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The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago

Supplementary Methods, Tables 1-3, Figures 1-4 from Hedgehog Overexpression Is Associated with Stromal Interactions and Predicts for Poor Outcome in Breast Cancer

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

Photovoltaic-thermal (PVT) collectors can provide heat and electricity. A quasi-stationary model of a PVT collector is presented and validated based on measured data. Subsequently, the electrical and thermal performance of such a collector is simulated for different locations. These simulations focus on different flow rate control approaches with the aim of maximizing the electrical yield. The simulation results show that the two control approaches presented in this paper have the potential to increase the electrical output for all locations. The increase in additional electrical output at locations with higher solar radiation is significantly higher than the increase in solar radiation between the locations investigated. Additionally, the cooling fluids’ temperature differences for such PVT collectors vary between the investigated control approaches, where variable flow rates lead to a steadier increase in cooling fluids’ temperature. In conclusion, the flow rate needs to be adopted to the pre-defined main goal of the respective PVT collector. The use of variable flow rates by providing low-temperature heat at a more constant temperature level appears to be advantageous in this context.

Humans lack the capacity to produce the Galα1–3Galβ1–4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

DNA i-motif structures are formed in the nucleus of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by characterisation of select model genes, the abundance and distribution of such structures in the human genome has remained unclear. Here we utilize high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in human genomic DNA. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified over 650,000 i-motif structures in human genomic DNA. The i-motif structures are widely distributed throughout the human genome and are common among highly expressed genes and in genes upregulated in G0/G1 cell cycle phase. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA.

Background Drug resistance is a major obstacle in cancer therapy. To elucidate the genetic factors that regulate sensitivity to anti-cancer drugs, we performed CRISPR-Cas9 knockout screens for resistance to a spectrum of drugs. Results In addition to known drug targets and resistance mechanisms, this study revealed novel insights into drug mechanisms of action, including cellular transporters, drug target effectors, and genes involved in target-relevant pathways. Importantly, we identified ten multi-drug resistance genes, including an uncharacterized gene C1orf115, which we named Required for Drug-induced Death 1 (RDD1). Loss of RDD1 resulted in resistance to five anti-cancer drugs. Finally, targeting RDD1 leads to chemotherapy resistance in mice and low RDD1 expression is associated with poor prognosis in multiple cancers. Conclusions Together, we provide a functional landscape of resistance mechanisms to a broad range of chemotherapeutic drugs and highlight RDD1 as a new factor controlling multi-drug resistance. This information can guide personalized therapies or instruct rational drug combinations to minimize acquisition of resistance.

The purpose of this study was to investigate the dosimetric impact of weight loss in head and neck (H&N) patients and examine the effectiveness of adaptive planning. Data was collected from 22 H&N cancer patients who experienced weight loss during their course of radiotherapy. The robustness of Intensity Modulated Radiation Therapy (IMRT) and Volumetric Modulated Arc Therapy (VMAT) treatment plans were compared including the potential need for replanning. The dosimetric impact of weight loss was evaluated by calculating a verification plan for each patient on an assessment CT scan taken during the course of treatment. Using a regression analysis, significance was tested for the dosimetric change in target volumes and 10 specific organs at risk (OAR) using an anatomical separation difference in the H&N at corresponding levels. For both the IMRT and VMAT plans, a significant correlation was found for the dose to 5% of the high risk Planning Target Volume (PTV) (D5), dose to 95% of the intermediate risk PTV and Clinical Target Volume (CTV) (D95), and the percentage of the pharynx receiving 65 Gy. An independent t-test was also performed for each metric in the VMAT and IMRT plans showing the dose to 95% of the intermediate risk PTV as significant. No quantitative method for finding the threshold of anatomical separation difference requiring a replan was established. Based on the increase in dose to organs at risk and increased target coverage due to separation loss, it was concluded that adaptive radiotherapy may not always be necessary when alignment of bony anatomy and remaining soft tissue is within tolerance. Physician judgment and preference is needed in such situations.

Das Jahr 2019 war durch eine öffentlich sehr sichtbare Bewegung für mehr Klimaschutz geprägt. Gesellschaftliche Gruppen wie „Fridays for Future“ haben die nationalen wie europäischen Bemühungen der Politik um die Minderung der Treibhausgas-Emissionen beschleunigt. Die Vision von Netto-Null-Klimagasemissionen bis zum Jahr 2050 wurde dadurch zu einem ausgiebig diskutierten und auch akzeptierten Ziel und damit auch von immer breiteren Kreisen der Politik als Handlungsleitlinie und primäre Zielvorgabe akzeptiert. Im Kontext dieses gesellschaftlichen Settings und dieser übergeordneten Zielstellung wurde die Nutzung des erneuerbaren Energieangebots in Deutschland im Jahr 2019 verhalten weiter ausgebaut – und das mit deutlichen Unterschieden zwischen einzelnen Sektoren und Technologien.

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.

Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. Over this time global vaccine programs have been introduced, contributing to lowered COVID-19 hospitalisation and mortality rates, particularly in the first world. In late 2021, the Omicron (B.1.1.529) virus variant emerged, with significant genetic differences and clinical effects from other variants of concern (VOC). This variant a demonstrated higher number of polymorphisms in the gene encoding the Spike (S) protein, and there has been displacement of the dominant Delta variant. We assessed the impact of Omicron infection on the ability of: serum from vaccinated and/or previously infected individuals; concentrated human IgG from plasma donors, and licensed monoclonal antibody therapies to neutralise the virus in vitro. There was a 17 to 27-fold reduction in neutralisation titres across all donors who had a detectable neutralising antibody titre to the Omicron variant. Concentrated pooled human IgG from convalescent and vaccinated donors had greater breadth of neutralisation, although the potency was still reduced 16-fold. Of all therapeutic antibodies tested, significant neutralisation of the Omicron variant was only observed for Sotrovimab, with other monoclonal antibodies unable to neutralise Omicron in vitro. These results have implications for ongoing therapy of individuals infected with the Omicron variant.

SUMMARYChildren infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV- 2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. More naïve interferon-activated CD4+ T cells were recruited into the memory compartment and recovery was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.HIGHLIGHTSChildren have diverse polyclonal SARS-CoV-2-specific naïve T cellsAdults have clonally expanded exhausted SARS-CoV-2-specific memory T cellsInterferon-activated naïve T cells differentiate into memory T cells in adults but not childrenAdults but not children develop robust memory T cell responses to SARS-CoV-2

The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Graphical Abstract, Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

Most breast cancer deaths are caused by estrogen receptor-α–positive (ER+) disease. Preclinical progress is hampered by a shortage of therapy-naive ER+ tumor models that recapitulate metastatic pro...

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) form the evolutionarily conserved pancreatic polypeptide family. While the fold is widely utilized in nature, crystal structures remain elusive, particularly for the human forms, with only the structure of a distant avian form of PP reported. Here we utilize a crystallization chaperone (antibody Fab fragment), specifically recognizing the amidated peptide termini, to solve the structures of human NPY and human PYY. Intriguingly, and despite limited sequence identity (~50%), the structure of human PYY closely resembles that of avian PP, highlighting the broad structural conservation of the fold throughout evolution. Specifically, the PYY structure is characterized by a C-terminal amidated α-helix, preceded by a backfolded poly-proline N-terminus, with the termini in close proximity to each other. In contrast, in the structure of human NPY the N-terminal component is disordered, while the helical component of the peptide is observed in a four-helix bundle type arrangement, consistent with a propensity for multimerization suggested by NMR studies.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.

We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.

