Search

Abstract Introduction Implementation of patient‐centred care (PCC) practices in HIV treatment depends on healthcare workers’ (HCWs) perceptions of the acceptability, appropriateness and feasibility of such practices (e.g. use of intentional, metric‐driven activities to improve patient experiences). Methods We applied rapid, rigorous formative research methods to refine a PCC intervention for future trial. In 2018, we conducted focus group discussions (FGDs) with 46 HCWs purposefully selected from two pilot sites. We elicited HCW perceptions of HIV service delivery, HCW motivation and perceived value of patient experience measures intended to improve PCC. FGDs utilized participatory methods to understand HCW responses to patient‐reported care engagement challenges and Scholl's PCC Framework principles (e.g. seeing a patient as a unique person), enablers (e.g. care coordination) and activities (e.g. patient involvement). Our rapid analysis used analytic memos, thematic analysis, research team debriefs and HCW feedback to inform time‐sensitive trial implementation. Results While HCWs nearly universally identified with and supported principles of PCC in both facilities, they raised practical barriers given the practice environment. HCWs described motivation to help patients, attached value to seeing positive health outcomes and the importance of teamwork. However, HCWs reported challenges with enablers needed to deliver PCC. HCWs cited a work culture characterized by differential power dynamics between cadres and departments restricting HCW autonomy and resource access. Barriers included inflexibility in accommodating individual patient needs due to high patient volumes, limited human resources, laboratory capacity, infrastructure and skills translating patient perspectives into practice. HCW motivation was negatively influenced by encounters with “difficult patients,” and feeling “unappreciated” by management, resulting in cognitive dissonance between HCW beliefs and behaviours. However, the enactment of PCC values also occurred. Results suggested that PCC interventions should reduce practice barriers, highlighting the value of mentors who could help HCWs dynamically engage with health system constraints, to facilitate PCC. Conclusions While HCWs perceived PCC principles as acceptable, they did not think it universally appropriate or feasible given the practice environment. Participatory and rapid methods provided timely insight that PCC interventions must provide clear and effective systems enabling PCC activities by measuring and mitigating relational and organizational constraints amenable to change such as inter‐cadre coordination.

Objectives We sought to assess depression among healthcare workers (HCWs) in the context of COVID-19 in Lusaka Province, Zambia.Design This cross-sectional study is nested within a larger study, the Person-Centred Public Health for HIV Treatment in Zambia (PCPH), a cluster-randomised trial to assess HIV care and outcomes.Setting The research was conducted in 24 government-run health facilities from 11 August to 15 October 2020 during the first wave of the COVID-19 pandemic in Lusaka, Zambia.Participants We used convenience sampling to recruit HCW participants who were previously enrolled in the PCPH study, had more than 6 months’ experience working at the facility and were voluntarily willing to participate.Primary outcome measures We implemented the well-validated 9-question Patient Health Questionnaire (PHQ-9) to assess HCW depression. We used mixed-effects, adjusted Poisson regression to estimate the marginal probability of HCWs experiencing depression that may warrant intervention (PHQ-9 score ≥5) by healthcare facility.Results We collected PHQ-9 survey responses from 713 professional and lay HCWs. Overall, 334 (46.8%, 95% CI 43.1%, 50.6%) HCWs recorded a PHQ-9 score ≥5, indicating the need for further assessment and potential intervention for depression. We identified significant heterogeneity across facilities and observed a greater proportion of HCWs with symptoms of depression in facilities providing COVID-19 testing and treatment services.Conclusions Depression may be a concern for a large proportion of HCWs in Zambia. Further work to understand the magnitude and aetiologies of depression among HCWs in the public sector is needed to design effective prevention and treatment interventions to meet the needs for mental health support and to minimise poor health outcomes.

Abstract Background State-of-the-art care involving the utilisation of multiple health care interventions is the basis for an optimal long-term clinical prognosis for HIV-patients. We evaluated health care for HIV patients based on four key indicators. Methods Four indicators of health care were assessed: Compliance with current guidelines on initiation of: 1) combination antiretroviral therapy (cART); 2) chemoprophylaxis; 3) frequency of laboratory monitoring; and 4) virological response to cART (proportion of patients with HIV-RNA < 500copies/ml for >90% of time on cART). Results 7097 EuroSIDA patients were included from Northern (n = 923), Southern (n = 1059), West Central (n = 1290) East Central (n = 1366), Eastern (n = 1964) Europe, and Argentina (n = 495). Patients in Eastern Europe with a CD4 < 200cells/mm3 were less likely to initiate cART and Pneumocystis jiroveci-chemoprophylaxis compared to patients from all other regions, and less frequently had a laboratory assessment of their disease status. The proportion of patients with virological response was highest in Northern, 89% vs. 84%, 78%, 78%, 61%, 55% in West Central, Southern, East Central Europe, Argentina and Eastern Europe, respectively (p < 0.0001). Compared to Northern, patients from other regions had significantly lower odds of virological response; the difference was most pronounced for Eastern Europe and Argentina (adjusted OR 0.16 [95%CI 0.11-0.23, p < 0.0001]; 0.20[0.14-0.28, p < 0.0001] respectively). Conclusions This assessment of HIV health care utilization revealed pronounced regional differences in adherence to guidelines and can help to identify gaps and direct target interventions. It may serve as a tool for the assessment and benchmarking of the clinical management of HIV patients in any setting worldwide.

Background: For persons with suspected pulmonary tuberculosis, the guidelines of the Centers for Disease Control and Prevention recommend collecting 3 respiratory specimens 8 to 24 hours apart for acid-fast bacilli (AFB) smear and culture, in addition to 1 nucleic acid amplification test (NAAT). However, data supporting this approach are limited. Our objective was to estimate the performance of 1, 2, or 3 AFB smears with or without NAATs to detect pulmonary tuberculosis in a low-prevalence setting., Methods: We conducted a retrospective study of hospitalized persons at 8 Massachusetts acute care facilities who underwent mycobacterial culture on 1 or more respiratory specimens between July 2016 and December 2022. We evaluated percentage positivity and yield on serial AFB smears and NAATs among people with growth of Mycobacterium tuberculosis on mycobacterial cultures., Results: Among 104 participants with culture-confirmed pulmonary tuberculosis, the first AFB smear was positive in 41 cases (39%). A second AFB smear was positive in 11 (22%) of the 49 cases in which it was performed. No third AFB smears were positive following 2 initial negative smears. Of 52 smear-negative cases, 36 had a NAAT performed, leading to 23 additional diagnoses. Overall sensitivity to detect tuberculosis prior to culture positivity was higher in any strategy involving 1 or 2 NAATs (74%-79%), even without AFB smears, as compared with 3 smears alone (60%)., Conclusions: Tuberculosis diagnostic testing with 2 AFB smears offered the same yield as 3 AFB smears while potentially reducing laboratory burden and duration of airborne infection isolation. Use of 1 or 2 NAATs increased sensitivity to detect culture-positive pulmonary tuberculosis when added to AFB smear-based diagnostic testing alone., Competing Interests: Potential conflicts of interest. J. A. B. has received research funding for other studies from Zeus Scientific, Pfizer, and Analog Devices Inc and has been a paid consultant to Roche Diagnostics, Flightpath Biosciences, and Tarsus Pharmaceuticals. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The chromosomally encoded AmpC beta-lactamase is widely distributed throughout the Enterobacterales. When expressed at high levels through transient induction or stable de-repression, resistance to ceftriaxone, a commonly used antibiotic, can develop. Recent clinical guidance suggests, based on limited evidence, that resistance may be less likely to develop in Serratia marcescens compared to the better-studied Enterobacter cloacae and recommends that ceftriaxone may be used if the clinical isolate tests susceptible. We sought to generate additional data relevant to this recommendation. AmpC de-repression occurs predominantly because of mutation in the ampD peptidoglycan amidohydrolase. We find that, in contrast to E. cloacae , where deletion of ampD results in high-level ceftriaxone resistance (with ceftriaxone MIC = 96 µg/mL), in S. marcescens deletion of two amidohydrolases ( ampD and amiD2 ) is necessary for AmpC de-repression, and the resulting ceftriaxone MIC is 1 µg/mL. Two mechanisms for this difference were identified. We find both a higher relative increase in ampC transcript level in E. cloacae Δ ampD compared to S. marcescens ΔampDΔamiD2 , as well as higher in vivo efficiency of ceftriaxone hydrolysis by the E. cloacae AmpC enzyme compared to the S. marcescens AmpC enzyme. We also observed higher relative levels of transient AmpC induction in E. cloacae vs S. marcescens when exposed to ceftriaxone. In time-kill curves, this difference translates into the survival of E. cloacae but not S. marcescens at clinically relevant ceftriaxone concentrations. In summary, our findings can explain the decreased propensity for on-treatment ceftriaxone resistance development in S. marcescens , thereby supporting recently issued clinical guidance., Competing Interests: The authors declare no conflict of interest.

