Bovine tuberculosis (TB), caused by Mycobacterium bovis, remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (~95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.

Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log₂; linear over a range of 4.27 to 9.65 log₂ 50% inhibitory concentration (IC₅₀); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.

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.

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.

Lyme borreliosis is a tick-borne disease caused by the Borrelia burgdorferi sensu lato complex. Bio-Rad Laboratories has developed a fully automated multiplex bead-based assay for the detection of IgM and IgG antibodies to B. burgdorferi. The BioPlex 2200 Lyme Total assay exhibits an improved rate of seropositivity in patients with early Lyme infection. Asymptomatic subjects from endemic and nonendemic origins demonstrated a seroreactivity rate of approximately 4% that was similar to other commercial assays evaluated in this study. Coupled to this result was the observation that the Lyme Total assay retained a high first-tier specificity of 96% while demonstrating a relatively high sensitivity of 91% among a well-characterized CDC Premarketing Lyme serum panel. The Lyme Total assay also performs well under a modified two-tier algorithm (sensitivity, 84.4 to 88.9%; specificity, 98.4 to 99.5%). Furthermore, the new assay is able to readily detect early Lyme infection in patient samples from outside North America.

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.

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.

Human herpesvirus 6 (HHV-6) is an important cause of meningitis and meningoencephalitis. As testing for HHV-6 in cerebrospinal fluid (CSF) is more readily available using the FilmArray Meningitis/Encephalitis panel (FA-ME; BioFire Diagnostics, Salt Lake City, UT), we aimed to determine the clinical significance of detecting HHV-6 in order to identify true infections and to ensure appropriate antiviral initiation. Chart review on 25 patients positive for HHV-6 by FA-ME was performed to determine clinical presentation, comorbidity, treatment, and outcome. The presence of chromosomally integrated HHV-6 (ciHHV-6) DNA was also investigated. Of 1,005 children tested by FA-ME, HHV-6 was detected in 25 (2.5%). Five patients were diagnosed with either HHV-6 meningitis or meningoencephalitis based on HHV-6 detection in CSF, clinical presentation, and radiographic findings. Detection of HHV-6 by FA-ME led to discontinuation of acyclovir within 12.0 h in all 12 patients empirically treated with acyclovir. Six of the 12 patients were started on ganciclovir therapy within 6.8 h; 4 of these were treated specifically for HHV-6 infection, whereas therapy was discontinued in the remaining 2 patients. CSF parameters were not generally predictive of HHV-6 positivity. The presence of ciHHV-6 was confirmed in 3 of 18 patients who could be tested. Five of the 25 patients included in the study were diagnosed with HHV-6 meningitis/meningoencephalitis. FA-ME results led to discontinuation of empirical antiviral treatment in 12 patients and appropriate initiation of ganciclovir in 4 patients. In our institution, detection of HHV-6 using FA-ME led to faster establishment of disease etiology and optimization of antimicrobial therapy.

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 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.

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.

Annelies Wouters, Jan-Pieter Ploem, Sabine A. S. Langie, Tom Artois, Aziz Aboobaker, and Karen Smeets

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Kristi McElmurry, Jessica E. Stone, Donghan Ma, Phillip Lamoureux, Yueyun Zhang, Michelle Steidemann, Lucas Fix, Fang Huang, Kyle E. Miller, and Daniel M. Suter

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

Yi Liu, Jaeyoun Kim, Reuben Philip, Vaishali Sridhar, Megha Chandrashekhar, Jason Moffat, Mark van Breugel, and Laurence Pelletier

PLK4 has emerged as a prime target for cancer therapeutics, and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind the role of PLK4 in these processes still remains only partly understood. Our previous work has demonstrated that an intact CEP85–STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here, we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL to the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 (also known as ACTR2) phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85–STIL complex in modulating PLK4-driven cancer cell migration.

This article has an associated First Person interview with the first author of the paper.

¹⁸F-rhPSMA-7 (radiohybrid prostate-specific membrane antigen [PSMA]) is a novel ligand for PET imaging. Here, we present data from a retrospective analysis using PET/CT and PET/MRI examinations to investigate the efficacy of ¹⁸F-rhPSMA-7 PET for primary N-staging of patients with prostate cancer (PC) compared with morphologic imaging (CT or MRI) and validated by histopathology. Methods: Data from 58 patients with high-risk PC (according to the D’Amico criteria) who were staged with ¹⁸F-rhPSMA-7 PET/CT or PET/MRI at our institution between July 2017 and June 2018 were reviewed. The patients had a median prescan prostate-specific antigen value of 12.2 ng/mL (range, 1.2–81.6 ng/mL). The median injected activity of ¹⁸F-rhPSMA-7 was 327 MBq (range, 132–410 MBq), with a median uptake time of 79.5 min (range, 60–153 min). All patients underwent subsequent radical prostatectomy and extended pelvic lymph node dissection. The presence of lymph node metastases was determined by an experienced reader independently for both the PET and the morphologic datasets using a template-based analysis on a 5-point scale. Patient-level and template-based results were both compared with histopathologic findings. Results: Lymph node metastases were present in 18 patients (31.0%) and were located in 52 of 375 templates (13.9%). Receiver-operating-characteristic analyses showed ¹⁸F-rhPSMA-7 PET to perform significantly better than morphologic imaging on both patient-based and template-based analyses (areas under curve, 0.858 vs. 0.649 [P = 0.012] and 0.765 vs. 0.589 [P < 0.001], respectively). On patient-based analyses, the sensitivity, specificity, and accuracy of ¹⁸F-rhPSMA-7 PET were 72.2%, 92.5%, and 86.2%, respectively, and those of morphologic imaging were 50.0%, 72.5%, and 65.5%, respectively. On template-based analyses, the sensitivity, specificity, and accuracy of ¹⁸F-rhPSMA-7 PET were 53.8%, 96.9%, and 90.9%, respectively, and those of morphologic imaging were 9.6%, 95.0%, and 83.2%, respectively. Conclusion: ¹⁸F-rhPSMA-7 PET is superior to morphologic imaging for N-staging of high-risk primary PC. The efficacy of ¹⁸F-rhPSMA-7 is similar to published data for ⁶⁸Ga-PSMA-11.

Prostate-specific membrane antigen (PSMA)–targeting α-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer. There is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The aim of this work was to characterize PSMA-TAT–nonresponding lesions by targeted next-generation sequencing. Methods: Of 60 patients treated with ²²⁵Ac-PSMA-617, we identified 10 patients who presented with a poor response despite sufficient tumor uptake in PSMA PET/CT. We were able to perform CT-guided biopsies with histologic validation of the nonresponding lesions in 7 of these nonresponding patients. Specimens were analyzed by targeted next-generation sequencing interrogating 37 DNA damage-repair–associated genes. Results: In the 7 tumor samples analyzed, we found a total of 15 whole-gene deletions, deleterious or presumably deleterious mutations affecting TP53 (n = 3), CHEK2 (n = 2), ATM (n = 2), and BRCA1, BRCA2, PALB2, MSH2, MSH6, NBN, FANCB, and PMS1 (n = 1 each). The average number of deleterious or presumably deleterious mutations was 2.2 (range, 0–6) per patient. In addition, several variants of unknown significance in ATM, BRCA1, MSH2, SLX4, ERCC, and various FANC genes were detected. Conclusion: Patients with resistance to PSMA-TAT despite PSMA positivity frequently harbor mutations in DNA damage-repair and checkpoint genes. Although the causal role of these alterations in the patient outcome remains to be determined, our findings encourage future studies combining PSMA-TAT and DNA damage-repair–targeting agents such as poly(ADP-ribose)-polymerase inhibitors.

The incidence of abnormal cerebrospinal fluid (CSF) flow dynamics in children with central nervous system (CNS) tumors before intraventricular therapy has not been described. Methods: We performed a single-institution, retrospective review of patients with primary or metastatic CNS tumors treated between 2003 and 2018 (15 y). Patients underwent ¹¹¹In-diethylenetriaminepentaacetic acid injection into the CSF intraventricular space followed by nuclear medicine imaging at 90 min, 4 h, 24 h, and 48 h (if required). CSF flow was classified as normal, delayed, asymmetric, or obstructed. Results: In total, 278 CSF flow studies were performed on 224 patients, 202 of whom (90%) were less than 18 y old. Of these, 116 patients (52%) had metastatic CNS neuroblastoma, 57 (25%) had medulloblastoma, and 51 (23%) had other histologic types of CNS tumors. Of the 278 studies, 237 (85%) were normal, 9 (3%) required neurosurgical intervention, 25 (9%) were delayed, and 7 (3%) were asymmetric. Conclusion: Abnormal CSF flow and the necessity for neurosurgical intervention must be considered when attempting to ensure appropriate intraventricular therapy in the pediatric population.

Background and objective

Exercise is a cornerstone of management for type 2 diabetes; however, little is known about the cardiovascular (CV) response to submaximal functional exercise in people with type 2 diabetes. The aim of this study was to compare performance and CV response during a 6-minute walk test (6MWT) between people with type 2 diabetes and matched control subjects.

Editor’s Note: This article was adapted from the address Dr. Snoek delivered as the recipient of the American Diabetes Association’s Richard R. Rubin Award for 2019. This award recognizes a behavioral researcher who has made outstanding, innovative contributions to the study and understanding of the behavioral aspects of diabetes in diverse populations. Dr. Snoek delivered the address in June 2019 at the Association’s 79th Scientific Sessions in San Francisco, CA.

Editor’s Note: This article is adapted from the address Ms. Valentine delivered as the recipient of the American Diabetes Association’s (ADA’s) Outstanding Educator in Diabetes Award for 2019. She delivered the address in June 2019 at the Association’s 79th Scientific Sessions in San Francisco, CA. A webcast of this speech is available for viewing at the ADA website (professional.diabetes.org/webcast/outstanding-educator-diabetes-award-lecture%E2%80%94-most-important-thing-we-give-people-hope).

A multidisciplinary endocrinologist-led shared medical appointment (SMA) model showed statistically significant reductions in A1C from baseline over 3 years that were not significantly different from appointments with endocrinologists or primary care providers alone within a resource-poor population. Similarly, the SMA model achieved clinical outcomes on par with endocrinologist-only visits with the added benefit of improving endocrine provider productivity and specialty access for patients. Greater patient engagement with the SMA model was associated with significantly lower A1C.

Background

Advances in information communications technology (ICT) provide opportunities for enhanced diabetes care. Knowledge of the more acceptable communication modalities in patients of different ages will help to inform the direction of future innovations.

Background

Diabetes is associated with poor oral health, as well as reduced access to dental care. A large percentage of patients hospitalized in the United States carry a diagnosis of diabetes; however, the oral health status of patients with diabetes who are hospitalized is unknown.

Therapeutic inertia is a prevalent problem in people with type 2 diabetes in primary care and affects clinical outcomes. It arises from a complex interplay of patient-, clinician-, and health system–related factors. Ultimately, clinical practice guidelines have not made an impact on improving glycemic targets over the past decade. A more proactive approach, including focusing on optimal combination agents for early glycemic durability, may reduce therapeutic inertia and improve clinical outcomes.

Factors contributing to therapeutic inertia related to patients’ medication experiences include concerns about side effects and out-of-pocket costs, stigmatization for having diabetes, confusion about frequent changes in evidence-based guidelines, low health literacy, and social determinants of health. A variety of solutions to this multifactorial problem may be necessary, including integrating pharmacists into interprofessional care teams, using medication refill synchronization programs, maximizing time with patients to discuss fears and concerns, being cognizant of language used to discuss diabetes-related topics, and avoiding stigmatizing patients. Managing diabetes successfully is a team effort, and the full commitment of all team members (including patients) is required to achieve desired outcomes through an individualized approach.

Despite significant advances in therapies for pediatric type 1 diabetes, achievement of glycemic targets remains elusive, and management remains burdensome for patients and their families. This article identifies common challenges in diabetes management at the patient-provider and health care system levels and proposes practical approaches to overcoming therapeutic inertia to enhance health outcomes for youth with type 1 diabetes.

Diabetes educators can be challenged by therapeutic inertia, as has been documented with other health care providers. There are many contributing factors related to the educators themselves, their patients, and the health care system in which they operate. To address this potentially significant barrier to quality patient care, diabetes educators can adopt numerous strategies to maximize their impact and address the factors contributing to therapeutic inertia in their practices.

Many people with diabetes do not achieve individualized treatment targets. Therapeutic inertia, the underuse of effective therapies in preventing serious clinical end points, is a frequent, important contributor to this failure. Clinicians, patients, health systems, payors, and producers of medications, devices, and other products for those with diabetes all play a role in the development of therapeutic inertia and can all help to reduce it.

Background and objectives

The effects of active case finding (ACF) models that mobilise community networks for early identification and treatment of tuberculosis (TB) remain unknown. We investigated and compared the effect of community-based ACF using a seed-and-recruit model with one-off roving ACF and passive case finding (PCF) on the time to treatment initiation and identification of bacteriologically confirmed TB.

Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5' or 3' half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon.

IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.

Diadenosine tetraphosphate (Ap₄A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap₄A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap₄A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap₄A in B. subtilis. YqeK shows a catalytic efficiency toward Ap₄A similar to that of the symmetrically cleaving Ap₄A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.

IMPORTANCE Elevation of Ap₄A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap₄A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap₄A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae. By identifying the player contributing to Ap₄A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

An exceptionally well-preserved specimen of the articulated rhodophyte Permocalculus, compared with P. tenellus sensu Elliott, 1955, is described from fine-grained Upper Permian limestones of the Khuff Formation of Saudi Arabia. Longitudinal medullary and sheaf-like cortical filaments extend through the uniserial series of elongate-globular, concave- and convex-terminating, interlocking segments for which they are interpreted to have functioned in articulation. The filaments tend to splay and branch laterally into the cortex where they terminate at the pores. At the terminal aperture, the filaments extend as bifurcating and possibly trifurcating branches and may serve as the origin of a new segment. Numerous elongate-globular chambers, up to five in each row and intimately involved with the filaments, are developed in the outer medulla and are considered to represent reproductive sporangia. The specimen is considered to have occupied predominantly low-energy, normal to slightly elevated salinity, shallow conditions within the subtidal regime of a lagoon.

ABSTRACTBackground:Coaching can improve the quality of care in primary-level birth facilities and promote birth attendant adherence to essential birth practices (EBPs) that reduce maternal and perinatal mortality. The intensity of coaching needed to promote and sustain behavior change is unknown. We investigated the relationship between coaching intensity, EBP adherence, and maternal and perinatal health outcomes using data from the BetterBirth Trial, which assessed the impact of a complex, coaching-based implementation of the World Health Organization's Safe Childbirth Checklist in Uttar Pradesh, India.Methods:For each birth, we defined multiple coaching intensity metrics, including coaching frequency (coaching visits per month), cumulative coaching (total coaching visits accrued during the intervention), and scheduling adherence (coaching delivered as scheduled). We considered coaching delivered at both facility and birth attendant levels. We assessed the association between coaching intensity and birth attendant adherence to 18 EBPs and with maternal and perinatal health outcomes using regression models.Results:Coaching frequency was associated with modestly increased EBP adherence. Delivering 6 coaching visits per month to facilities was associated with adherence to 1.3 additional EBPs (95% confidence interval [CI]=0.6, 1.9). High-frequency coaching delivered with high coverage among birth attendants was associated with greater improvements: providing 70% of birth attendants at a facility with at least 1 visit per month was associated with adherence to 2.0 additional EBPs (95% CI=1.0, 2.9). Neither cumulative coaching nor scheduling adherence was associated with EBP adherence. Coaching was generally not associated with health outcomes, possibly due to the small magnitude of association between coaching and EBP adherence.Conclusions:Frequent coaching may promote behavior change, especially if delivered with high coverage among birth attendants. However, the effects of coaching were modest and did not persist over time, suggesting that future coaching-based interventions should explore providing frequent coaching for longer periods.

ABSTRACTBackground:The focused assessment with sonography for HIV-associated tuberculosis (TB) (FASH) ultrasound protocol has been increasingly used to help clinicians diagnose TB. We sought to quantify the diagnostic utility of FASH for TB among individuals with HIV in Malawi.Methods:Between March 2016 and August 2017, 210 adults with HIV who had 2 or more signs and symptoms that were concerning for TB (fever, cough, night sweats, weight loss) were enrolled from a public HIV clinic in Lilongwe, Malawi. The treating clinicians conducted a history, physical exam, FASH protocol, and additional TB evaluation (laboratory diagnostics and chest radiography) on all participants. The clinician made a final treatment decision based on all available information. At the 6-month follow-up visit, we categorized participants based on clinical outcomes and diagnostic tests as having probable/confirmed TB or unlikely TB; association of FASH with probable/confirmed TB was calculated using Fisher's exact tests. The impact of FASH on empiric TB treatment was determined by asking the clinicians prospectively about whether they would start treatment at 2 time points in the baseline visit: (1) after the initial history and physical exam; and (2) after history, physical exam, and FASH protocol.Results:A total of 181 participants underwent final analysis, of whom 56 were categorized as probable/confirmed TB and 125 were categorized as unlikely TB. The FASH protocol was positive in 71% (40/56) of participants with probable/confirmed TB compared to 24% (30/125) of participants with unlikely TB (odds ratio=7.9, 95% confidence interval=3.9,16.1; P<.001). Among those classified as confirmed/probable TB, FASH increased the likelihood of empiric TB treatment before obtaining any other diagnostic studies from 9% (5/56) to 46% (26/56) at the point-of-care. For those classified as unlikely TB, FASH increased the likelihood of empiric treatment from 2% to 4%.Conclusion:In the setting of HIV coinfection in Malawi, FASH can be a helpful tool that augments the clinician's ability to make a timely diagnosis of TB.

BACKGROUND

Sex differences have been described in diabetes cardiovascular outcome trials (CVOTs).

BACKGROUND

Continuous glucose monitoring (CGM) provides important information to aid in achieving glycemic targets in people with diabetes.

OBJECTIVE

To investigate factors related to glycemic management among members of a professional cycling team with type 1 diabetes over a 7-day Union Cycliste Internationale World Tour stage race.