Insgesamt wurde in 2018 so viel Energie aus erneuerbaren Energien genutzt wie niemals zuvor. Dies gilt neben der (festen) Biomasse zur Bereitstellung insbesondere thermischer Energie primär für die Erzeugung elektrischer Energie. Stromerzeugungsanlagen – unter anderem auf der Basis von Wasserkraft, Biomasse, Windenergie und Solarstrahlung – waren 2018 für etwa ein Viertel der globalen Stromerzeugung verantwortlich. Demgegenüber zeigte die Wärme- und Kraftstoffbereitstellung aus erneuerbaren Energien auch 2018 nach wie vor nur einen unterdurchschnittlichen beziehungsweise stagnierenden Anstieg. Diese Entwicklung dürfte auch in den kommenden Jahren weitergehen.

Mehr denn je ist der zentrale Treiber für den Ausbau der regenerativen Energien der Klimaschutz mit dem im Pariser Klimavertrag vereinbarten Ziel, den maximalen anthropogenen globalen Temperaturanstieg im Mittel unter 2,0 °C zu halten und sogar möglichst auf 1,5 °C zu begrenzen. Letzterer Wert wird von immer mehr Akteuren als der Relevantere erachtet, sodass das Erreichen von Netto-Null-Emissionen an Treibhausgasen bis zum Jahr 2050 allmählich zum anerkannten Ziel nach dem aktuellen Stand des Wissens und damit auch von immer mehr Politiker als Handlungsleitlinie akzeptiert wird. Im Kontext dieser übergeordneten Zielstellung wurde die Nutzung des erneuerbaren Energieangebots in Deutschland im Jahr 2018 weiter ausgebaut – wenn auch mit deutlichen Unterschieden zwischen den einzelnen Sektoren des Energiesystems. Lediglich im Strombereich kam es 2018 zu einer weitergehenden Nutzung des erneuerbaren Energieangebots – und hier vor allem durch die Photovoltaik und die Windenergie. Dahingegen stagniert der Ausbau der „erneuerbaren“ Wärme und des klimaneutralen Mobilitätssektors; bei letzterem kam es 2018 vor allem im Biodieselbereich zu einer begrenzten Zunahme. Insgesamt reicht die bisherige Ausbaudynamik aber bei weitem nicht aus, die für 2030 beziehungsweise 2050 anvisierten Ziele zu erreichen.

Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.

The continued threat of SARS-CoV-2 to global health necessitates development of improved research tools and vaccines. We present an improved SARS-CoV-2 spike ectodomain, “VFLIP”, bearing five proline substitutions, a flexible cleavage site linker, and an inter-protomer disulfide bond. VFLIP displays significantly improved stability, high-yield production and retains its trimeric state without exogenous trimerization motifs. High-resolution cryo-EM and glycan profiling reveal that the VFLIP quaternary structure and glycosylation mimic the native spike on the viral surface. Further, VFLIP has enhanced affinity and binding kinetics relative to other stabilized spike proteins for antibodies in the Coronavirus Immunotherapeutic Consortium (CoVIC), and mice immunized with VFLIP exhibit potent neutralizing antibody responses against wild-type and B.1.351 live SARS-CoV-2. Taken together, VFLIP represents an improved tool for diagnostics, structural biology, antibody discovery, and vaccine design.Competing Interest StatementThe authors have declared no competing interest.

Considerable concerns relating to the duration of protective immunity against SARS-CoV-2 exist, with evidence of antibody titres declining rapidly after infection and reports of reinfection. Here we monitor the antibody responses against SARS-CoV-2 receptor binding domain (RBD) for up to six months after infection. While antibody titres are maintained, about 13% of the cohort’s neutralising responses return to background. However, encouragingly in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralising capacity from these memory B cells. Overall our study suggests that the loss of neutralising antibodies in plasma may be countered by the maintenance of neutralising capacity in the memory B cell repertoire., Graphical Abstract, Abayasingam et al. report that despite the declining anti-RBD antibody titres and neutralising capacity of antibodies in the serum at six months, the memory B cells still contain RBD-specific reactivity that have the capacity to generate antibodies that can neutralise SARS-CoV-2 in vitro.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus–cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)–confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of “high responders” maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.