Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

Combined In-Solution Fragment Screening and Crystallographic Binding-Mode Analysis with a Two-Domain Hsp70 Construct.

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP.

The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.

The Vaccinia Virus DNA Helicase Structure from Combined Single-Particle Cryo-Electron Microscopy and AlphaFold2 Prediction.

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

COVID-19 Intensive Care-Evaluation of Public Information Sources and Current Standards of Care in German Intensive Care Units: A Cross Sectional Online Survey on Intensive Care Staff in Germany.

Backround: In February 2021, the first formal evidence and consensus-based (S3) guidelines for the inpatient treatment of patients with COVID-19 were published in Germany and have been updated twice during 2021. The aim of the present study is to re-evaluate the dissemination pathways and strategies for ICU staff (first evaluation in December 2020 when previous versions of consensus-based guidelines (S2k) were published) and question selected aspects of guideline adherence of standard care for patients with COVID-19 in the ICU. Methods: We conducted an anonymous online survey among German intensive care staff from 11 October 2021 to 11 November 2021. We distributed the survey via e-mail in intensive care facilities and requested redirection to additional intensive care staff (snowball sampling). Results: There was a difference between the professional groups in the number, selection and qualitative assessment of information sources about COVID-19. Standard operating procedures were most frequently used by all occupational groups and received a high quality rating. Physicians preferred sources for active information search (e.g., medical journals), while nurses predominantly used passive consumable sources (e.g., every-day media). Despite differences in usage behaviour, the sources were rated similarly in terms of the quality of the information on COVID-19. The trusted organizations have not changed over time. The use of guidelines was frequently stated and highly recommended. The majority of the participants reported guideline-compliant treatment. Nevertheless, there were certain variations in the use of medication as well as the criteria chosen for discontinuing non-invasive ventilation (NIV) compared to guideline recommendations. Conclusions: An adequate external source of information for nursing staff is lacking, the usual sources of physicians are only appropriate for the minority of nursing staff. The self-reported use of guidelines is high.

Early spontaneous breathing for acute respiratory distress syndrome in individuals with COVID-19.

BACKGROUND: Acute respiratory distress syndrome (ARDS) represents the most severe course of COVID-19 (caused by the SARS-CoV-2 virus), usually resulting in a prolonged stay in an intensive care unit (ICU) and high mortality rates. Despite the fact that most affected individuals need invasive mechanical ventilation (IMV), evidence on specific ventilation strategies for ARDS caused by COVID-19 is scarce. Spontaneous breathing during IMV is part of a therapeutic concept comprising light levels of sedation and the avoidance of neuromuscular blocking agents (NMBA). This approach is potentially associated with both advantages (e.g. a preserved diaphragmatic motility and an optimised ventilation-perfusion ratio of the ventilated lung), as well as risks (e.g. a higher rate of ventilator-induced lung injury or a worsening of pulmonary oedema due to increases in transpulmonary pressure). As a consequence, spontaneous breathing in people with COVID-19-ARDS who are receiving IMV is subject to an ongoing debate amongst intensivists. OBJECTIVES: To assess the benefits and harms of early spontaneous breathing activity in invasively ventilated people with COVID-19 with ARDS compared to ventilation strategies that avoid spontaneous breathing. SEARCH METHODS: We searched the Cochrane COVID-19 Study Register (which includes CENTRAL, PubMed, Embase, Clinical Trials.gov WHO ICTRP, and medRxiv) and the WHO COVID-19 Global literature on coronavirus disease to identify completed and ongoing studies from their inception to 2 March 2022. SELECTION CRITERIA: Eligible study designs comprised randomised controlled trials (RCTs) that evaluated spontaneous breathing in participants with COVID-19-related ARDS compared to ventilation strategies that avoided spontaneous breathing (e.g. using NMBA or deep sedation levels). Additionally, we considered controlled before-after studies, interrupted time series with comparison group, prospective cohort studies and retrospective cohort studies. For these non-RCT studies, we considered a minimum total number of 50 participants to be compared as necessary for inclusion. Prioritised outcomes were all-cause mortality, clinical improvement or worsening, quality of life, rate of (serious) adverse events and rate of pneumothorax. Additional outcomes were need for tracheostomy, duration of ICU length of stay and duration of hospitalisation. DATA COLLECTION AND ANALYSIS: We followed the methods outlined in the Cochrane Handbook for Systematic Reviews of Interventions. Two review authors independently screened all studies at the title/abstract and full-text screening stage. We also planned to conduct data extraction and risk of bias assessment in duplicate. We planned to conduct meta-analysis for each prioritised outcome, as well as subgroup analyses of mortality regarding severity of oxygenation impairment and duration of ARDS. In addition, we planned to perform sensitivity analyses for studies at high risk of bias, studies using NMBA in addition to deep sedation level to avoid spontaneous breathing and a comparison of preprints versus peer-reviewed articles. We planned to assess the certainty of evidence using the GRADE approach. MAIN RESULTS: We identified no eligible studies for this review. AUTHORS' CONCLUSIONS: We found no direct evidence on whether early spontaneous breathing in SARS-CoV-2-induced ARDS is beneficial or detrimental to this particular group of patients.  RCTs comparing early spontaneous breathing with ventilatory strategies not allowing for spontaneous breathing in SARS-CoV-2-induced ARDS are necessary to determine its value within the treatment of severely ill people with COVID-19. Additionally, studies should aim to clarify whether treatment effects differ between people with SARS-CoV-2-induced ARDS and people with non-SARS-CoV-2-induced ARDS.

Cytoplasmic gene expression: lessons from poxviruses.

In eukaryotic cells, the process of gene expression is confined to the nucleus and enabled by multisubunit RNA polymerases (RNAPs). Many viruses make use of the host cellular gene expression apparatus during infection, and hence transfer their genome at least transiently to the host nucleus. However, poxviruses have evolved a different strategy to propagate. Their double-stranded DNA genome is transcribed in the host cytoplasm by a virus-encoded RNAP (vRNAP), which is evolutionarily related to eukaryotic RNA polymerase II. In this Review, we highlight recent high-resolution structures of the poxviral transcription apparatus in different phases of action. These structures, along with biochemical data, now allow the definition of a comprehensive model of poxviral gene expression and its regulation.

A generic protocol for the affinity-purification of native macromolecular complexes from poxvirus-infected cells.

The functional and structural characterization of macromolecular complexes requires protocols for their native isolation. Here, we describe a protocol for this task based on the recombinant poxvirus Vaccinia expressing tagged proteins of interest in infected cells. Tagged proteins and their interactors can then be isolated via affinity chromatography. The procedure is illustrated for the Vaccinia virus encoded multi-subunit RNA polymerase. Our protocol also allows the expression and isolation of heterologous proteins and hence is suitable for a broader application. For complete details on the use and execution of this profile, please refer to Grimm et al. (2019).

[COVID-19 pandemic: preferences and barriers for dissemination of evidence syntheses : Survey of intensive care personnel in Germany]. / COVID-19-Pandemie: Präferenzen und Barrieren für die Disseminierung von Evidenzsynthesen : Befragung des intensivmedizinischen Personals in Deutschland.

BACKGROUND: In the context of COVID-19, the German CEOsys project (COVID-19 Evidenz Ökosystem, www.covid-evidenz.de ) identifies, evaluates and summarizes the results of scientific studies to obtain evidence on this disease. The evidence syntheses are used to derive specific recommendations for clinical practice and to contribute to national guidelines. Besides the necessity of conducting good quality evidence syntheses during a pandemic, just as important is that the dissemination of evidence needs to be quick and efficient, especially in a health crisis. The CEOsys project has set itself this challenge. OBJECTIVE: Preparing the most suitable distribution of evidence syntheses as part of the CEOsys project tasks. METHODS: Intensive care unit (ICU) personnel in Germany were surveyed via categorical and free text questions. The survey focused on the following topics: evidence syntheses, channels and strategies of distribution, possibility of feedback, structure and barriers of dissemination and trustworthiness of various organizations. Profession, qualification, setting and size of the facility were recorded. Questionnaires were pretested throughout the queried professions (physician, nurse, others). The survey was anonymously carried out online through SosciSurvey® and an e­mail was sent directly to 940 addresses. The survey was launched on 3 December, a reminder was sent after 14 days and it ended on 31 December. The survey was also announced via e­mail through DIVI. RESULTS: Of 317 respondents 200 completed the questionnaire. All information was analyzed including the responses from incomplete questionnaires. The most stated barriers were lack of time and access. Especially residents and nurses without specialization in intensive care mentioned uncertainty or insufficient experience in dealing with evidence syntheses as a barrier. Active distribution of evidence syntheses was clearly preferred. More than half of the participants chose websites of public institutions, medical journals, professional societies and e­mail newsletters for drawing attention to new evidence syntheses. Short versions, algorithms and webinars were the most preferred strategies for dissemination. Trust in organizations supplying information on the COVID-19 pandemic was given to professional societies and the Robert Koch Institute (RKI) as the German governmental institute for infections and public health. The respondents' prioritized topics are long-term consequences of the disease, protection of medical personnel against infection and possibilities of ventilation treatment. CONCLUSION: Even though universally valid, evidence syntheses should be actively brought to the target audience, especially during a health crisis such as the COVID-19 pandemic with its exceptional challenges including lack of time and uncertainties in patient care. The contents should be clear, short (short versions, algorithms) and with free access. E­mail newsletters, websites or medical journals should continuously report on new evidence syntheses. Professional societies and the governmental institute for infections and public health should be involved in dissemination due to their obvious trustworthiness.

A Comparison and Evaluation of International Guidelines on the Treatment of Severe SARS-CoV-2 Infection.

BACKGROUND: When the SARS-CoV-2 pandemic began, no uniform treatment and care strategies for critically ill COVID-19 patients were yet available. National and international treatment recommendations were formulated under time pressure, initially on the basis of indirect evidence from the treatment of similar diseases. In this article, we give an overview of the content, currency, and methodological quality of the existing national and international guidelines, with special attention to the care of critically ill patients. METHODS: Guidelines were identified by a comprehensive search, the included guidelines were assessed in standardized fashion with the AGREE II guideline assessment instrument and according to the AMWF rulebook criteria, and the core recommendations of the included and methodologically high-quality guidelines were compared. RESULTS: Nine of the 97 guidelines that were identified fulfilled the content criteria for inclusion, and 6 of these fulfilled the qualitative criteria; these 6 guidelines still differed, however, in the topics to which they devoted the most attention, as well as in their methodological quality and currency. The treatment strategies for patients with severe respiratory failure (lung-protective ventilation strategies and rescue measures) deviated little from established standards. Uniform recommendations were made, among other things, for the administration of dexamethasone, which was recommended in all of the guidelines for patients requiring oxygen treatment, as well as for antithrombotic drug prophylaxis and for the prone positioning of ventilated patients. Many recommendations were based on insufficient evidence, and some were contradictory, e.g., those regarding antibiotic treatment or the choice between high-flow oxygen administration via nasal canula (HFNC) and noninvasive ventilation (NIV). CONCLUSION: The consultation of multiple high-quality international guidelines and guideline recommendations shared in online portals such as MagicApp are helpful sources of information for clinicians. In view of the continuing lack of strong evidence, further research on intensive care treatments is needed (aspects of ventilation, positioning therapy, and the role of extracorporeal membrane oxygenation [ECMO]).

Structure and function of the poxvirus transcription machinery.

Members of the Poxviridae family are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of their hosts. This goes in hand with a high level of independence from the host cell, which supports transcription and replication events only in the nucleus or in DNA-containing organelles. Consequently, virus specific, rather than cellular enzymes mediate most processes involving DNA replication and mRNA synthesis. Recent technological advances allowed a detailed functional and structural investigation of the transcription machinery of the prototypic poxvirus vaccinia. The DNA-dependent RNA polymerase (RNAP) at its core displays distinct similarities to eukaryotic RNAPs. Strong idiosyncrasies, however, are apparent for viral factors that are associated with the viral RNAP during mRNA production. We expect that future studies will unravel more key aspects of poxvirus gene expression, helping also the understanding of nuclear transcription mechanisms.

Case Report: Interferon- γ Rescues Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) Function in a COVID-19 Patient With ARDS and Superinfection With Multiple MDR 4MRGN Bacterial Strains.

Background: CD14+ monocytes present antigens to adaptive immune cells via monocytic human leukocyte antigen receptor (mHLA-DR), which is described as an immunological synapse. Reduced levels of mHLA-DR can display an acquired immune defect, which is often found in sepsis and predisposes for secondary infections and fatal outcomes. Monocytic HLA-DR expression is reliably induced by interferon- γ (IFNγ) therapy. Case Report: We report a case of multidrug-resistant superinfected COVID-19 acute respiratory distress syndrome (ARDS) on extracorporeal membrane oxygenation (ECMO) support. The resistance profiles of the detected Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Citrobacter freundii isolates were equipped with resistance to all four antibiotic classes including carbapenems (4MRGN) and Cefiderocol in the case of K. pneumoniae. A causal therapeutic antibiotic strategy was not available. Therefore, we measured the immune status of the patient aiming to identify a potential acquired immune deficiency. Monocyte HLA-DR expression identified by FACS analysis revealed an expression level of 34% positive monocytes and suggested severe immunosuppression. We indicated IFNγ therapy, which resulted in a rapid increase in mHLA-DR expression (96%), rapid resolution of invasive bloodstream infection, and discharge from the hospital on day 70. Discussion: Superinfection is a dangerous complication of COVID-19 pneumonia, and sepsis-induced immunosuppression is a risk factor for it. Immunosuppression is expressed by a disturbed antigen presentation of monocytes to cells of the adaptive immune system. The case presented here is remarkable as no validated antibiotic regimen existed against the detected bacterial pathogens causing bloodstream infection and severe pneumonia in a patient suffering from COVID-19 ARDS. Possible restoration of the patient's own immunity by IFNγ was a plausible option to boost the patient's immune system, eliminate the identified 4MRGNs, and allow for lung recovery. This led to the conclusion that immune status monitoring is useful in complicated COVID-19-ARDS and that concomitant IFNγ therapy may support antibiotic strategies. Conclusion: After a compromised immune system has been detected by suppressed mHLA-DR levels, the immune system can be safely reactivated by IFNγ.

Structural basis of the complete poxvirus transcription initiation process.

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

A Nationwide Cross-Sectional Online Survey on the Treatment of COVID-19-ARDS: High Variance in Standard of Care in German ICUs.

INTRODUCTION: Coronavirus disease (COVID-19) has recently dominated scientific literature. Incomplete understanding and a lack of data concerning the pathophysiology, epidemiology, and optimal treatment of the disease has resulted in conflicting recommendations. Adherence to existing guidelines and actual treatment strategies have thus far not been studied systematically. We hypothesized that capturing the variance in care would lead to the discovery of aspects that need further research and-in case of proven benefits of interventions not being performed-better communication to care providers. METHODS: This article is based on a quantitative and qualitative cross-sectional mixed-methods online survey among intensive-care physicians in Germany during the COVID-19 pandemic by the CEOsys (COVID-19 Evidence Ecosystem) network, endorsed by the German Interdisciplinary Association for Intensive Care and Emergency Medicine (DIVI) conducted from December 3 to 31 December 2020. RESULTS: We identified several areas of care with an especially high variance in treatment among hospitals in Germany. Crucially, 51.5% of the participating ICUs (n = 205) reported using intubation as a last resort for respiratory failure in COVID-19 patients, while 21.8% used intubation early after admission. Furthermore, 11.5% considered extracorporeal membrane oxygenation (ECMO) in awake patients. Finally, 72.3% of respondents used the ARDS-network-table to titrate positive end-expiratory-pressure (PEEP) levels, with 36.9% choosing the low-PEEP table and 41.8% the high-PEEP table. CONCLUSIONS: We found that significant differences exist between reported treatment strategies and that adherence to published guidelines is variable. We describe necessary steps for future research based on our results highlighting significant clinical variability in care.

Infection control, prophylactic antibiotics, and testing for SARS-CoV-2 and PPE on German intensive care units: results from a national mixed methods survey.

Aim: Recommendations on hygiene measures, personal protective equipment (PPE), isolation, and antibiotic prophylaxis were developed during the coronavirus 2019 disease (COVID-19) pandemic and have been revised several times to date. Some of the underlying literature indicates a large evidence gap. We suspect that this leads to a large variance of measures on German intensive care units (ICU). Methods: A mixed methods online survey among intensive-care specialists in Germany caring for COVID-19 patients was conducted in December 2020. Results: We received responses from 205 German ICUs that had treated COVID-19 patients to date. There was wide variation in the use of PPE. Polymerase Chain reaction (PCR) testing for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) was used by 94.8% of the units, with an average waiting time of 12 hours for the result. 18.7% of the respondents prescribed antibiotic prophylaxis in COVID-19 patients. Conclusion: We found a high variance in essential care strategies for COVID-19 patients on German intensive care units. This included differences in infection prophylaxis, personal protective equipment, and the indication of prophylactic antibiotic therapy. Based on our results, we recommend further studies to quantify and improve guideline adherence.

Targeted Minimal Staff-to-Patient Ratios Are Unachievable - A Nationwide Survey in German ICUs During the COVID-19 Pandemic.

Introduction Adequate staffing in the intensive care units (ICUs) is the most important factor to provide optimal care and ensure favorable outcomes in critically ill patients. Recently, the need for ICU beds has reached unprecedented levels and the management and treatment of critically ill patients has been in focus. The aim of the study was to assess the targeted and actual nurse-to-patient (NPR) and physician-to-patient ratios (PPR) regarding patients with and without COVID-19. Methods We conducted a nationwide online survey assessing the standard of care in German ICUs treating patients with COVID-19. We asked questions regarding targeted PPR and NPR and their implementation in daily clinical practice to heads of German ICU departments. Results We received 244 responses of which 171 were eligible for final analysis. Targeted median PPR ratio was 8 [interquartile range (IQR) = 4] and targeted NPR was 2 (IQR = 1). For COVID-19 patients, the median targeted PPR was 6 (IQR = 2) and the median targeted NPR was 2 (IQR = 0). Targeted PPRs were rarely met by 15.2% and never met by 3.5% of responding institutions. Targeted NPRs were rarely met in 32.2% and never in 5.3% of responding institutions. Conclusion In contrast to PPR, targeted NPRs were largely unattainable in German ICUs. Our results raise concern in view of studies linking worse outcomes in critically ill patients to suboptimal NPRs. This warrants further health policy efforts regarding optimal staffing in the ICU.

A novel zebrafish model for intermediate type spinal muscular atrophy demonstrates importance of Smn for maintenance of mature motor neurons.

A deficiency in Survival Motor Neuron (SMN) protein results in motor neuron loss in spinal muscular atrophy (SMA) patients. Human SMN is encoded by SMN1 and SMN2 that differ by a single C6T transition in a splice regulatory region of exon 7. In SMN2, exon 7 is skipped leading to an unstable protein, which cannot compensate for SMN1 loss in SMA patients. The disease severity of human SMA (Types 1-4) depends on the levels of SMN protein, with intermediate levels leading to delayed disease onset and extended life expectancy in Type 2 patients. We used homology directed repair (HDR) to generate a zebrafish mutant with intermediate Smn levels, to mimic intermediate, hSMN2 dependent forms of SMA. In the obtained smnA6Tind27 mutant zebrafish, Smn protein formed oligomers but protein levels dropped significantly at juvenile stages. Motor neurons and neuromuscular junctions (NMJ) also formed normally initially but motor neuron loss and locomotor deficiencies became evident at 21 days. Subsequent muscle wasting and early adult lethality also phenocopied intermediate forms of human SMA. Together, our findings are consistent with the interpretation that Smn is required for neuromuscular maintenance, and establish the smnA6Tind27 zebrafish mutant as a novel model for intermediate types of SMA. As this mutant allows studying the effect of late Smn loss on motor neurons, neuromuscular junctions, and muscle at advanced stages of the disease, it will be a valuable resource for testing new drugs targeted towards treating intermediate forms of SMA.

Case Report: Interferon-γ Restores Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) in Severe COVID-19 With Acquired Immunosuppression Syndrome.

Background: The major histocompatibility complex (MHC) class II characterized by monocytes CD14+ expression of human leukocyte antigen receptors (HLA-DR), is essential for the synapse between innate and adaptive immune response in infectious disease. Its reduced expression is associated with a high risk of secondary infections in septic patients and can be safely corrected by Interferon-y (IFNy) injection. Coronavirus disease (COVID-19) induces an alteration of Interferon (IFN) genes expression potentially responsible for the observed low HLA-DR expression in circulating monocytes (mHLA-DR). Methods: We report a case of one-time INFy injection (100 mcg s.c.) in a superinfected 61-year-old man with COVID-19-associated acute respiratory distress syndrome (ARDS), with monitoring of mHLA-DR expression and clinical tolerance. Observations: Low mHLA-DR pretreatment expression (26.7%) was observed. IFNy therapy leading to a rapid increase in mHLA-DR expression (83.1%). Conclusions: Severe ARDS in a COVID-19 patient has a deep reduction in mHLA-DR expression concomitantly with secondary infections. The unique IFNy injection was safe and led to a sharp increase in the expression of mHLA-DR. Based on immune and infection monitoring, more cases of severe COVID-19 patients with low mHLA-DR should be treated by IFNy to test the clinical effectiveness.

Identification and structural analysis of the Schizosaccharomyces pombe SMN complex.

The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.