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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.

Background Isolation of hospitalized persons under investigation (PUIs) for coronavirus disease 2019 (COVID-19) reduces nosocomial transmission risk. Efficient evaluation of PUIs is needed to preserve scarce healthcare resources. We describe the development, implementation, and outcomes of an inpatient diagnostic algorithm and clinical decision support system (CDSS) to evaluate PUIs. Methods We conducted a pre-post study of CORAL (COvid Risk cALculator), a CDSS that guides frontline clinicians through a risk-stratified COVID-19 diagnostic workup, removes transmission-based precautions when workup is complete and negative, and triages complex cases to infectious diseases (ID) physician review. Before CORAL, ID physicians reviewed all PUI records to guide workup and precautions. After CORAL, frontline clinicians evaluated PUIs directly using CORAL. We compared pre- and post-CORAL frequency of repeated severe acute respiratory syndrome coronavirus 2 nucleic acid amplification tests (NAATs), time from NAAT result to PUI status discontinuation, total duration of PUI status, and ID physician work hours, using linear and logistic regression, adjusted for COVID-19 incidence. Results Fewer PUIs underwent repeated testing after an initial negative NAAT after CORAL than before CORAL (54% vs 67%, respectively; adjusted odd ratio, 0.53 [95% confidence interval, .44–.63]; P Conclusions CORAL is an efficient and effective CDSS to guide frontline clinicians through the diagnostic evaluation of PUIs and safe discontinuation of precautions.

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.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.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.The HR-B/V test quantified the expression of 45 host messenger RNAs in approximately 45 minutes to derive a probability of bacterial infection.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 score0.19], viral likely [probability score of 0.19-0.40], bacterial likely [probability score of 0.41-0.73], and bacterial very likely [probability score0.73]) and compared with procalcitonin measurement.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.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.

Summary 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., Graphical Abstract, Highlights • FACS-based CRISPR screen to identify host factors required for C. trachomatis invasion • Candidate genes comprise heparan sulfate biosynthesis, actin remodeling, and COPI • COPI regulates heparan sulfate cell surface presentation and C. trachomatis attachment • COPI is also required for efficient C. trachomatis T3SS translocation, Molecular Mechanism of Behavior; Medical Microbiology; Methodology in Biological Sciences; Cell Biology; Host-pathogen Interactions; Molecular Microbiology; Genetic Engineering; Genetic Screens

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.

Paecilomyces variotii is a ubiquitous environmental saprophyte with worldwide distribution. Commonly found in soil and decomposing organic material [1, 2], P. variotii can also be isolated from drinking water [3] and indoor and outdoor air [4–6]. In immunocompetent hosts, P. variotii has been reported as a cause of locally invasive disease including prosthetic valve endocarditis [7, 8], endophthalmitis [9, 10], rhinosinusitis [11, 12], and dialysis-associated peritonitis [13, 14]. In contrast, disseminated infections are more commonly reported in immunocompromised patients, including those with chronic granulomatous disease [15], solid malignancy [16], acute leukemia [17], lymphoma [18], multiple myeloma [19], and after stem cell transplant for myelodysplasia [20]. In 1 case series examining invasive infections by non-Aspergillus molds, P. variotii was the most common cause after Fusarium spp. [21]. Here, we present the case of an immunocompetent patient with extensive intravascular infection involving prosthetic material. We describe successful induction therapy with combination antifungals and extended suppression with posaconazole with clinical quiescence and eventual normalization of serum fungal biomarkers., Paecilomyces variotii is a ubiquitous environmental saprophyte that rarely causes invasive disease. We report a case of P. variotii infection in an immunocompetent patient with extensive intravascular infection involving prosthetic material and review key features of previously reported cases.

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.

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.

A 48-year-old woman was transferred to our service with confusion and a change in her personality. She had presented initially with several days of malaise, abdominal pain, and nonproductive cough. She had also experienced multiple mechanical falls at home and reported weight loss of 20 pounds over 1 month. Her examination was notable for mild epigastric tenderness without guarding, a blunted and indifferent affect, difficulty with multistep commands, and recall of 0/3 objects after 5 minutes. She was otherwise alert and oriented with normal cranial nerve, upper extremity, and lower extremity examinations. CT of the head was performed as part of her initial evaluation (figure, A–D). She had additional imaging given her cough and abdominal pain: CT of the chest demonstrated a right breast mass, and CT of the abdomen and pelvis showed peripancreatic, upper periaortic, and mesenteric masses.

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.

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.

Cytoplasmic dynein plays important roles in mitosis and the intracellular transport of organelles, proteins, and mRNAs. Dynein function is particularly critical for survival of neurons, as mutations in dynein are linked to neurodegenerative diseases. Dynein function is also implicated in neuronal regeneration, driving the active transport of signaling molecules following injury of peripheral neurons. To enhance our understanding of dynein function and regulation in neurons, we established a novel knock-in mouse line in which the neuron-specific cytoplasmic dynein 1 intermediate chain 1 (IC-1) is tagged with both GFP and a 3xFLAG tag at its C-terminus. The fusion gene is under the control of IC-1's endogenous promoter and is integrated at the endogenous locus of the IC-1-encoding gene Dync1i1. The IC-1-GFP-3xFLAG fusion protein is incorporated into the endogenous dynein complex, and movements of GFP-labeled dynein expressed at endogenous levels can be observed in cultured neurons for the first time. The knock-in mouse line also allows isolation and analysis of dynein-bound proteins specifically from neurons. Using this mouse line we have found proteins, including 14-3-3 zeta, which physically interact with dynein upon injury of the brain cortex. Thus, we have created a useful tool for studying dynein function in the central nervous system under normal and pathologic conditions.

SummaryMotor-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.

Fibroblast growth factor (FGF) signaling is essential for endochondral bone formation. Mutations cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. Most previous work in this area has focused on embryonic chondrogenesis. To explore the role of FGF signaling in the postnatal growth plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Toward this aim, rat proximal tibial growth plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. Growth plate expressed FGFs 2, 7, 18, and 22. Perichondrial expression was generally greater than growth plate expression, supporting the concept that perichondrial FGFs regulate growth plate chondrogenesis. Nevertheless, FGFs synthesized by growth plate chondrocytes may be physiologically important because of their proximity to target receptors. In growth plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL1, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing growth velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF1, FGF7, FGF18, and FGF22 were upregulated. In summary, we have analyzed the expression of all known FGFs and FGFRs in the postnatal growth plate using a method that is quantitative and highly sensitive. This approach identified ligands and receptors not previously known to be expressed in growth plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the growth plate. We also found temporal changes in FGF and FGFR expression which may contribute to growth plate senescence and thus help determine the size of the adult skeleton.

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 drives vesicular transport from the periphery to the cell body of neurons. Missense mutations in the dynein tail domain cause neurodegenerative disease in mouse models. new data on the effect of one such dynein mutation provide insight into the intramolecular communication and flexible stepping of this essential cellular motor.

The dynein partner dynactin not only binds to microtubules, but is found to potently influence microtubule dynamics in neurons., 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 p150Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150Glued may also modulate the dynamics of microtubules, we hypothesized that p150Glued 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 p150Glued is a potent anti-catastrophe factor for microtubules. p150Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150Glued are required in tandem for this activity. p150Glued is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150Glued 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., Author Summary Microtubules are polymers of tubulin that undergo successive cycles of growth and shrinkage so that the cell can maintain a stable yet adaptable cytoskeleton. In neurons, the microtubule motor protein dynein and its partner complex dynactin drive retrograde transport along microtubules from the distal axon towards the cell body. In addition to binding to dynein, the p150Glued subunit of dynactin independently binds directly to microtubules. We hypothesized that by binding to microtubules, p150Glued might also alter microtubule dynamics. We demonstrate using biochemistry and microscopy in vitro and in cells that p150Glued stabilizes microtubules by inhibiting the transition from growth to shrinkage. We show that specific domains of p150Glued encoded by neuronally enriched splice-forms are necessary for this activity. Although depletion of p150Glued in nonpolarized cells does not alter microtubule dynamics, depletion of endogenous p150Glued in neurons leads to dramatic microtubule instability. Strikingly, a mutation in p150Glued known to cause the neurodegenerative disorder Perry syndrome abolishes this activity. In summary, we identified a previously unappreciated function of dynactin in direct regulation of the microtubule cytoskeleton. This activity may enhance generic microtubule stability in the cell, but could be especially important in specific areas of the cell where dynactin and dynein are loaded onto microtubules.

SummaryMicrotubules 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 [1–3]. 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 [3–7]. 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.

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

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.

Regulation of microtubule dynamics is critical, particularly in neurons, where defects may contribute to neurodegeneration. Here, we demonstrate in vitro using TIRF microscopy and in primary neurons using live-cell imaging that the p150Glued subunit of dynactin regulates microtubule dynamics. To modify dynamics, p150Glued must be dimerized, and it must bind soluble tubulin, an interaction that we find requires tandem CAP-Gly and basic domains. p150Glued is alternatively spliced in vivo, with the full length isoform including both these domains expressed primarily in neurons. Accordingly, depletion of p150Glued in a non-polarized cell line does not alter microtubule dynamics, while p150Glued RNAi in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a Parkinson syndrome-associated mutation blocks this microtubule-stabilizing activity both in vitro and in neurons. Together, our data reveal that p150Glued plays a crucial role in promoting microtubule stability in neurons, and that defects in this function may lead to neurodegeneration.

Abstract 83 Protein quality control (PQC) pathways, including the ubiquitin-proteasome system, play critical roles in tissue development, including erythropoiesis. PQC mechanisms also regulate mitosis by facilitating the degradation of cell cycle stage-specific proteins. During erythropoiesis, committed progenitors undergo specialized cell divisions that facilitate terminal maturation and culminate in nuclear extrusion. We identified the tri partite motif-containing protein family member Trim58 as a putative ubiquitin ligase that is specifically expressed during late erythroid maturation as cells undergo their final round of division. Genome-wide ChIP-Seq and transcriptome analyses indicate that the Trim58 gene is activated by the essential erythroid nuclear proteins GATA1 and SCL/TAL1 (Cheng et al. Genome Research 2009). In human genome-wide association studies (GWAS), TRIM58 gene polymorphisms correlate with altered erythrocyte size (Ganesh et al. Nature Genetics 2009; Kamatani et al. Nature Genetics 2010). To study the function of Trim58, we used shRNAs to inhibit its expression in primary erythroid cultures of murine fetal liver hematopoietic progenitors. Four Trim58 shRNAs knocked down mRNA and protein expression from 60–90%. Compared to controls, Trim58 knockdown had no effect on erythroblast proliferation, survival, hemoglobinization, or expression of the maturation markers Ter119 and CD44. However, Trim58-deficient erythroblasts exhibited defective enucleation to the reticulocyte stage after 48 hours in culture (54% reticulocytes vs. 69% in controls, p Figure 1. Compared to control shRNA treatment (shLuciferase), Trim58 knockdown (shTrim58) in differentiating primary murine erythroblasts causes a late maturation block with reduced reticulocytes and accumulation of multinuclear erythroblasts. Figure 1. Compared to control shRNA treatment (shLuciferase), Trim58 knockdown (shTrim58) in differentiating primary murine erythroblasts causes a late maturation block with reduced reticulocytes and accumulation of multinuclear erythroblasts. Disclosures: No relevant conflicts of interest to declare.

While the myosin and kinesin superfamilies achieve diverse functions through a large number of motors, each specialized to its role, cytoplasmic dynein accomplishes varied roles in the cell through diverse interacting partners. We hypothesized that in addition to its role in bidirectional organelle transport, mammalian dynein might also play role in tethering microtubules at the cortex, analogous to the well-characterized function of dynein in S. cerevisiae. We identified a direct interaction between dynein and the neuronal cell adhesion molecule NCAM180 that recruits dynein to the cortex. To test the ability of dynein to tether microtubule plus ends, microtubules containing short biotinylated segments at the minus end were attached to the coverslip, allowing the non-biotinylated plus-ends to exhibit diffusive movements. When dynein-coated beads were brought near to the plus-end using an optical trap, the microtubule became tethered, while beads coated with BSA or kinesin-1 did not effectively restrict lateral fluctuations. In dynamic microtubule assays, plus-ends tended to associate with dynein-coated beads and continue to grow, remaining in contact with a bead for 94+/-18 s (mean+/-SEM). In contrast, encounters between growing plus-ends and BSA-coated beads often led to catastrophe, decreasing the contact time to 45+/-16 s. In live cell assays we also measured a significant increase in microtubule dwell time in the vicinity of NCAM patches (189+/-12 s, compared to 100+/-7 s in controls). Together, these data support a mechanism in which dynein is anchored to the cell cortex by NCAM180 and transiently tethers the plus-ends of microtubules. Tethered microtubules likely provide a direct connection between the cell center and sites of cell-cell interaction, either mechanically or as preferred routes for transport, creating a powerful mechanism for cells to respond to or produce stimuli in the extracellular environment.