Monkeypox virus was historically rare outside of West and Central Africa until the current 2022 global outbreak, which has required clinicians to be alert to identify individuals with possible monkeypox, institute isolation, and take appropriate next steps in evaluation and management. Clinical decision support systems (CDSS), which have been shown to improve adherence to clinical guidelines, can support frontline clinicians in applying the most current evaluation and management guidance in the setting of an emerging infectious disease outbreak when those guidelines are evolving over time. Here, we describe the rapid development and implementation of a CDSS tool embedded in the electronic health record to guide frontline clinicians in the diagnostic evaluation of monkeypox infection and triage patients with potential monkeypox infection to individualized infectious disease physician review. We also present data on the initial performance of this tool in a large integrated healthcare system., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A large, ongoing multicountry outbreak of human monkeypox has the potential to cause considerable morbidity and mortality. Therapeutics for the treatment of smallpox, a related Orthopoxvirus , may be used and affect the natural history of monkeypox. We present 3 patients from our hospitals treated with tecovirimat, a pan- Orthopoxvirus inhibitor currently available under an expanded access investigational new drug protocol for monkeypox., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Importance: Bacterial and viral causes of acute respiratory illness (ARI) are difficult to clinically distinguish, resulting in the inappropriate use of antibacterial therapy. The use of a host gene expression-based test that is able to discriminate bacterial from viral infection in less than 1 hour may improve care and antimicrobial stewardship., Objective: To validate the host response bacterial/viral (HR-B/V) test and assess its ability to accurately differentiate bacterial from viral infection among patients with ARI., Design, Setting, and Participants: This prospective multicenter diagnostic study enrolled 755 children and adults with febrile ARI of 7 or fewer days' duration from 10 US emergency departments. Participants were enrolled from October 3, 2014, to September 1, 2019, followed by additional enrollment of patients with COVID-19 from March 20 to December 3, 2020. Clinical adjudication of enrolled participants identified 616 individuals as having bacterial or viral infection. The primary analysis cohort included 334 participants with high-confidence reference adjudications (based on adjudicator concordance and the presence of an identified pathogen confirmed by microbiological testing). A secondary analysis of the entire cohort of 616 participants included cases with low-confidence reference adjudications (based on adjudicator discordance or the absence of an identified pathogen in microbiological testing). Thirty-three participants with COVID-19 were included post hoc., Interventions: The HR-B/V test quantified the expression of 45 host messenger RNAs in approximately 45 minutes to derive a probability of bacterial infection., Main Outcomes and Measures: Performance characteristics for the HR-B/V test compared with clinical adjudication were reported as either bacterial or viral infection or categorized into 4 likelihood groups (viral very likely [probability score <0.19], viral likely [probability score of 0.19-0.40], bacterial likely [probability score of 0.41-0.73], and bacterial very likely [probability score >0.73]) and compared with procalcitonin measurement., Results: Among 755 enrolled participants, the median age was 26 years (IQR, 16-52 years); 360 participants (47.7%) were female, and 395 (52.3%) were male. A total of 13 participants (1.7%) were American Indian, 13 (1.7%) were Asian, 368 (48.7%) were Black, 131 (17.4%) were Hispanic, 3 (0.4%) were Native Hawaiian or Pacific Islander, 297 (39.3%) were White, and 60 (7.9%) were of unspecified race and/or ethnicity. In the primary analysis involving 334 participants, the HR-B/V test had sensitivity of 89.8% (95% CI, 77.8%-96.2%), specificity of 82.1% (95% CI, 77.4%-86.6%), and a negative predictive value (NPV) of 97.9% (95% CI, 95.3%-99.1%) for bacterial infection. In comparison, the sensitivity of procalcitonin measurement was 28.6% (95% CI, 16.2%-40.9%; P < .001), the specificity was 87.0% (95% CI, 82.7%-90.7%; P = .006), and the NPV was 87.6% (95% CI, 85.5%-89.5%; P < .001). When stratified into likelihood groups, the HR-B/V test had an NPV of 98.9% (95% CI, 96.1%-100%) for bacterial infection in the viral very likely group and a positive predictive value of 63.4% (95% CI, 47.2%-77.9%) for bacterial infection in the bacterial very likely group. The HR-B/V test correctly identified 30 of 33 participants (90.9%) with acute COVID-19 as having a viral infection., Conclusions and Relevance: In this study, the HR-B/V test accurately discriminated bacterial from viral infection among patients with febrile ARI and was superior to procalcitonin measurement. The findings suggest that an accurate point-of-need host response test with high NPV may offer an opportunity to improve antibiotic stewardship and patient outcomes.

Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing health care environments and is an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because, in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-MICs of two cephalosporins, cefoxitin, and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single-gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH , a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of y dgH in S. marcescens resulted in decreased susceptibility to multiple third-generation cephalosporins and, in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm, and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

Background: Concerns about false-negative (FN) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid amplification tests (NAATs) have prompted recommendations for repeat testing if suspicion for coronavirus disease 2019 (COVID-19) infection is moderate to high. However, the frequency of FNs and patient characteristics associated with FNs are poorly understood., Methods: We retrospectively reviewed test results from 15 011 adults who underwent ≥1 SARS-CoV-2 NAATs; 2699 had an initial negative NAAT and repeat testing. We defined FNs as ≥1 negative NAATs followed by a positive NAAT within 14 days during the same episode of illness. We stratified subjects with FNs by duration of symptoms before the initial FN test (≤5 days versus >5 days) and examined their clinical, radiologic, and laboratory characteristics., Results: Sixty of 2699 subjects (2.2%) had a FN result during the study period. The weekly frequency of FNs among subjects with repeat testing peaked at 4.4%, coinciding with peak NAAT positivity (38%). Most subjects with FNs had symptoms (52 of 60; 87%) and chest radiography (19 of 32; 59%) consistent with COVID-19. Of the FN NAATs, 18 of 60 (30%) were performed early (ie, ≤1 day of symptom onset), and 18 of 60 (30%) were performed late (ie, >7 days after symptom onset) in disease. Among 17 subjects with 2 consecutive FNs on NP NAATs, 9 (53%) provided lower respiratory tract (LRT) specimens for testing, all of which were positive., Conclusions: Our findings support repeated NAATs among symptomatic patients, particularly during periods of higher COVID-19 incidence. The LRT testing should be prioritized to increase yield among patients with high clinical suspicion for COVID-19., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB -based vector systems are often used for allelic exchange, but counterselection escape, which prevents isolation of cells with the desired mutation, occasionally limits their utility. To circumvent this, we engineered a series of "pTOX" allelic-exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome-binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the gene encoding blue AmilCP or magenta TsPurple nonfluorescent chromoprotein facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens , Escherichia coli O157:H7, Enterobacter cloacae , and Shigella flexneri Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 paralogous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC β-lactamase activity and the corresponding β-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high-level ampC derepression and β-lactam resistance. These data suggest why β-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens, like E. cloacae Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria. IMPORTANCE Targeted modification of bacterial genomes is critical for genetic analysis of microorganisms. Allelic exchange is a technique that relies on homologous recombination to replace native loci with engineered sequences. However, current allelic-exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic-exchange vectors, pTOX, which were validated in several medically important proteobacteria. They encode visible nonfluorescent chromoproteins that enable easy identification of colonies bearing integrated vectors and permit stringent selection for the second step of homologous recombination. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on β-lactam antibiotic resistance., (Copyright © 2019 American Society for Microbiology.)

The invasion of Chlamydia trachomatis, an obligate intracellular bacterium, into epithelial cells is driven by a complex interplay of host and bacterial factors. To comprehensively define the host genes required for pathogen invasion, we undertook a fluorescence-activated cell sorting (FACS)-based CRISPR screen in human cells. A genome-wide loss-of-function library was infected with fluorescent C. trachomatis and then sorted to enrich for invasion-deficient mutants. The screen identified heparan sulfate, a known pathogen receptor, as well as coatomer complex I (COPI). We found that COPI, through a previously unappreciated role, promotes heparan sulfate cell surface presentation, thereby facilitating C. trachomatis attachment. The heparan sulfate defect does not fully account for the resistance of COPI mutants. COPI also promotes the activity of the pathogen's type III secretion system. Together, our findings establish the requirement for COPI in C. trachomatis invasion and the utility of FACS-based CRISPR screening for the elucidation of host factors required for pathogen invasion., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen's key virulence factors., (Copyright © 2018 Pacheco et al.)

Biologic agents are effective treatments for rheumatoid arthritis but are associated with important risks, including severe infections. Tumor Necrosis Factor (TNF) α inhibitors are known to increase the risk of systemic fungal infections such as disseminated histoplasmosis. Abatacept is a biologic agent with a mechanism different from that of TNFα inhibitors: It suppresses cellular immunity by competing for the costimulatory signal on antigen-presenting cells. The risk of disseminated histoplasmosis for patients on abatacept is not known. We report a case of abatacept-associated disseminated histoplasmosis and review the known infectious complications of abatacept. While the safety of resuming biologic agents following treatment for disseminated histoplasmosis is also not known, abatacept is recommended over TNFα inhibitors for rheumatoid arthritis patients with a prior serious infection. We discuss the evidence supporting this recommendation and discuss alternative treatments for rheumatoid arthritis patients with a history of a serious infection.

Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single-molecule resolution, live-cell transport assays in primary neurons, computational image analysis, and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

TRIM58 is an E3 ubiquitin ligase superfamily member implicated by genome-wide association studies to regulate human erythrocyte traits. Here, we show that Trim58 expression is induced during late erythropoiesis and that its depletion by small hairpin RNAs (shRNAs) inhibits the maturation of late-stage nucleated erythroblasts to anucleate reticulocytes. Imaging flow cytometry studies demonstrate that Trim58 regulates polarization and/or extrusion of erythroblast nuclei. In vitro, Trim58 directly binds and ubiquitinates the intermediate chain of the microtubule motor dynein. In cells, Trim58 stimulates proteasome-dependent degradation of the dynein holoprotein complex. During erythropoiesis, Trim58 expression, dynein loss, and enucleation occur concomitantly, and all are inhibited by Trim58 shRNAs. Dynein regulates nuclear positioning and microtubule organization, both of which undergo dramatic changes during erythroblast enucleation. Thus, we propose that Trim58 promotes this process by eliminating dynein. Our findings identify an erythroid-specific regulator of enucleation and elucidate a previously unrecognized mechanism for controlling dynein activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dynactin is an essential cofactor for most cellular functions of the microtubule motor cytoplasmic dynein, but the mechanism by which dynactin activates dynein remains unclear. Here we use single molecule approaches to investigate dynein regulation by the dynactin subunit p150(Glued). We investigate the formation and motility of a dynein-p150(Glued) co-complex using dual-colour total internal reflection fluorescence microscopy. p150(Glued) recruits and tethers dynein to the microtubule in a concentration-dependent manner. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of p150(Glued) decreases the detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

Cytoplasmic dynein is well characterized as an organelle motor, but dynein also acts to tether and stabilize dynamic microtubule plus-ends in vitro. Here we identify a novel and direct interaction between dynein and the 180-kDa isoform of the neural cell adhesion molecule (NCAM). Optical trapping experiments indicate that dynein bound to beads via the NCAM180 interaction domain can tether projecting microtubule plus-ends. Live cell assays indicate that the NCAM180-dependent recruitment of dynein to the cortex leads to the selective stabilization of microtubules projecting to NCAM180 patches at the cell periphery. The dynein-NCAM180 interaction also enhances cell-cell adhesion in heterologous cell assays. Dynein and NCAM180 co-precipitate from mouse brain extract and from synaptosomal fractions, consistent with an endogenous interaction in neurons. Thus, we examined microtubule dynamics and synaptic density in primary cortical neurons. We find that depletion of NCAM, inhibition of the dynein-NCAM180 interaction, or dampening of microtubule dynamics with low dose nocodazole all result in significantly decreased in synaptic density. Based on these observations, we propose a working model for the role of dynein at the synapse, in which the anchoring of the motor to the cortex via binding to an adhesion molecule mediates the tethering of dynamic microtubule plus-ends to potentiate synaptic stabilization.

Long-range retrograde axonal transport in neurons is driven exclusively by the microtubule motor cytoplasmic dynein. The efficient initiation of dynein-mediated transport from the distal axon is critical for normal neuronal function, and neurodegenerative disease-associated mutations have been shown to specifically disrupt this process. Here, we examine the role of dynamic microtubules and microtubule plus-end binding proteins (+TIPs) in the initiation of dynein-mediated retrograde axonal transport using live-cell imaging of cargo motility in primary mouse dorsal root ganglion neurons. We show that end-binding (EB)-positive dynamic microtubules are enriched in the distal axon. The +TIPs EB1, EB3, and cytoplasmic linker protein-170 (CLIP-170) interact with these dynamic microtubules, recruiting the dynein activator dynactin in an ordered pathway, leading to the initiation of retrograde transport by the motor dynein. Once transport has initiated, however, neither the EBs nor CLIP-170 are required to maintain transport flux along the mid-axon. In contrast, the +TIP Lis1 activates transport through a distinct mechanism and is required to maintain processive organelle transport along both the distal and mid-axon. Further, we show that the EB/CLIP-170/dynactin-dependent mechanism is required for the efficient initiation of transport from the distal axon for multiple distinct cargos, including mitochondria, Rab5-positive early endosomes, late endosomes/lysosomes, and TrkA-, TrkB-, and APP-positive organelles. Our observations indicate that there is an essential role for +TIPs in the regulation of retrograde transport initiation in the neuron.

Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150(Glued) subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150(Glued) may also modulate the dynamics of microtubules, we hypothesized that p150(Glued) might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150(Glued) is a potent anti-catastrophe factor for microtubules. p150(Glued) alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150(Glued) are required in tandem for this activity. p150(Glued) is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150(Glued) in nonpolarized cells does not alter microtubule dynamics, while depletion of p150(Glued) in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150(Glued) causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease., Competing Interests: The authors have declared that no competing interests exist.

Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The "search and capture" model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mammalian somatic growth is rapid in early postnatal life but then slows and eventually ceases in multiple tissues. We hypothesized that there exists a postnatal gene expression program that is common to multiple tissues and is responsible for this coordinate growth deceleration. Consistent with this hypothesis, microarray analysis identified more than 1600 genes that were regulated with age (1 vs. 4 wk) coordinately in kidney, lung, and heart of male mice, including many genes that regulate proliferation. As examples, we focused on three growth-promoting genes, Igf2, Mest, and Peg3, that were markedly down-regulated with age. In situ hybridization revealed that expression occurred in organ-specific parenchymal cells and suggested that the decreasing expression with age was due primarily to decreased expression per cell rather than a decreased number of expressing cells. The declining expression of these genes was slowed during hypothyroidism and growth inhibition (induced by propylthiouracil at 0-5 wk of age) in male rats, suggesting that the normal decline in expression is driven by growth rather than by age per se. We conclude that there exists an extensive genetic program occurring during postnatal life. Many of the involved genes are regulated coordinately in multiple organs, including many genes that regulate cell proliferation. At least some of these are themselves apparently regulated by growth, suggesting that, in the embryo, a gene expression pattern is established that allows for rapid somatic growth of multiple tissues, but then, during postnatal life, this growth leads to negative-feedback changes in gene expression that in turn slow and eventually halt somatic growth, thus imposing a fundamental limit on adult body size.

Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (+TIPs) localize to and track with growing microtubule plus-ends in the cell. +TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the +TIP tracking activity are not well understood, however. We reconstituted the +TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our single-molecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170-EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for +TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5'-[alpha,beta-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian +TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.

Cells transformed by Harvey or Moloney sarcoma virus secrete at least 40 times as much type beta transforming growth factor as their respective untransformed control cells. The transformed cells bind only 20 to 50% as much type beta transforming growth factor as the control cells, suggesting that transformation causes down-regulation of the type beta transforming growth factor receptor.