Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans.

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a ß-l-rhamnosyltransferase; domain 2 is an α-l-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α-l-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α-l-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N-glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α-l-Rha-(1→2)-ß-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

Intra-articular injections of platelet-rich plasma in symptomatic knee osteoarthritis: a consensus statement from French-speaking experts.

PURPOSE: There has been much debate regarding the use of intra-articular injections of platelet-rich plasma (PRP) as symptomatic treatment for knee osteoarthritis. The objective of this consensus was to develop guidelines for PRP injections in knee osteoarthritis according to the French National Authority for Health recommendations. METHODS: Fifteen physicians from different French-speaking countries (10 rheumatologists, 4 specialists in rehabilitation and sports medicine and 1 radiologist) were selected for their expertise in the areas of PRP and osteoarthritis. A comprehensive literature review was conducted on Medline including all published therapeutic trials, open studies, meta-analysis and systematic reviews focusing on the effects of PRP in knee OA, as well as fundamental studies concerning the characteristics of the various types of PRP and their mechanisms, indexed before April 2019. Using the method recommended by the French National Authority for Health inspired by the Delphi consensus process, 25 recommendations were finally retained and evaluated. The recommendations were classified as appropriate or not appropriate, with strong or relative agreement, or uncertain if a consensus was not achieved. RESULTS: Among the 25 recommendations selected, the main ones are the following: (1) Intra-articular injections of PRP are an effective symptomatic treatment for early to moderate knee osteoarthritis. This recommendation was considered appropriate with a relative agreement (Median = 8; rank = 6-9). Level of evidence 1A. (2) A PRP treatment sequence in knee osteoarthritis may include 1-3 injections. This recommendation was considered appropriate with a strong agreement (Median = 9; rank = 7-9). Level of evidence 1A. (3) Leucocytes-poor PRP should be preferred in knee osteoarthritis. This recommendation was considered appropriate with a relative agreement (Median = 8; rank = 5-9). Level of evidence 5. (4) Intra-articular PRP knee injections should be performed under ultrasound or fluoroscopic guidance. This recommendation was considered uncertain with no consensus (Median = 8; rank = 3-9). Level of evidence 5. (5) PRP should not be mixed with an anesthetic or intra-articular corticosteroid. This recommendation was considered appropriate with a relative agreement (Median = 9; rank = 6-9). Level of evidence 5 CONCLUSION: Those 25 recommendations should standardize and facilitate the use of IA PRP injections, which are considered by experts as an effective treatment especially in early or moderate knee OA. Although a strong or relative agreement from the experts was obtained for most of the recommendations, many of them had a very low level of evidence (Level 5) and were principally based on the clinical experience of the experts.

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases.

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a ß-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a ß-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.

Influence of Muscle Fatty Degeneration on Functional Outcomes After Endoscopic Gluteus Medius Repair.

PURPOSE: To report the early outcomes of endoscopic repair of tears of the gluteus medius tendon and to determine whether the fatty degeneration had an influence on clinical results. METHODS: Between October 2012 and June 2014, data were prospectively collected and retrospectively reviewed for all patients who underwent endoscopic gluteus medius repair. Patients were assessed pre- and postoperatively using the modified Harris hip score, the nonarthritic hip score, and visual analog scale for pain. The gluteus minimus and the 3 distinct parts of the gluteus medius (anterior, middle, and posterior) were assigned a grade of fatty degeneration on preoperative magnetic resonance imaging scans. RESULTS: Twenty-two hips (in 20 patients) were assessed with the mean follow-up of 31.7 months (range: 24 to 47 months). There were 15 partial-thickness and 7 full-thickness tears. No patient was lost to follow-up. The mean age at the time of surgery was 66 years (range: 45 to 82 years). Of the 20 magnetic resonance imaging-assessed hips included in the study, 14 had fatty degeneration of the gluteus medius (partial-thickness tears: n = 8, full-thickness tears: n = 6). The mean gluteus medius fatty degeneration index was 1.57 (range: 0.33 to 3.33). Postoperative improvement was seen in modified Harris hip score (33.7 points vs 80.2 points, P = .0001), nonarthritic hip score (47.7 points vs 76.8 points, P = .0001), and in the visual analog scale for pain (7.2 vs 3.2, P < .05). Increasing preoperative fatty degeneration index of the gluteus medius correlated with decreased postoperative functional hip score values (regression coefficient, 0.5839; P < .0001). Tear characteristics (partial or full-thickness) did not correlate with fatty degeneration or muscular atrophy and did not affect postoperative outcomes. CONCLUSIONS: Endoscopic surgical repair can be an effective treatment of gluteus medius tears in the short term. Fatty degeneration of the gluteus medius and minimus has a negative impact on clinical outcomes of endoscopic gluteus medius repair. LEVEL OF EVIDENCE: Level IV, therapeutic case series (no control group).

Chlorovirus Skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.

UNLABELLED: The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE: Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.

