Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody Responses Impact the Estimates of Infections in Population-Based Seroprevalence Studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses to the spike (S) protein monomer, S protein native trimeric form, or the nucleocapsid (N) proteins were evaluated in cohorts of individuals with acute infection (n = 93) and in individuals enrolled in a postinfection seroprevalence population study (n = 578) in Switzerland. Commercial assays specific for the S1 monomer, for the N protein, or within a newly developed Luminex assay using the S protein trimer were found to be equally sensitive in antibody detection in the acute-infection-phase samples. Interestingly, compared to anti-S antibody responses, those against the N protein appear to wane in the postinfection cohort. Seroprevalence in a "positive patient contacts" group (n = 177) was underestimated by N protein assays by 10.9 to 32.2%, while the "randomly selected" general population group (n = 311) was reduced by up to 45% relative to the S protein assays. The overall reduction in seroprevalence targeting only anti-N antibodies for the total cohort ranged from 9.4 to 31%. Of note, the use of the S protein in its native trimer form was significantly more sensitive compared to monomeric S proteins. These results indicate that the assessment of anti-S IgG antibody responses against the native trimeric S protein should be implemented to estimate SARS-CoV-2 infections in population-based seroprevalence studies.IMPORTANCE In the present study, we have determined SARS-CoV-2-specific antibody responses in sera of acute and postinfection phase subjects. Our results indicate that antibody responses against viral S and N proteins were equally sensitive in the acute phase of infection, but that responses against N appear to wane in the postinfection phase where those against the S protein persist over time. The most sensitive serological assay in both acute and postinfection phases used the native S protein trimer as the binding antigen, which has significantly greater conformational epitopes for antibody binding compared to the S1 monomer protein used in other assays. We believe these results are extremely important in order to generate correct estimates of SARS-CoV-2 infections in the general population. Furthermore, the assessment of antibody responses against the trimeric S protein will be critical to evaluate the durability of the antibody response and for the characterization of a vaccine-induced antibody response.

Tomographic Task-Related Functional Near-Infrared Spectroscopy in Acute Sport-Related Concussion: An Observational Case Study.

Making decisions regarding return-to-play after sport-related concussion (SRC) based on resolution of symptoms alone can expose contact-sport athletes to further injury before their recovery is complete. Task-related functional near-infrared spectroscopy (fNIRS) could be used to scan for abnormalities in the brain activation patterns of SRC athletes and help clinicians to manage their return-to-play. This study aims to show a proof of concept of mapping brain activation, using tomographic task-related fNIRS, as part of the clinical assessment of acute SRC patients. A high-density frequency-domain optical device was used to scan 2 SRC patients, within 72 h from injury, during the execution of 3 neurocognitive tests used in clinical practice. The optical data were resolved into a tomographic reconstruction of the brain functional activation pattern, using diffuse optical tomography. Moreover, brain activity was inferred using single-subject statistical analyses. The advantages and limitations of the introduction of this optical technique into the clinical assessment of acute SRC patients are discussed.

Cognitive behavioural therapy plus standard care versus standard care for people with schizophrenia.

