumorigenesis. Viral integration requires double-stranded breaks on the DNA; therefore, regions around the integration event are prone to structural alterations and themselves are targets of genome instability. In this review, we present the mechanisms by which HPV-dependent and -independent genome instability is initiated and maintained in HPV-driven cancers, both across the genome and at regions of HPV integration.

Conformationally restricted analogs of the direct thrombin inhibitor FM 19.

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme's active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure-activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH(2)). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC(50) values of 0.51 and 0.45 µM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.

Redesign of cosubstrate specificity and identification of important residues for substrate binding to hChAT.

In eukaryotes, choline acetyltransferase (ChAT) catalyzes the reversible formation of the neurotransmitter acetylcholine from choline and acetyl-CoA. ChAT belongs to a family of CoA-dependent enzymes that also includes the carnitine acyltransferases CrAT, CrOT, and CPTs. In contrast to CrOT and CPTs that are very active toward medium- and long-chain acyl-CoAs, respectively, CrAT and ChAT display activity toward only short-chain acyl-CoAs. We recently demonstrated the substrate and cosubstrate promiscuity of the wild-type human ChAT (hChAT). To extend the flexibility of this enzyme, we have generated a series of single, double, and triple hChAT mutants. Here we report the conversion of hChAT into choline octanoyltransferase (ChOT) and choline palmitoyltransferase (ChPT). The E337 and C550 residues (numbering from hChAT) were previously shown to dictate the acyl-CoA cosubstrate specificity in the carnitine series. Here we identify and demonstrate the importance of C551, in addition to E337 and C550, in contributing to the acyl-CoA specificity of hChAT. We also show that either C550 or C551 needs to be present for the transfer of medium- and long-chain acyl-CoAs by hChAT. By exploring the potential expansion of the tunnel on the substrate side, we demonstrate that residues M84, Y436, and Y552 play a critical role in binding and holding the choline substrate in the ChAT active site.

Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4').

Aminoglycosides are important antibiotics used against a wide range of pathogens. As a mechanism of defense, bacteria have evolved enzymes able to inactivate these drugs by regio-selectively adding a variety of functionalities (acetyl, phospho, and nucelotidyl groups) to their scaffolds. The aminoglycoside nucleotidyltransferase ANT(4') is one of the most prevalent and unique modifying-enzymes. Here, by TLC, HRMS, and colorimetric assays, we demonstrate that the resistance enzyme ANT(4') from Staphylococcus aureus is highly substrate and cosubstrate promiscuous. We show that deoxy-ribonucleotide triphosphates (dNTPs) are better cosubstrates than NTPs. We demonstrate that the position of the triphosphate group (5' and not 3') on the ribose/deoxyribose ring is important for recognition by ANT(4'), and that NTPs with larger substituents at the 3'-position of the ribose ring are not cosubstrates for ANT(4'). We confirm that for all aminoglycosides tested, the respective nucleotidylated products are completely inactive. These results provide valuable insights into the development of strategies to combat the ever-growing bacterial resistance problem.

Analysis of Ugandan cervical carcinomas identifies human papillomavirus clade-specific epigenome and transcriptome landscapes.

Cervical cancer is the most common cancer affecting sub-Saharan African women and is prevalent among HIV-positive (HIV+) individuals. No comprehensive profiling of cancer genomes, transcriptomes or epigenomes has been performed in this population thus far. We characterized 118 tumors from Ugandan patients, of whom 72 were HIV+, and performed extended mutation analysis on an additional 89 tumors. We detected human papillomavirus (HPV)-clade-specific differences in tumor DNA methylation, promoter- and enhancer-associated histone marks, gene expression and pathway dysregulation. Changes in histone modification at HPV integration events were correlated with upregulation of nearby genes and endogenous retroviruses.

Community- and Healthcare-Associated Methicillin-Resistant Staphylococcus aureus Strains: An Investigation Into Household Transmission, Risk Factors, and Environmental Contamination.

OBJECTIVE To measure transmission frequencies and risk factors for household acquisition of community-associated and healthcare-associated (HA-) methicillin-resistant Staphylococcus aureus (MRSA). DESIGN Prospective cohort study from October 4, 2008, through December 3, 2012. SETTING Seven acute care hospitals in or near Toronto, Canada. PARTICIPANTS Total of 99 MRSA-colonized or MRSA-infected case patients and 183 household contacts. METHODS Baseline interviews were conducted, and surveillance cultures were collected monthly for 3 months from household members, pets, and 8 prespecified high-use environmental locations. Isolates underwent pulsed-field gel electrophoresis and staphylococcal cassette chromosome mec typing. RESULTS Overall, of 183 household contacts 89 (49%) were MRSA colonized, with 56 (31%) detected at baseline. MRSA transmission from index case to contacts negative at baseline occurred in 27 (40%) of 68 followed-up households. Strains were identical within households. The transmission risk for HA-MRSA was 39% compared with 40% (P=.95) for community-associated MRSA. HA-MRSA index cases were more likely to be older and not practice infection control measures (P=.002-.03). Household acquisition risk factors included requiring assistance and sharing bath towels (P=.001-.03). Environmental contamination was identified in 78 (79%) of 99 households and was more common in HA-MRSA households. CONCLUSION Household transmission of community-associated and HA-MRSA strains was common and the difference in transmission risk was not statistically significant. Infect Control Hosp Epidemiol 2016;1-7.

Development of a bioavailable µ opioid receptor (MOPr) agonist, δ opioid receptor (DOPr) antagonist peptide that evokes antinociception without development of acute tolerance.

We have previously described a cyclic tetrapeptide, 1, that displays µ opioid receptor (MOPr) agonist and δ opioid receptor (DOPr) antagonist activity, a profile associated with a reduced incidence of opioid tolerance and dependence. Like many peptides, 1 has poor bioavailability. We describe here an analogue of 1 with an added C-terminal ß-glucosylserine residue, Ser(ß-Glc)NH2, a modification that has previously been shown to improve bioavailability of opioid peptides. The resulting peptide, 4, exhibits full antinociceptive efficacy in the mouse warm water tail withdrawal assay after intraperitoneal administration with potency similar to that of morphine. Further, 4 does not give rise to acute tolerance and thus represents a promising lead for the development of opioid analgesics with reduced side effects.

Antimalarial therapy selection for quinolone resistance among Escherichia coli in the absence of quinolone exposure, in tropical South America.

BACKGROUND: Bacterial resistance to antibiotics is thought to develop only in the presence of antibiotic pressure. Here we show evidence to suggest that fluoroquinolone resistance in Escherichia coli has developed in the absence of fluoroquinolone use. METHODS: Over 4 years, outreach clinic attendees in one moderately remote and five very remote villages in rural Guyana were surveyed for the presence of rectal carriage of ciprofloxacin-resistant gram-negative bacilli (GNB). Drinking water was tested for the presence of resistant GNB by culture, and the presence of antibacterial agents and chloroquine by HPLC. The development of ciprofloxacin resistance in E. coli was examined after serial exposure to chloroquine. Patient and laboratory isolates of E. coli resistant to ciprofloxacin were assessed by PCR-sequencing for quinolone-resistance-determining-region (QRDR) mutations. RESULTS: In the very remote villages, 4.8% of patients carried ciprofloxacin-resistant E. coli with QRDR mutations despite no local availability of quinolones. However, there had been extensive local use of chloroquine, with higher prevalence of resistance seen in the villages shortly after a Plasmodium vivax epidemic (p<0.01). Antibacterial agents were not found in the drinking water, but chloroquine was demonstrated to be present. Chloroquine was found to inhibit the growth of E. coli in vitro. Replica plating demonstrated that 2-step QRDR mutations could be induced in E. coli in response to chloroquine. CONCLUSIONS: In these remote communities, the heavy use of chloroquine to treat malaria likely selected for ciprofloxacin resistance in E. coli. This may be an important public health problem in malarious areas.