Wearable transcutaneous electrical nerve stimulation (actiTENS®) is effective and safe for the treatment of knee osteoarthritis pain: a randomized controlled trial versus weak opioids.

INTRODUCTION: Despite their poor tolerance, especially in the elderly, weak opioids (WO) remain commonly prescribed for patients with knee osteoarthritis (KOA). We compared the efficacy and safety of a new wearable transcutaneous electrical nerve stimulation (W-TENS) device with WO for the treatment of moderate-to-severe, nociceptive KOA chronic pain. METHODS: The study was a non-inferiority, multicentric, prospective, randomized, single-blind, controlled, 2-parallel groups Trial. A total of 110 patients with KOA were included (Kellgren-Lawrence radiographic grade ⩾2; American College of Rheumatology criteria), with chronic moderate-to-severe nociceptive pain (mean 8-day pain intensity (PI) ⩾ 4 on an 11-point numerical rating scale), in failure to non-opioid analgesics, including nonsteroidal anti-inflammatory drugs (NSAIDs). Patients with neuropathic pain were excluded. The co-primary endpoints were mean PI at 3 months (M3) and number of potentially treatment-related adverse events (TRAEs). Secondary outcomes included Western Ontario MAC Master University function subscale (range, 0-68), additional pain and quality of life measures, and responder rates. RESULTS: The non-inferiority of W-TENS was demonstrated in both the per protocol (PP) and intent-to-treat (ITT) populations. At M3, PI in PP population was 3.87 (2.12) compared with 4.66 (2.37) [delta: -0.79 (0.44); 95% CI (-1.65, 0.08)] in W-TENS and WO groups, respectively. A planned superiority analysis showed a significant superiority of W-TENS over WO on PI at M3 (p = 0.0124). The number of TRAEs was significantly lower in the W-TENS group (n = 7) than in the WO group (n = 36) (p < 0.001). Other secondary outcomes also favored W-TENS. CONCLUSION: W-TENS was more effective and better tolerated than WO in the treatment of chronic nociceptive KOA pain and offers an interesting non-pharmacological analgesic alternative in the management of KOA.Trial Registration: ClinicalTrials.gov: NCT03902340.

Near-atomic, non-icosahedrally averaged structure of giant virus Paramecium bursaria chlorella virus 1.

Giant viruses are a large group of viruses that infect many eukaryotes. Although components that do not obey the overall icosahedral symmetry of their capsids have been observed and found to play critical roles in the viral life cycles, identities and high-resolution structures of these components remain unknown. Here, by determining a near-atomic-resolution, five-fold averaged structure of Paramecium bursaria chlorella virus 1, we unexpectedly found the viral capsid possesses up to five major capsid protein variants and a penton protein variant. These variants create varied capsid microenvironments for the associations of fibers, a vesicle, and previously unresolved minor capsid proteins. Our structure reveals the identities and atomic models of the capsid components that do not obey the overall icosahedral symmetry and leads to a model for how these components are assembled and initiate capsid assembly, and this model might be applicable to many other giant viruses.

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing.

Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans.

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.

The rheumatoid shoulder: current consensus on diagnosis and treatment.

Shoulder involvement is usually inconspicuous in patients with rheumatoid arthritis, and the clinical manifestations are nonspecific. Nevertheless, shoulder involvement should be sought routinely and detected early. Range of motion at the shoulder should be evaluated. Although normal radiographic findings do not rule out shoulder involvement, radiographs are crucial for detecting micro- and macro-geodes during follow-up. The development of glenohumeral joint space narrowing is a turning point that indicates a risk of rapid joint destruction. Magnetic resonance imaging is useful for assessing the lesions and guiding the treatment strategy. Stepwise use of local interventions as indicated by imaging findings is recommended. Joint replacement should not be left too late, and surgical procedures on the shoulder should be built into the overall treatment plan.

[Diagnostic strategy for shoulder pathology?]. / Comment s'orienter devant une épaule douloureuse?

Faced to shoulder pathology, the way of investigating must be very systematic. Before doing sophisticated explorations (arthroCT, MRI), we first have to use the triad "interrogatory-clinical examination-standard X Rays". This first step is absolutely necessary; it must allow to make the diagnosis to which the therapy must be adapted. The rotator cuff pathology is the most frequent one. Its frequency depends on patient's age, precessive trauma or not and the pratice of activities using the arm up (sports, leasures, works). A stiff shoulder must never be neglected, the diagnosis is done by clinical examination and its origine is very often precised by standard Xrays. Others diagnosis are done by the conjunction of several elements (pathologic context, clinical examination, standard Xrays). In some complex situations, other investigations (US, arthroCT, MRI) will be needed.

Endoscopic Management of Gluteus Medius Tendon Tears.

Tears in the gluteus medius and minimus tendons have been recognized as an important cause of recalcitrant greater trochanteric pain syndrome. Because of the frequency of partial-thickness undersurface tears, this relatively unknown pathology is often misdiagnosed and left untreated. Surgery is indicated in case of 4 associated conditions: (i) Failure of conservative treatment with duration of symptoms >6 months; (ii) magnetic resonance imaging showing a tendon tear; (iii) positive ultrasound-guided infiltration test; and (iv) the absence of an evolved fatty degeneration or atrophy of the gluteus medius and minimus muscle. Endoscopic repair of partial or full-thickness tears, with systematic resection of the bony structures implicated in the impingement, and a complete bursectomy appear to give satisfactory results, although these results remain to be confirmed by clinical studies with longer follow-up. The degree of tendon degeneration may compromise the tissue left for reattachment, raising concerns over its healing capacity, durability, and ultimate strength of the repair.

[Rheumatoid arthritis: a general disease and local diseases]. / Polyarthrite rhumatoïde: une maladie générale et des maladies locales.

The rheumatoid synovitis affects the joints by destroying the cartilage, the sub-chondral bone and the articular capsule. The tendons and ligaments can be degraded by proximity or by the means of the affected synovial sheaths. This conjunction of effects involves a foreseeable degradation on the complex articulations whose clinician must know the stages to interfere effectively into a preventive way by local interventions when the general treatments of the disease are insufficient and before recourse to the repairing surgery. This management can only be considered with a team where the general practitioner has a central place of alarm. Extraarticular symptoms (Sjogren's syndrome, cardiac, pulmonary or renal involvement) are specific local diseases and should be managed appropriately by the general practitioner and referred specialists.

Comparison of biomass and lipid production under ambient carbon dioxide vigorous aeration and 3% carbon dioxide condition among the lead candidate Chlorella strains screened by various photobioreactor scales.

Chlorella species from the UTEX collection, classified by rDNA-based phylogenetic analysis, were screened based on biomass and lipid production in different scales and modes of culture. The lead candidate strains of C. sorokiniana UTEX 1230 and C. vulgaris UTEX 395 and 259 were compared between conditions of vigorous aeration with filtered atmospheric air and 3% CO2 shake-flask cultivation. The biomass of UTEX 1230 produced 2 times higher at 652 mg L(-1) dry weight under both ambient CO2 vigorous aeration and 3% CO2 conditions, while UTEX 395 and 259 under 3% CO2 increased to 3 times higher at 863 mg L(-1) dry weight than ambient CO2 vigorous aeration. The triacylglycerol contents of UTEX 395 and 259 increased more than 30 times to 30% dry weight with 3% CO2, indicating that additional CO2 is essential for both biomass and lipid accumulation in UTEX 395 and 259.

Results of arthroscopic treatment of posterosuperior glenoid impingement in tennis players.

Twenty-eight tennis players with symptomatic posterosuperior glenoid impingement limiting their participation underwent arthroscopic debridement of the supraspinatus tendon and glenoid lesions associated with this diagnosis after nonoperative treatment had failed. The dominant extremity was affected in all patients; the patients' average age was 26.9 years. Eighteen patients participated at the highest level of competition for their age, and the remaining patients participated at the intermediate level. Patients were evaluated at an average of 45.7 months after surgery by physical examination, an activities questionnaire, a subjective result questionnaire, and a questionnaire regarding their return to activity. Postoperatively, the patients averaged 26.9 of 30 possible points on the activities questionnaire. Twenty-three of the patients were subjectively satisfied with the surgical result. Twenty-two patients had returned to tennis. Despite their return, 20 of the 22 patients reported some persistent pain with participation. To our knowledge, this is the first report detailing the results of operative treatment for posterosuperior glenoid impingement in a population limited to tennis players. Even though the results are encouraging in terms of the high number of patients returning to tennis, the effects of this persistent pain with activity, although diminished in severity, on long-term participation is unknown.

Rotator cuff tears in middle-aged tennis players: results of surgical treatment.

BACKGROUND: Tennis players, like participants in other overhead sports, are vulnerable to rotator cuff tears. In players who continue to play into their middle-age years, the incidence of such injury increases. HYPOTHESIS: Surgical treatment of rotator cuff tears in middle-aged tennis players is largely successful in allowing return to tennis. STUDY DESIGN: Retrospective review. METHODS: We evaluated the results of surgical treatment of 51 middle-aged tennis players (average age, 51 years) with a rotator cuff tear in their dominant shoulder. Tennis participation among the group had averaged 3.5 hours per week for an average of 25 years. Forty-two patients underwent open repair of the tear with or without biceps tenodesis, whereas 9 patients underwent arthroscopic debridement of the tear with or without a biceps tenotomy. Patients were reviewed at an average of 57 months after surgery with an activities score, a subjective questionnaire, and a questionnaire regarding their postoperative participation in tennis. RESULTS: The activities score averaged 26.6 of 30 possible points. Forty-seven patients were satisfied with their result, and 40 patients were able to return to tennis at an average of 9.8 months after surgery. No difference was found in the ability to return to tennis between the open repair group and the arthroscopic debridement group. CONCLUSIONS: The results of this study indicate that it is possible for nearly 80% of middle-aged tennis players to return to participation after operative treatment of rotator cuff tears.