BACKGROUND: Cognitive behavioural therapy (CBT) is a psychosocial treatment that aims to re-mediate distressing emotional experiences or dysfunctional behaviour by changing the way in which a person interprets and evaluates the experience or cognates on its consequence and meaning. This approach helps to link the person's feelings and patterns of thinking which underpin distress. CBT is now recommended by the National Institute for Health and Care Excellence (NICE) as an add-on treatment for people with a diagnosis of schizophrenia. This review is also part of a family of Cochrane CBT reviews for people with schizophrenia. OBJECTIVES: To assess the effects of cognitive behavioural therapy added to standard care compared with standard care alone for people with schizophrenia. SEARCH METHODS: We searched the Cochrane Schizophrenia Group's Trials Register (up to March 6, 2017). This register is compiled by systematic searches of major resources (including AMED, BIOSIS CINAHL, Embase, MEDLINE, PsycINFO, PubMed, and registries of clinical trials) and their monthly updates, handsearches, grey literature, and conference proceedings, with no language, date, document type, or publication status limitations for inclusion of records into the register. SELECTION CRITERIA: We selected all randomised controlled clinical trials (RCTs) involving people diagnosed with schizophrenia or related disorders, which compared adding CBT to standard care with standard care given alone. Outcomes of interest included relapse, rehospitalisation, mental state, adverse events, social functioning, quality of life, and satisfaction with treatment.We included studies fulfilling the predefined inclusion criteria and reporting useable data. DATA COLLECTION AND ANALYSIS: We complied with the Cochrane recommended standard of conduct for data screening and collection. Where possible, we calculated relative risk (RR) and its 95% confidence interval (CI) for binary data and mean difference (MD) and its 95% confidence interval for continuous data. We assessed risk of bias for included studies and created a 'Summary of findings' table using GRADE. MAIN RESULTS: This review now includes 60 trials with 5,992 participants, all comparing CBT added to standard care with standard care alone. Results for the main outcomes of interest (all long term) showed no clear difference between CBT and standard care for relapse (RR 0.78, 95% CI 0.61 to 1.00; participants = 1538; studies = 13, low-quality evidence). Two trials reported global state improvement. More participants in the CBT groups showed clinically important improvement in global state (RR 0.57, 95% CI 0.39 to 0.84; participants = 82; studies = 2 , very low-quality evidence). Five trials reported mental state improvement. No differences in mental state improvement were observed (RR 0.81, 95% CI 0.65 to 1.02; participants = 501; studies = 5, very low-quality evidence). In terms of safety, adding CBT to standard care may reduce the risk of having an adverse event (RR 0.44, 95% CI 0.27 to 0.72; participants = 146; studies = 2, very low-quality evidence) but appears to have no effect on long-term social functioning (MD 0.56, 95% CI -2.64 to 3.76; participants = 295; studies = 2, very low-quality evidence, nor on long-term quality of life (MD -3.60, 95% CI -11.32 to 4.12; participants = 71; study = 1, very low-quality evidence). It also has no effect on long-term satisfaction with treatment (measured as 'leaving the study early') (RR 0.93, 95% CI 0.77 to 1.12; participants = 1945; studies = 19, moderate-quality evidence). AUTHORS' CONCLUSIONS: Relative to standard care alone, adding CBT to standard care appears to have no effect on long-term risk of relapse. A very small proportion of the available evidence indicated CBT plus standard care may improve long term global state and may reduce the risk of adverse events. Whether adding CBT to standard care leads to clinically important improvement in patients' long-term mental state, quality of life, and social function remains unclear. Satisfaction with care (measured as number of people leaving the study early) was no higher for participants receiving CBT compared to participants receiving standard care. It should be noted that although much research has been carried out in this area, the quality of evidence available is poor - mostly low or very low quality and we still cannot make firm conclusions until more high quality data are available.

Cognitive behavioural therapy plus standard care versus standard care plus other psychosocial treatments for people with schizophrenia.

BACKGROUND: Cognitive behavioural therapy (CBT) is a psychosocial treatment that aims to help individuals re-evaluate their appraisals of their experiences that can affect their level of distress and problematic behaviour. CBT is now recommended by the National Institute for Health and Care Excellence (NICE) as an add-on treatment for people with a diagnosis of schizophrenia. Other psychosocial therapies that are often less expensive are also available as an add-on treatment for people with schizophrenia. This review is also part of a family of Cochrane Reviews on CBT for people with schizophrenia. OBJECTIVES: To assess the effects of CBT compared with other psychosocial therapies as add-on treatments for people with schizophrenia. SEARCH METHODS: We searched the Cochrane Schizophrenia Group's Study Based Register of Trials (latest 6 March, 2017). This register is compiled by systematic searches of major resources (including AMED, BIOSIS CINAHL, Embase, MEDLINE, PsycINFO, PubMed, and registries of clinical trials) and their monthly updates, handsearches, grey literature, and conference proceedings, with no language, date, document type, or publication status limitations for inclusion of records into the register. SELECTION CRITERIA: We selected randomised controlled trials (RCTs) involving people with schizophrenia who were randomly allocated to receive, in addition to their standard care, either CBT or any other psychosocial therapy. Outcomes of interest included relapse, global state, mental state, adverse events, social functioning, quality of life and satisfaction with treatment. We included trials meeting our inclusion criteria and reporting useable data. DATA COLLECTION AND ANALYSIS: We reliably screened references and selected trials. Review authors, working independently, assessed trials for methodological quality and extracted data from included studies. We analysed dichotomous data on an intention-to-treat basis and continuous data with 60% completion rate. Where possible, for binary data we calculated risk ratio (RR), for continuous data we calculated mean difference (MD), all with 95% confidence intervals (CIs). We used a fixed-effect model for analyses unless there was unexplained high heterogeneity. We assessed risk of bias for the included studies and used the GRADE approach to produce a 'Summary of findings' table for our main outcomes of interest. MAIN RESULTS: The review now includes 36 trials with 3542 participants, comparing CBT with a range of other psychosocial therapies that we classified as either active (A) (n = 14) or non active (NA) (n = 14). Trials were often small and at high or unclear risk of bias. When CBT was compared with other psychosocial therapies, no difference in long-term relapse was observed (RR 1.05, 95% CI 0.85 to 1.29; participants = 375; studies = 5, low-quality evidence). Clinically important change in global state data were not available but data for rehospitalisation were reported. Results showed no clear difference in long term rehospitalisation (RR 0.96, 95% CI 0.82 to 1.14; participants = 943; studies = 8, low-quality evidence) nor in long term mental state (RR 0.82, 95% CI 0.67 to 1.01; participants = 249; studies = 4, low-quality evidence). No long-term differences were observed for death (RR 1.57, 95% CI 0.62 to 3.98; participants = 627; studies = 6, low-quality evidence). Only average endpoint scale scores were available for social functioning and quality of life. Social functioning scores were similar between groups (long term Social Functioning Scale (SFS): MD 8.80, 95% CI -4.07 to 21.67; participants = 65; studies = 1, very low-quality evidence), and quality of life scores were also similar (medium term Modular System for Quality of Life (MSQOL): MD -4.50, 95% CI -15.66 to 6.66; participants = 64; studies = 1, very low-quality evidence). There was a modest but clear difference favouring CBT for satisfaction with treatment - measured as leaving the study early (RR 0.86, 95% CI 0.75 to 0.99; participants = 2392; studies = 26, low quality evidence). AUTHORS' CONCLUSIONS: Evidence based on data from randomised controlled trials indicates there is no clear and convincing advantage for cognitive behavioural therapy over other - and sometimes much less sophisticated and expensive - psychosocial therapies for people with schizophrenia. It should be noted that although much research has been carried out in this area, the quality of evidence available is mostly low or of very low quality. Good quality research is needed before firm conclusions can be made.

Improved process conditions for increasing expression of MHC class II protein from a stable Drosophila S2 cell line.

OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification.

Due to the lowered pKa of 4-fluorohistidine relative to histidine, peptides and proteins containing this amino acid are potentially endowed with novel properties. We report here the optimized synthesis of 4-fluorohistidine and show that it can efficiently replace histidine in in vitro translation reactions. Moreover, peptides containing 6×-fluorohistidine tags are able to be selectively captured and eluted from nickel resin in the presence of his-tagged protein mixtures.

Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension.

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.

Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines.

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc.

Production of active glycosylation-deficient γ-secretase complex for crystallization studies.

Alzheimer's disease (AD)-associated γ-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although γ-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. γ-Secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only γ-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of γ-secretase as a functional complex. We purified glycosylation-deficient γ-secretase from this recombinant cell line (CL1-9) and fully glycosylated γ-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient γ-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of γ-secretase and to structure-based drug discovery for AD.

Small-Scale Cultivation and Transfection of ExpiCHO and HEK293E Cells in Single-Use Orbitally Shaken Bioreactors.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most important mammalian hosts for the production of recombinant proteins. In this chapter, the suspension cultivation and transfection of these cells in small-scale, single-use orbitally shaken bioreactors, TubeSpin™ bioreactor 50 [orbitally shaken reactor 50 (OSR50)], and TubeSpin™ bioreactor 600 [orbitally shaken reactor 600 (OSR600)] are described. These are conical centrifuge tubes with nominal volumes of 50 mL and 600 mL, respectively, that have been redesigned with a ventilated cap for the cultivation of animal cells in suspension at working volumes up to 20 mL and 400 mL, respectively.

Examining the validity of the Delis-Kaplan Executive Function System (D-KEFS) in traumatic brain injury.

This study examines the validity of the Delis-Kaplan Executive Function System (D-KEFS) in a traumatic brain injury (TBI) population compared to participants with orthopaedic injuries and normative controls. The utility of the D-KEFS was examined using a between groups design. One hundred patients with mild uncomplicated to severe TBI were recruited from a consecutive cohort of patients admitted as inpatients to a UK Major Trauma Centre and compared to 823 participants from the D-KEFS normative sample and 26 participants with orthopaedic injuries. Data were filtered for performance validity. Sample discrimination was calculated from D-KEFS subtest scores and derived index scores. Sensitivity to TBI severity was established. The TBI participants performed significantly lower on the D-KEFS Trail Making Test, Colour Word Interference, Colour Word Switching, Letter Fluency and Verbal Fluency Category Switching Total Words Correct. The D-KEFS index scores discriminated between TBI, orthopaedic and normative participants with large and moderate effect sizes, respectively. The D-KEFS demonstrated a dose-response relationship with TBI severity. These effects were robust to differences in premorbid intellectual functioning; however, D-KEFS performance was sensitive to performance on tests of mental processing speed. The use of a D-KEFS index score provides a robust and reliable discrimination of TBI patients from healthy control participants. This discrimination is not accounted for by premorbid intellect or the non-specific effects of trauma. The clinical and conceptual implications of these findings are considered.

Highly efficient production of the Alzheimer's γ-secretase integral membrane protease complex by a multi-gene stable integration approach.

Inefficient production of membrane-embedded multi-protein complexes by conventional methods has largely prevented the generation of high-resolution structural information and the performance of high-throughput drug discovery screens for this class of proteins. Not exempt from this rule is γ-secretase, an intramembrane-cleaving protease complex regulating a multitude of signaling pathways and biological processes by influencing gene transcription. γ-Secretase is also implicated in the pathogenesis of Alzheimer's disease and several types of cancer. As an additional challenge, the reconstitution of the protease complex in its active form requires an intricate assembly and maturation process, including a highly regulated endoproteolytic processing of its catalytic component. In this article we report the application of a transposon-mediated multigene stable integration technology to produce active γ-secretase in mammalian cells in amounts adequate for crystallization studies and drug screening. Our strategy is expected to help elucidate the molecular mechanisms of intramembrane proteolysis. It is further expected to be widely used for the production of other multi-protein complexes for applications in structural biology and drug development.

Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells.

Transient gene expression (TGE) from mammalian cells is an increasingly important tool for the rapid production of recombinant proteins for research applications in biochemistry, structural biology, and biomedicine. Here we review methods for the transfection of human embryo kidney (HEK-293) and Chinese hamster ovary (CHO) cells in suspension culture using the cationic polymer polyethylenimine (PEI) for gene delivery.

An evaluation of the convergent validity of a face-to-face and virtual neuropsychological assessment counter balanced.

The COVID-19 pandemic has highlighted the need for further research evaluating the validity of conducting a battery of neuropsychological assessments virtually compared with face-to-face administration. Previous research has suggested that some neuropsychological assessments yield valid results when administered virtually, however, much of the previous research focused on older adults. To determine the validity of virtually administered neuropsychological tests, 28 healthy participants were assessed using a within-subjects, counter-balanced design. Participants completed a neuropsychological assessment battery covering tests of general intellectual functioning, memory and attention, executive functioning, language and information processing speed, as well as effort. There was no significant difference between face-to-face administration of the neuropsychological battery compared with virtual administration for the majority of the tests used. However, there were significant differences in the Colour Naming Task, with participants making fewer errors on the colour naming task and inhibition/switching task when administered virtually compared with face-to-face administration. There was also a significant age cohort effect in the inhibition/switching task. There was also a trending significant difference in mode of administration for the Verbal Fluency Task. Virtually administered neuropsychological assessments largely provide a valid alternative to face-to-face assessments; however, consideration must be given to test selection as well as the population of participants that are being assessed. Other important considerations must focus on preserving the security and integrity of test materials, as well as administration in a medico-legal setting. Future research should focus on validating assessments with specific patient populations and developing a neuropsychological assessment battery using information technology.

A meta-analytic review examining the validity of executive functioning tests to predict functional outcomes in individuals with a traumatic brain injury.

OBJECTIVES: Deficits in executive functioning are a common consequence of Traumatic Brain Injury (TBI) and the severity of TBI is known to predict functional outcomes. In this review, the authors examine the ability of three commonly used tests of executive functioning [The Trail Making Test (TMT-B), The Wisconsin Card Sorting Test (WCST), and Verbal Fluency (VF)] to predict domains of function. METHODS: Seven hundred and twenty articles were identified and twenty-four met inclusion criteria (original articles published in English examining an adult TBI population). Data were subject to a study quality analysis and then meta-analyzed to assess whether tests of executive functioning (TMT-B, WCST, and VF) can predict functional, employment, and driving outcomes following a TBI. RESULTS: The TMT-B (r = 0.29; 95% CI 0.17-0.41) and the WCST (r = 0.20; 95% CI 0.02-0.37) were significantly associated with functional outcomes. The TMT-B was also associated with a person's ability to return to driving (r = 0.3890; 95% CI 0.2678-0.5103). No test of executive functioning was associated with employment outcomes following a TBI. CONCLUSION: These findings are important to guide rehabilitation strategies and future planning. This review has also highlighted the scarcity of research on specific outcomes.

Cognitive Outcome After Complicated Mild Traumatic Brain Injury: A Literature Review and Meta-Analysis.

Cognitive outcome for mild traumatic brain injury (mTBI) with positive brain imaging (complicated mTBI) was compared with that for mTBI with normal imaging (uncomplicated mTBI) and with moderate to severe TBI, using meta-analysis. Twenty-three studies utilizing objective neurocognitive tests were included in the analysis. At less than 3 months post-injury, complicated mTBI was associated with poorer cognitive outcomes than uncomplicated mTBI, but deficits were not comparable to those with moderate-severe TBI. After 3 months post-injury, a similar pattern was detected. Beyond 3 months, deficits in complicated mTBI relative to those with uncomplicated mTBI were present in processing speed, memory, executive function, and language, although the latter may be the result of reduced semantic fluency. The effect size of deficits in these domains was more marked in moderate-severe TBI. The available data support the use of complicated mTBI as a distinct classification in the prediction of cognitive outcome. The extent of cognitive deficit in complicated mTBI was small and unlikely to cause significant disability. However, patients with complicated mTBI constitute a broad category encompassing individuals who may differ markedly in the nature and extent of intracranial imaging abnormality, and further studies are warranted. Limitations of the available studies include small, selected samples; variations in TBI severity classification; absence of validity ("effort") testing; differing imaging methodology; and lack of long-term follow-up.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Chemical macrocyclization of peptides fused to antibody Fc fragments.

To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (K(i) = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody-drug conjugates.

A NanoDrop-based method for rapid determination of viability decline in suspension cultures of animal cells.

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280nm (A(280)) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A(280) was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension.