Chlamydia psittaci Screening of Animal Workers from Argentina Exposed to Carrier Birds.

Different syndromes are involved in human psittacosis (flu-like syndrome, atypical pneumonia up to lacrimal gland lymphoma). Diagnostic methods include serology, culture, and PCR. The rate of Chlamydia psittaci (Cp) positive tests among exposed workers is still unknown. Our study aimed to assess the rate of positive tests among workers who have contact with carrier birds in natural reserves from Buenos Aires, Argentina. Secondary aims were to analyze risk factors linked to these outcomes and the occurrence of signs that suggest psittacosis. Nasopharyngeal swabs and serum pairs were collected from employees who had interacted with confirmed carrier birds. Those with detectable DNA of Cp and/or anti-Chlamydia spp. antibody baseline titer ≥ 160 mUI/ml, or at least quadruplicating, were considered positive. Activities performed with or near birds, personal protective equipment use, and previous chronic conditions were assessed. Possible Cp-related pathologies were evaluated during follow-up. A total of 63 exposed workers (71.4% men) with a median age of 35.7 years (IQR 26-39) were evaluated to detect 28.6% positives. Respiratory chronic conditions were the unique factor associated with positive tests (OR 5.2 [1.5-18.5] p < .05). Surprisingly, about a third of the workers resulted positive and all responded to medical treatment, none developing an acute atypical pneumonia syndrome associated with classical presentation of psittacosis. Active testing for early diagnosis and proper treatment in zoological workers exposed to carrier or potentially carrier birds is strongly suggested as part of zoonotic diseases preventive measures.

Comparison of diclofenac with tramadol, tizanidine or placebo in the treatment of acute low back pain and sciatica: multi-center randomized controlled trial.

BACKGROUND: Low back pain (LBP) is a leading cause of disability worldwide and has posed numerous health and socioeconomic challenges. This study compared whether nonsteroidal anti-inflammatory drugs (NSAIDs) in combination with tramadol, tizanidine or placebo would be the best treatment regime to improve the Roland Morris Disability Questionnaire (RMDQ) scores at 1 week. METHODS: This was a multi-center, double-blind, randomized, and placebo-controlled trial including adult patients with acute LBP and sciatica in three emergency departments in Hong Kong. Patients were randomized to the receive tramadol 50 mg, tizanidine 2 mg, or placebo every 6 hours for 2 weeks in a 1:1:1 ratio. The RMDQ and other secondary outcomes were measured at baseline, Day 2, 7, 14, 21, and 28. Data were analyzed on an intention to treat basis. Crude and adjusted mean differences in the changes of RMDQ and NRS scores from baseline to Day 7 between tizanidine/tramadol and placebo were determined with 95% confidence intervals. RESULTS: Two hundred and ninety-one patients were analyzed with the mean age of 47.4 years and 57.7% were male. The primary outcome of mean difference in RMDQs on Day 7 (compared with baseline) was non-significant for tizanidine compared with placebo (adjusted mean difference - 0.56, 95% CI -2.48 to 1.37) and tramadol compared with placebo (adjusted mean difference - 0.85, 95% CI -2.80 to 1.10). Only 23.7% were fully compliant to the treatment allocated. Complier Average Causal Effect analysis also showed no difference in the primary outcome for the tizanidine and tramadol versus placebo. CONCLUSION: Among patients with acute LBP and sciatica presenting to the ED, adding tramadol or tizanidine to diclofenac did not improve functional recovery.

Patient burden and joint-inflammation during development of RA from arthralgia: is it similar in ACPA-positive and ACPA-negative disease?

OBJECTIVES: Anti-citrullinated protein antibody(ACPA)-positive and ACPA-negative rheumatoid arthritis(RA) differ in underlying risk factors but have a similar clinical presentation at RA-diagnosis. It is unknown what the ACPA-associated differences or similarities are during the symptomatic at-risk stage of RA, clinically suspect arthralgia(CSA). To deepen insights into these differences/similarities, we compared the course of symptoms/impairments and subclinical joint-inflammation in the CSA-phase during progression to inflammatory arthritis(IA) or to CSA-resolution. METHODS: 845 CSA-patients were followed for median 24 months; 136 patients developed IA and additional 355/505 patients had resolution of CSA according to rheumatologists. Patient burden (pain/morning stiffness/fatigue/functional disabilities/presenteeism) was assessed at baseline, 4/12/24 months and IA-development. Subclinical joint-inflammation in hands/feet was assessed over time with 1.5 T-MRI. Linear/Poisson mixed models were used. RESULTS: Both in ACPA-positive and ACPA-negative patients, patient burden increased towards IA-development and decreased towards CSA-resolution. However, patient burden was lower in ACPA-positive than ACPA-negative disease on all timepoints. Conversely, subclinical joint-inflammation tended to increase more rapidly during development of ACPA-positive IA (IRR = 1.52,95%CI = 0.94-2.47, p= 0.089), and remained higher over time in ACPA-positive CSA-patients achieving resolution compared with ACPA-negative patients (IRR = 1.52,95%CI = 1.07-2.15, p= 0.018). Although correlation coefficients between changes in patient burden and subclinical joint-inflammation during progression to IA were weak, they were consistently higher in ACPA-positive than ACPA-negative disease, e.g. ρ = 0.29 vs ρ = 0.12 for functional disabilities. CONCLUSION: During RA-development and CSA-resolution, ACPA-positive CSA-patients have lower patient burden, but more subclinical joint-inflammation than ACPA-negative CSA-patients. These data strengthen the notion that the development of ACPA-positive and ACPA-negative RA is pathophysiologically different, and encourage further research on these differences.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

Human proteins curing yeast prions.

Recognition that common human amyloidoses are prion diseases makes the use of the Saccharomyces cerevisiae prion model systems to screen for possible anti-prion components of increasing importance. [PSI+] and [URE3] are amyloid-based prions of Sup35p and Ure2p, respectively. Yeast has at least six anti-prion systems that together cure nearly all [PSI+] and [URE3] prions arising in their absence. We made a GAL-promoted bank of 14,913 human open reading frames in a yeast shuttle plasmid and isolated 20 genes whose expression cures [PSI+] or [URE3]. PRPF19 is an E3 ubiquitin ligase that cures [URE3] if its U-box is intact. DNAJA1 is a J protein that cures [PSI+] unless its interaction with Hsp70s is defective. Human Bag5 efficiently cures [URE3] and [PSI+]. Bag family proteins share a 110 to 130 residue "BAG domain"; Bag 1, 2, 3, 4, and 6 each have one BAG domain while Bag5 has five BAG domains. Two BAG domains are necessary for curing [PSI+], but one can suffice to cure [URE3]. Although most Bag proteins affect autophagy in mammalian cells, mutations blocking autophagy in yeast do not affect Bag5 curing of [PSI+] or [URE3]. Curing by Bag proteins depends on their interaction with Hsp70s, impairing their role, with Hsp104 and Sis1, in the amyloid filament cleavage necessary for prion propagation. Since Bag5 curing is reduced by overproduction of Sis1, we propose that Bag5 cures prions by blocking Sis1 access to Hsp70s in its role with Hsp104 in filament cleavage.

Insights into a viral motor: the structure of the HK97 packaging termination assembly.

Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

Molecular mechanisms of stress-induced reactivation in mumps virus condensates.

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

Measuring intramolecular connectivity in long RNA molecules using two-dimensional DNA patch-probe arrays.

We describe a simple method to infer intramolecular connections in a population of long RNA molecules in vitro. First we add DNA oligonucleotide "patches" that perturb the RNA connections, then we use a microarray containing a complete set of DNA oligonucleotide "probes" to record where perturbations occur. The pattern of perturbations reveals couplings between different regions of the RNA sequence, from which we infer connections as well as their prevalences in the population. We validate this patch-probe method using the 1,058-nucleotide RNA genome of satellite tobacco mosaic virus (STMV), which has previously been shown to have multiple long-range connections. Our results not only indicate long duplexes that agree with previous structures but also reveal the prevalence of competing connections. Together, these results suggest that globally-folded and locally-folded structures coexist in solution. We show that the prevalence of connections changes when pseudouridine, an important component of natural and synthetic RNA molecules, is substituted for uridine in STMV RNA.

Anti-Prion Systems Block Prion Transmission, Attenuate Prion Generation, Cure Most Prions as They Arise and Limit Prion-Induced Pathology in Saccharomyces cerevisiae.

All variants of the yeast prions [PSI+] and [URE3] are detrimental to their hosts, as shown by the dramatic slowing of growth (or even lethality) of a majority, by the rare occurrence in wild isolates of even the mildest variants and by the absence of reproducible benefits of these prions. To deal with the prion problem, the host has evolved an array of anti-prion systems, acting in normal cells (without overproduction or deficiency of any component) to block prion transmission from other cells, to lower the rates of spontaneous prion generation, to cure most prions as they arise and to limit the damage caused by those variants that manage to elude these (necessarily) imperfect defenses. Here we review the properties of prion protein sequence polymorphisms Btn2, Cur1, Hsp104, Upf1,2,3, ribosome-associated chaperones, inositol polyphosphates, Sis1 and Lug1, which are responsible for these anti-prion effects. We recently showed that the combined action of ribosome-associated chaperones, nonsense-mediated decay factors and the Hsp104 disaggregase lower the frequency of [PSI+] appearance as much as 5000-fold. Moreover, while Btn2 and Cur1 are anti-prion factors against [URE3] and an unrelated artificial prion, they promote [PSI+] prion generation and propagation.

Screening of BRCA1 (c.5177_5180delGAAA rs80357867 and c.4986+6T>C rs80358086) and the BRCA2 (c.6445_6446delAT rs80359592) Genes for Breast Cancer Prevention in Burkina Faso.

Background: The objective of this study is to search for mutations in the BRCA1 (c.5177_5180delGAAA and c.4986+6T>C) and BRCA2 genes (c.6445_6446delAT) in a population of women diagnosed with breast cancer. Methods: This is a case-control study that involved 140 participants, including 70 patients with histologically diagnosed breast cancer and 70 healthy women without breast cancer. Mutations in the BRCA1 (rs80357867, rs80358086) and BRCA2 (rs80359592) genes were tested by real-time PCR. The 95% confidence interval Odds Ratio (OR) was used to estimate the associations between specific genotypes and breast cancer. Results: The study revealed that no mutations were detected for rs80359592. Similarly, no reference allele (TTTC/TTTC) of rs80357867 was found in this study. However, the homozygous double mutant (-/) genotype of this rs80357867 was observed in 11.43% and 1.43% of patients and controls respectively, while 88.57% of patients and 98.57% of controls had a heterozygous deletion (TTTC/-). Concerning rs80358086, 8.57% of the patients had a heterozygous mutation (A/G) with no significantly risk association with occurrence of breast cancer (OR = 6.46; 95% CI: 0.75-55.21; p = 0.11). In addition, this heterozygous mutation was significantly associated with a family history of breast cancer (OR=128; 95% CI: 9.46-1730.93) and breast cancer risk in nonmultiparous women (OR=6; 95% CI: 1-35.90; p= 0.05) but no association with overweight/obesity (OR=1.66; 95% CI: 0.18-15.35; p=1). Conclusion: This study shows high frequencies of heterozygous mutation of rs80357867 and rs80358086 from patients. In Burkina Faso, these results could help with early diagnosis of breast cancer in patients.

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

Mechanism of RNA polymerase I selection by transcription factor UAF.

Preribosomal RNA is selectively transcribed by RNA polymerase (Pol) I in eukaryotes. The yeast transcription factor upstream activating factor (UAF) represses Pol II transcription and mediates Pol I preinitiation complex (PIC) formation at the 35S ribosomal RNA gene. To visualize the molecular intermediates toward PIC formation, we determined the structure of UAF in complex with native promoter DNA and transcription factor TATA-box-binding protein (TBP). We found that UAF recognizes DNA using a hexameric histone-like scaffold with markedly different interactions compared with the nucleosome and the histone-fold-rich transcription factor IID (TFIID). In parallel, UAF positions TBP for Core Factor binding, which leads to Pol I recruitment, while sequestering it from DNA and Pol II/III-specific transcription factors. Our work thus reveals the structural basis of RNA Pol selection by a transcription factor.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.

Innate immunity to prions: anti-prion systems turn a tsunami of prions into a slow drip.

The yeast prions (infectious proteins) [URE3] and [PSI+] are essentially non-functional (or even toxic) amyloid forms of Ure2p and Sup35p, whose normal function is in nitrogen catabolite repression and translation termination, respectively. Yeast has an array of systems working in normal cells that largely block infection with prions, block most prion formation, cure most nascent prions and mitigate the toxic effects of those prions that escape the first three types of systems. Here we review recent progress in defining these anti-prion systems, how they work and how they are regulated. Polymorphisms of the prion domains partially block infection with prions. Ribosome-associated chaperones ensure proper folding of nascent proteins, thus reducing [PSI+] prion formation and curing many [PSI+] variants that do form. Btn2p is a sequestering protein which gathers [URE3] amyloid filaments to one place in the cells so that the prion is often lost by progeny cells. Proteasome impairment produces massive overexpression of Btn2p and paralog Cur1p, resulting in [URE3] curing. Inversely, increased proteasome activity, by derepression of proteasome component gene transcription or by 60S ribosomal subunit gene mutation, prevents prion curing by Btn2p or Cur1p. The nonsense-mediated decay proteins (Upf1,2,3) cure many nascent [PSI+] variants by associating with Sup35p directly. Normal levels of the disaggregating chaperone Hsp104 can also cure many [PSI+] prion variants. By keeping the cellular levels of certain inositol polyphosphates / pyrophosphates low, Siw14p cures certain [PSI+] variants. It is hoped that exploration of the yeast innate immunity to prions will lead to discovery of similar systems in humans.

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity.

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.

Proteasome Control of [URE3] Prion Propagation by Degradation of Anti-Prion Proteins Cur1 and Btn2 in Saccharomyces cerevisiae.

[URE3] is a prion of the nitrogen catabolism controller, Ure2p, and [PSI+] is a prion of the translation termination factor Sup35p in S. cerevisiae. Btn2p cures [URE3] by sequestration of Ure2p amyloid filaments. Cur1p, paralogous to Btn2p, also cures [URE3], but by a different (unknown) mechanism. We find that an array of mutations impairing proteasome assembly or MG132 inhibition of proteasome activity result in loss of [URE3]. In proportion to their prion-curing effects, each mutation affecting proteasomes elevates the cellular concentration of the anti-prion proteins Btn2 and Cur1. Of >4,600 proteins detected by SILAC, Btn2p was easily the most overexpressed in a pre9Δ (α3 core subunit) strain. Indeed, deletion of BTN2 and CUR1 prevents the prion-curing effects of proteasome impairment. Surprisingly, the 15 most unstable yeast proteins are not increased in pre9Δ cells suggesting altered proteasome specificity rather than simple inactivation. Hsp42, a chaperone that cooperates with Btn2 and Cur1 in curing [URE3], is also necessary for the curing produced by proteasome defects, although Hsp42p levels are not substantially altered by a proteasome defect. We find that pre9Δ and proteasome chaperone mutants that most efficiently lose [URE3], do not destabilize [PSI+] or alter cellular levels of Sup35p. A tof2 mutation or deletion likewise destabilizes [URE3], and elevates Btn2p, suggesting that Tof2p deficiency inactivates proteasomes. We suggest that when proteasomes are saturated with denatured/misfolded proteins, their reduced degradation of Btn2p and Cur1p automatically upregulates these aggregate-handling systems to assist in the clean-up.

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

North American Propolis Extracts From Upstate New York Decrease Nosema ceranae (Microsporidia) Spore Levels in Honey Bees (Apis mellifera).

Nosema ceranae infections in honey bees (Apis mellifera) pose a severe threat to colony health. Beekeepers have used dicyclohexylammonium fumagillin to control Nosema apis, although it may be ineffective against N. ceranae. We investigated the ability of various propolis extracts collected from Upstate New York (United States) to decrease in vivo N. ceranae infection levels when fed ad libitum to N. ceranae-infected honey bees. Propolis extracts, most notably a dichloromethane extract, significantly lowered spore levels in a dose-dependent fashion 4 days post inoculation. When testing the in vitro anti-Nosema activity of propolis extracts, we report for the first time that spore viability was unaffected after a 24 h exposure to propolis extracts. These results present evidence that propolis extracts may effectively lower Microsporidia infections in honey bees, and that direct exposure of environmental spores to propolis alone does not kill N. ceranae.