In this paper, a decision tree is presented, constructed on the basis of hydrogeological characteristics (water table depth, freshwater thickness, surface area required and distance between wells), to choose the optimal groundwater extraction method in the case of a coastal unconfined aquifer. A comparison is made of the groundwater extraction methods in a freshwater aquifer of limited thickness occurring in coastal dunes in the eastern region of the Province of Buenos Aires (Argentina). The negative effects brought about by the wrong use of the groundwater extraction methods are analysed, because, as a result of excessive extraction, such methods lead to the dramatic decrease of the freshwater reserves. The decision tree is a useful tool to assist decision-makers as it suggests the most suitable groundwater extraction method options (vertical wells or wellpoints), as well as identifying areas that are unsuitable for sustainable groundwater extraction.

A logistic regression model and a bivariate statistical analysis were used in this paper to evaluate the groundwater recharge susceptibility. The approach is based on the assessment of the relationship involving groundwater recharge and parameters that influence this hydrological process. Surface parameters and aquifer-related parameters were evaluated as thematic map layers using ArcGIS. Then, a weighted-rating method was adopted to categorize each parameter's map. To assess the role of each parameter in the aquifer recharge, a logistic regression model and a bivariate statistical analysis were applied to the Guenniche phreatic aquifer (Tunisia). Models are explored to establish a map showing the aquifer recharge susceptibility. The code Modflow was used to simulate the consequence of the recharge. The recharge amount was introduced in the model and was tested to verify the recharge effect on the hydraulic head for the two models. The obtained results reveal that the recharge as mapped in the bivariate statistical model has a minor impact on the hydraulic head. Results of the logistic regression model are more significant as the hydraulic head is widely affected. This model provides good results in mapping the spatial distribution of the aquifer recharge susceptibility.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d’Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for the molecular detection of IAV using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5' and 3' ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed limits of detection similar to or lower than that of M gene RT-PCR and achieved 100% sensitivity and specificity in the detection of A(H1N1), A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV detected by M gene RT-PCR conversion in sequentially collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) of the initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates the identification of novel diagnostic targets.

Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples. A total of 866 clinical samples, 120 retrospectively and 21 prospectively collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of detection of Candida species (C. albicans, C. auris, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis) ranged from 97.1 to 100% and 99.8 to 100%, respectively. For the other organism targets, sensitivity and specificity were as follows: 100% each for Cryptococcus neoformans and C. gattii, 98.6% and 100% for Fusarium spp., and 96.2% and 99.9% for Rhodotorula spp., respectively. In 4 of the 141 clinical samples, the BCID-FP panel correctly identified an additional Candida species, undetected by standard-of-care methods. The BCID-FP panel offers a faster turnaround time for identification of fungal pathogens in positive blood cultures that may allow for earlier antifungal interventions and includes C. auris, a highly multidrug-resistant fungus.

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

Quantitative bacterial culture of bronchoalveolar lavage fluids (BALF) is labor-intensive, and the delay involved in performing culture, definitive identification, and susceptibility testing often results in prolonged use of broad-spectrum antibiotics. The Unyvero lower respiratory tract (LRT) panel (Curetis, Holzgerlingen, Germany) allows the multiplexed rapid detection and identification of 20 potential etiologic agents of pneumonia within 5 h of collection. In addition, the assay includes detection of gene sequences that confer antimicrobial resistance. We retrospectively compared the performance of the molecular panel to routine quantitative bacterial culture methods on remnant BALF. Upon testing 175 BALF, we were able to analyze positive agreement of 181 targets from 129 samples, and 46 samples were negative. The positive percent agreement (PPA) among the microbial targets was 96.5%, and the negative percent agreement (NPA) was 99.6%. The targets with a PPA of <100% were Staphylococcus aureus (34/37 [91.9%]), Streptococcus pneumoniae (10/11 [90.9%]), and Enterobacter cloacae complex (2/4 [50%]). For the analyzable resistance targets, concordance with phenotypic susceptibility testing was 79% (14/18). This study found the Unyvero LRT panel largely concordant with culture results; however, no outcome or clinical impact studies were performed.

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

Screening for Chlamydia trachomatis and Neisseria gonorrhoeae at the pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Combining the three individual-site samples into a single pooled sample could result in significant cost savings, provided there is no significant sensitivity reduction. The aim of this study was to examine the sensitivity of pooled samples for detecting chlamydia and gonorrhea in asymptomatic MSM using a nucleic acid amplification test. Asymptomatic MSM who tested positive for chlamydia or gonorrhoea were invited to participate. Paired samples were obtained from participants prior to administration of treatment. To form the pooled sample, the anorectal swab was agitated in the urine specimen transport tube and then discarded. The pharyngeal swab and 2 ml of urine sample were then added to the tube. The difference in sensitivity between testing of pooled samples and individual-site testing was calculated against an expanded gold standard, where an individual is considered positive if either pooled-sample or individual-site testing returns a positive result. All samples were tested using the Aptima Combo 2 assay. A total of 162 MSM were enrolled in the study. Sensitivities of pooled-sample testing were 86% (94/109; 95% confidence interval [CI], 79 to 92%]) for chlamydia and 91% (73/80; 95% CI, 83 to 96%) for gonorrhea. The sensitivity reduction was significant for chlamydia (P = 0.02) but not for gonorrhea (P = 0.34). Pooling caused 22 infections (15 chlamydia and 7 gonorrhoea) to be missed, and the majority were single-site infections (19/22). Pooling urogenital and extragenital samples from asymptomatic MSM reduced the sensitivity of detection by approximately 10% for chlamydia but not for gonorrhea.

Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (C_T), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median C_T was lower in toxin-positive samples than in toxin-negative samples; however, C_T ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

The number of onsite clinical microbiology laboratories in hospitals is decreasing, likely related to the business model for laboratory consolidation and labor shortages, and this impacts a variety of clinical practices, including that of banking isolates for clinical or epidemiologic purposes. To determine the impact of these trends, infectious disease (ID) physicians were surveyed regarding their perceptions of offsite services. Clinical microbiology practices for retention of clinical isolates for future use were also determined. Surveys were sent to members of the Infectious Diseases Society of America’s (IDSA) Emerging Infections Network (EIN). The EIN is a sentinel network of ID physicians who care for adult and/or pediatric patients in North America and who are members of IDSA. The response rate was 763 (45%) of 1,680 potential respondents. Five hundred forty (81%) respondents reported interacting with the clinical microbiology laboratory. Eighty-six percent of respondents thought an onsite laboratory very important for timely diagnostic reporting and ongoing communication with the clinical microbiologist. Thirty-five percent practiced in institutions where the core microbiology laboratory has been moved offsite, and an additional 7% (n = 38) reported that movement of core laboratory functions offsite was being considered. The respondents reported that only 24% of laboratories banked all isolates, with the majority saving isolates for less than 30 days. Based on these results, the trend toward centralized core laboratories negatively impacts the practice of ID physicians, potentially delays effective implementation of prompt and targeted care for patients with serious infections, and similarly adversely impacts infection control epidemiologic investigations.

Prosthetic joint infections are difficult to diagnose and treat due to biofilm formation by the causative pathogens. Pathogen identification relies on microbial culture that requires days to weeks, and in the case of chronic biofilm infections, lacks sensitivity. Diagnosis of infection is often delayed past the point of effective treatment such that only the removal of the implant is curative. Early diagnosis of an infection based on antibody detection might lead to less invasive, early interventions. Our study examined antibody-based assays against the Staphylococcus aureus biofilm-upregulated antigens SAOCOL0486 (a lipoprotein), glucosaminidase (a domain of SACOL1062), and SACOL0688 (the manganese transporter MntC) for detection of chronic S. aureus infection. We evaluated these antigens by enzyme-linked immunosorbent assay (ELISA) using sera from naive rabbits and rabbits with S. aureus-mediated osteomyelitis, and then we validated a proof of concept for the lateral flow assay (LFA). The SACOL0688 LFA demonstrated 100% specificity and 100% sensitivity. We demonstrated the clinical diagnostic utility of the SACOL0688 antigen using synovial fluid (SF) from humans with orthopedic implant infections. Elevated antibody levels to SACOL0688 in clinical SF specimens correlated with 91% sensitivity and 100% specificity for the diagnosis of S. aureus infection by ELISA. We found measuring antibodies levels to SACOL0688 in SF using ELISA or LFA provides a tool for the sensitive and specific diagnosis of S. aureus prosthetic joint infection. Development of the LFA diagnostic modality is a desirable, cost-effective option, potentially providing rapid readout in minutes for chronic biofilm infections.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

There are roughly 48,000 deaths caused by influenza annually and an estimated 200,000 people who have undiagnosed human immunodeficiency virus (HIV). These are examples of acute and chronic illnesses that can be identified by employing a CLIA-waived test. Pharmacies across the country have been incorporating CLIA-waived point-of-care tests (POCT) into disease screening and management programs offered in the pharmacy. The rationale behind these programs is discussed. Additionally, a summary of clinical data for some of these programs in the infectious disease arena is provided. Finally, we discuss the future potential for CLIA-waived POCT-based programs in community pharmacies.

This minireview focuses on the microbiologic evaluation of patients with asymptomatic bacteriuria, as well as indications for antibiotic treatment. Asymptomatic bacteriuria is defined as two consecutive voided specimens (preferably within 2 weeks) with the same bacterial species, isolated in quantitative counts of ≥10⁵ CFU/ml in women, including pregnant women; a single voided urine specimen with one bacterial species isolated in a quantitative count ≥10⁵ CFU/ml in men; and a single catheterized urine specimen with one or more bacterial species isolated in a quantitative count of ≥10⁵ CFU/ml in either women or men (or ≥10² CFU/ml of a single bacterial species from a single catheterized urine specimen). Any urine specimen with ≥10⁴ CFU/ml group B Streptococcus is significant for asymptomatic bacteriuria in a pregnant woman. Asymptomatic bacteriuria occurs, irrespective of pyuria, in the absence of signs or symptoms of a urinary tract infection. The two groups with the best evidence of adverse outcomes in the setting of untreated asymptomatic bacteriuria include pregnant women and patients who undergo urologic procedures with risk of mucosal injury. Screening and treatment of asymptomatic bacteriuria is not recommended in the following patient populations: pediatric patients, healthy nonpregnant women, older patients in the inpatient or outpatient setting, diabetic patients, patients with an indwelling urethral catheter, patients with impaired voiding following spinal cord injury, patients undergoing nonurologic surgeries, and nonrenal solid-organ transplant recipients. Renal transplant recipients beyond 1 month posttransplant should not undergo screening and treatment for asymptomatic bacteriuria. There is insufficient evidence to recommend for or against screening of renal transplant recipients within 1 month, patients with high-risk neutropenia, or patients with indwelling catheters at the time of catheter removal. Unwarranted antibiotics place patients at increased risk of adverse effects (including Clostridioides difficile diarrhea) and contribute to antibiotic resistance. Methods to reduce unnecessary screening for and treatment of asymptomatic bacteriuria aid in antibiotic stewardship.

On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID₅₀]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 x 10⁴ RNA copies/ml (range, 2.21 x 10² to 4.71 x 10⁵ RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.

The new decade of the 21^st century (2020) started with the emergence of a novel coronavirus known as SARS-CoV-2 that caused an epidemic of coronavirus disease (COVID-19) in Wuhan, China. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans, and mechanisms associated with the pathogenicity of SARS-CoV-2 are not yet clear, however, its resemblance to SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing-related studies. The development of therapeutic strategies is necessary in order to prevent further epidemics and cure infections. In this review, we summarize current information about the emergence, origin, diversity, and epidemiology of three pathogenic coronaviruses with a specific focus on the current outbreak in Wuhan, China. Furthermore, we discuss the clinical features and potential therapeutic options that may be effective against SARS-CoV-2.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae. This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper. The sample consisted of 18 isolates from 14 patients. Specimens were analyzed in the IR Biotyper using the default analysis settings, and spectra were analyzed using OPUS 7.5 software. The software contains a feature that automatically proposes a cutoff value to define clusters; the cutoff value defines up to which distance the spectra are considered to be in the same cluster. Based on FT-IR, the outbreak represented 1 dominant clone, 1 secondary clone, and several unrelated clones. FT-IR results, using the cutoff value generated by the accompanying software after 4 replicates, were concordant with WGS for all but 1 isolate. BOX-PCR was underdiscriminatory compared to the other two methods. Using the cutoff value generated after 12 replicates, the results of FT-IR and WGS were completely concordant. The IR Biotyper can achieve the same typeability and discriminatory power as genome-based methods. However, to attain this high performance requires either previous, strain-dependent knowledge about the optimal technical parameters to be used or validation by a second method.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases (n = 175) and matched controls (n = 175) were used for assay validation. Spearman’s correlation was calculated, and the diagnostic value of all multiplex markers was assessed independently using the area under the receiver operating characteristic curve (AUC). Two refined signatures were identified using stepwise logistic regression and internally validated with 10-fold cross validation. Array and multiplex serology showed strong correlation for each individual EBV marker, as well as for a 13-marker combined model on continuous data. Two refined signatures with either four (LF2 and BGLF2 IgG, LF2 and BMRF1 IgA) or two (LF2 and BGLF2 IgG) antibodies on dichotomous data were identified as the most parsimonious set of serological markers able to distinguish NPC cases from controls with AUCs of 0.992 (95% confidence interval [CI], 0.983 to 1.000) and 0.984 (95% CI, 0.971 to 0.997), respectively. Neither differed significantly from the 13-marker model (AUC, 0.992; 95% CI, 0.982 to 1.000). All models were internally validated. Multiplex serology successfully validated the original EBV proteome microarray data. Two refined signatures of four and two antibodies were capable of detecting NPC with 99.2% and 98.4% accuracy.

Large-scale metagenomic and metatranscriptomic data analyses are often restricted by their gene-centric approach, limiting the ability to understand organismal and community biology. De novo assembly of large and mosaic eukaryotic genomes from complex meta-omics data remains a challenging task, especially in comparison with more straightforward bacterial and archaeal systems. Here, we use a transcriptome reconstruction method based on clustering co-abundant genes across a series of metagenomic samples. We investigated the co-abundance patterns of ~37 million eukaryotic unigenes across 365 metagenomic samples collected during the Tara Oceans expeditions to assess the diversity and functional profiles of marine plankton. We identified ~12,000 co-abundant gene groups (CAGs), encompassing ~7 million unigenes, including 924 metagenomics-based transcriptomes (MGTs, CAGs larger than 500 unigenes). We demonstrated the biological validity of the MGT collection by comparing individual MGTs with available references. We identified several key eukaryotic organisms involved in dimethylsulfoniopropionate (DMSP) biosynthesis and catabolism in different oceanic provinces, thus demonstrating the potential of the MGT collection to provide functional insights on eukaryotic plankton. We established the ability of the MGT approach to capture interspecies associations through the analysis of a nitrogen-fixing haptophyte-cyanobacterial symbiotic association. This MGT collection provides a valuable resource for analyses of eukaryotic plankton in the open ocean by giving access to the genomic content and functional potential of many ecologically relevant eukaryotic species.

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell–specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein–protein relationships and protein functions at the chromatin template.

A time course experiment is a widely used design in the study of cellular processes such as differentiation or response to stimuli. In this paper, we propose time course regulatory analysis (TimeReg) as a method for the analysis of gene regulatory networks based on paired gene expression and chromatin accessibility data from a time course. TimeReg can be used to prioritize regulatory elements, to extract core regulatory modules at each time point, to identify key regulators driving changes of the cellular state, and to causally connect the modules across different time points. We applied the method to analyze paired chromatin accessibility and gene expression data from a retinoic acid (RA)–induced mouse embryonic stem cells (mESCs) differentiation experiment. The analysis identified 57,048 novel regulatory elements regulating cerebellar development, synapse assembly, and hindbrain morphogenesis, which substantially extended our knowledge of cis-regulatory elements during differentiation. Using single-cell RNA-seq data, we showed that the core regulatory modules can reflect the properties of different subpopulations of cells. Finally, the driver regulators are shown to be important in clarifying the relations between modules across adjacent time points. As a second example, our method on Ascl1-induced direct reprogramming from fibroblast to neuron time course data identified Id1/2 as driver regulators of early stage of reprogramming.

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100x higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor–recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

The regulation of transposable element (TE) activity by small RNAs is a ubiquitous feature of germlines. However, despite the obvious benefits to the host in terms of ensuring the production of viable gametes and maintaining the integrity of the genomes they carry, it remains controversial whether TE regulation evolves adaptively. We examined the emergence and evolutionary dynamics of repressor alleles after P-elements invaded the Drosophila melanogaster genome in the mid-twentieth century. In many animals including Drosophila, repressor alleles are produced by transpositional insertions into piRNA clusters, genomic regions encoding the Piwi-interacting RNAs (piRNAs) that regulate TEs. We discovered that ~94% of recently collected isofemale lines in the Drosophila melanogaster Genetic Reference Panel (DGRP) contain at least one P-element insertion in a piRNA cluster, indicating that repressor alleles are produced by de novo insertion at an exceptional rate. Furthermore, in our sample of approximately 200 genomes, we uncovered no fewer than 80 unique P-element insertion alleles in at least 15 different piRNA clusters. Finally, we observe no footprint of positive selection on P-element insertions in piRNA clusters, suggesting that the rapid evolution of piRNA-mediated repression in D. melanogaster was driven primarily by mutation. Our results reveal for the first time how the unique genetic architecture of piRNA production, in which numerous piRNA clusters can encode regulatory small RNAs upon transpositional insertion, facilitates the nonadaptive rapid evolution of repression.

Recent progress has been made in identifying genomic regions implicated in trait evolution on a microevolutionary scale in many species, but whether these are relevant over macroevolutionary time remains unclear. Here, we directly address this fundamental question using bird beak shape, a key evolutionary innovation linked to patterns of resource use, divergence, and speciation, as a model trait. We integrate class-wide geometric-morphometric analyses with evolutionary sequence analyses of 10,322 protein-coding genes as well as 229,001 genomic regions spanning 72 species. We identify 1434 protein-coding genes and 39,806 noncoding regions for which molecular rates were significantly related to rates of bill shape evolution. We show that homologs of the identified protein-coding genes as well as genes in close proximity to the identified noncoding regions are involved in craniofacial embryo development in mammals. They are associated with embryonic stem cell pathways, including BMP and Wnt signaling, both of which have repeatedly been implicated in the morphological development of avian beaks. This suggests that identifying genotype-phenotype association on a genome-wide scale over macroevolutionary time is feasible. Although the coding and noncoding gene sets are associated with similar pathways, the actual genes are highly distinct, with significantly reduced overlap between them and bill-related phenotype associations specific to noncoding loci. Evidence for signatures of recent diversifying selection on our identified noncoding loci in Darwin finch populations further suggests that regulatory rather than coding changes are major drivers of morphological diversification over macroevolutionary times.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Genome-wide association studies have implicated thousands of noncoding variants across common human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate that signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in cortical layer V excitatory neurons. We also show that differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in cortical layers II-III, IV, and V, as well as the dentate gyrus. Finally, we leverage these data to fine-map variants in 177 schizophrenia loci nominating variants in 104/177. We integrate these data with transcription factor binding site, chromatin interaction, and validated enhancer data, placing variants in the cellular context where they may modulate risk.

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A, and RAD21, cohesin in somatic cells contains one of two orthologous STAG subunits, STAG1 or STAG2. How these variable subunits affect the function of the cohesin complex is still unclear. STAG1- and STAG2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of STAG1 and STAG2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either STAG1 or STAG2. Following rapid depletion of either subunit, we perform high-resolution Hi-C, gene expression, and sequential ChIP studies to show that STAG1 and STAG2 do not co-occupy individual binding sites and have distinct ways by which they affect looping and gene expression. These findings are further supported by single-molecule localizations via direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging. Since somatic and congenital mutations of the STAG subunits are associated with cancer (STAG2) and intellectual disability syndromes with congenital abnormalities (STAG1 and STAG2), we verified STAG1-/STAG2-dependencies using human neural stem cells, hence highlighting their importance in particular disease contexts.

Breast cancer is a leading cause of death in women worldwide, but the underlying mechanisms of breast tumorigenesis remain unclear. Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor in breast cancer. However, the mechanisms of FBP1 as a tumor suppressor in breast cancer remain to be explored. Here we showed that FBP1 bound to Notch1 in breast cancer cells. Moreover, FBP1 enhanced ubiquitination of Notch1, further leading to proteasomal degradation via FBXW7 pathway. In addition, we found that FBP1 significantly repressed the transactivation of Notch1 in breast cancer cells. Functionally, Notch1 was involved in FBP1-mediated tumorigenesis of breast cancer cells in vivo and in vitro. Totally, these findings indicate that FBP1 inhibits breast tumorigenesis by regulating Notch1 pathway, highlighting FBP1 as a potential therapeutic target for breast cancer.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes in response to receptor tyrosine kinase (RTK)/Ras/ERK signaling. Following activation of RTK, ERK enters the nucleus and serine-phosphorylates CIC, releasing it from its targets to permit gene expression. We recently showed that ERK triggers ubiquitin-mediated degradation of CIC in glioblastoma (GBM). In this study, we examined whether another important downstream effector of RTK/EGFR, the non-RTK c-Src, affects CIC repressor function in GBM. We found that c-Src binds and tyrosine-phosphorylates CIC on residue 1455 to promote nuclear export of CIC. On the other hand, CIC-mutant allele (CIC-Y1455F), that escapes c-Src–mediated tyrosine phosphorylation, remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic transcription factors ETV1 and ETV5. Furthermore, we show that the orally available Src family kinase inhibitor, dasatinib, which prevents EGF-mediated tyrosine phosphorylation of CIC and attenuates elevated ETV1 and ETV5 levels, reduces viability of GBM cells and glioma stem cells (GSC), but not of their control cells with undetectable c-Src activity. In fact, GBM cells and GSC expressing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity to dasatinib, further endorsing the effect of dasatinib on Src-mediated tyrosine phosphorylation of CIC. These findings elucidate important mechanisms of CIC regulation and provide the rationale to target c-Src alongside ERK pathway inhibitors as a way to fully restore CIC tumor suppressor function in neoplasms such as GBM.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation.

Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttranslational modification enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hypercitrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps (NET). NETs were first described as antimicrobial fibers that bind and kill bacteria. However, it is not known whether PAD4 can mediate the release of chromatin DNA into the extracellular space of cancer cells. Here, we report that murine breast cancer 4T1 cells expressing high levels of PADI4 can release cancer extracellular chromatin networks (CECN) in vitro and in vivo. Deletion of Padi4 using CRISPR/Cas9 abolished CECN formation in 4T1 cells. Padi4 deletion from 4T1 cells also reduced the rate of tumor growth in an allograft model, and decreased lung metastasis by 4T1 breast cancers. DNase I treatment, which degrades extracellular DNA including CECNs, also reduced breast to lung metastasis of Padi4 wild-type 4T1 cells in allograft experiments in the Padi4-knockout mice. We further demonstrated that DNase I treatment in this mouse model did not alter circulating tumor cells but decreased metastasis through steps after intravasation. Taken together, our genetic studies show that PAD4 plays a cell autonomous role in cancer metastasis, thus revealing a novel strategy for preventing cancer metastasis by inhibiting cancer cell endogenous PAD4.

High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both in vitro and in vivo models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A.

The histone demethylase JMJD1A plays a key functional role in spermatogenesis, sex determination, stem cell renewal, and cancer via removing mono- and di-methyl groups from H3K9 to epigenetically control gene expression. However, its role in prostate cancer progression remains unclear. Here, we found JMJD1A was significantly elevated in prostate cancer tissue compared with matched normal tissue. Ectopic JMJD1A expression in prostate cancer cells promoted proliferation, migration, and invasion in vitro, and tumorigenesis in vivo; JMJD1A knockdown exhibited the opposite effects. Mechanically, we revealed that JMJD1A directly interacted with the Snail gene promoter and regulated its transcriptional activity, promoting prostate cancer progression both in vitro and in vivo. Furthermore, we found that JMJD1A transcriptionally activated Snail expression via H3K9me1 and H3K9me2 demethylation at its special promoter region. In summary, our studies reveal JMJD1A plays an important role in regulating proliferation and progression of prostate cancer cells though Snail, and thus highlight JMJD1A as potential therapeutic target for advanced prostate cancer.

Pancreatic cancer is one of the most lethal human malignancies, partly because of its propensity for metastasis. However, the mechanisms of metastasis in pancreatic cancer remain unclear. Oxidized low-density lipoprotein receptor 1 (OLR1), a lectin-like scavenger receptor that recognizes several ligands, such as oxidized low-density lipoprotein, was previously reported in cardiovascular and metabolic diseases. The role and mechanism of OLR1 in pancreatic cancer is unclear. In this study, we found that OLR1 expression was significantly higher in pancreatic cancer tissues than that in adjacent normal tissues and closely associated with reduced overall survival. OLR1 promoted proliferation and metastasis of pancreatic cancer cells in vitro and in vivo. Mechanistically, OLR1 increased HMGA2 transcription by upregulating c-Myc expression to promote the metastasis of pancreatic cancer cells. In addition, patients with pancreatic cancer with high expression of OLR1–c-Myc–HMGA2 axis showed worse prognosis compared with patients with low expression of OLR1–c-Myc–HMGA2 axis.

We recently reported that restoring the CYP27A1–27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti–prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6–JAK–STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6–JAK–STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation.

Pre-eclampsia (PE)-induced fetal programming predisposes offspring to health hazards in adult life. Here, we tested the hypothesis that pre-eclamptic fetal programming elicits sexually dimorphic inflammatory and cardiovascular complications to endotoxemia in adult rat offspring. PE was induced by oral administration of L-NAME (50 mg/kg per day for seven consecutive days) starting from day 14 of conception. Cardiovascular studies were performed in conscious adult male and female offspring preinstrumented with femoral indwelling catheters. Compared with non-PE male counterparts, intravenous administration of lipopolysaccharide (LPS, 5 mg/kg) to PE male offspring caused significantly greater 1) falls in blood pressure, 2) increases in heart rate, 3) rises in arterial dP/dtmax, a correlate of left ventricular contractility, and 4) decreases in time- and frequency-domain indices of heart rate variability (HRV). By contrast, the hypotensive and tachycardic actions of LPS in female offspring were independent of the pre-eclamptic state and no clear changes in HRV or dP/dtmax were noted. Measurement of arterial baroreflex activity by vasoactive method revealed no sex specificity in baroreflex dysfunction induced by LPS. Immunohistochemical studies showed increased protein expression of toll-like receptor 4 in heart as well as in brainstem neuronal pools of the nucleus of solitary tract and rostral ventrolateral medulla in endotoxic PE male, but not female, offspring. Enhanced myocardial, but not neuronal, expression of monocyte chemoattractant protein-1 was also demonstrated in LPS-treated male offspring. Together, pre-eclamptic fetal programming aggravates endotoxic manifestations of hypotension and autonomic dysfunction in male offspring via exacerbating myocardial and neuromedullary inflammatory pathways.

Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo ¹³C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases.