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Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism., Competing Interests: BW, ML, EM, PC, VE, CB, CA, SS, EM, PK, JR, HR, JL, RJ No competing interests declared, (© 2023, Wadsworth et al.)

Language is a universal human ability, acquired readily by young children, who otherwise struggle with many basics of survival. And yet, language ability is variable across individuals. Naturalistic and experimental observations suggest that children's linguistic skills vary with factors like socioeconomic status and children's gender. But which factors really influence children's day-to-day language use? Here, we leverage speech technology in a big-data approach to report on a unique cross-cultural and diverse data set: >2,500 d-long, child-centered audio-recordings of 1,001 2- to 48-mo-olds from 12 countries spanning six continents across urban, farmer-forager, and subsistence-farming contexts. As expected, age and language-relevant clinical risks and diagnoses predicted how much speech (and speech-like vocalization) children produced. Critically, so too did adult talk in children's environments: Children who heard more talk from adults produced more speech. In contrast to previous conclusions based on more limited sampling methods and a different set of language proxies, socioeconomic status (operationalized as maternal education) was not significantly associated with children's productions over the first 4 y of life, and neither were gender or multilingualism. These findings from large-scale naturalistic data advance our understanding of which factors are robust predictors of variability in the speech behaviors of young learners in a wide range of everyday contexts., Competing Interests: Competing interests statement:The authors declare no competing interest.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin β1, only some integrin β7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology.)

Advantageous inequity aversion (i.e., the tendency to respond negatively to unfairness that benefits oneself) usually develops in 6-8-year-olds. However, little is known about the selection pressures that might have shaped this phenomenon. Using data collected from 120 4-8-year-old Finnish children, we tested two evolutionary explanations for the development of advantageous inequity aversion: reciprocal altruism (i.e., benefiting from sharing when the roles are likely reversed in the future) and inclusive fitness (i.e., benefiting from sharing with biological relatives that carry the same alleles). We first successfully replicated a previous experiment, showing that 6-8-year-olds display advantageous inequity aversion by preferring to throw away a resource rather than keep it for themselves. Here, this behavior was also displayed in 5-year-olds. Using a novel experiment, we then asked children to distribute five erasers between themselves, a sibling, a peer, and a stranger. That is, an equal distribution was only possible if throwing away one eraser. We found no support for advantageous inequity aversion being shaped by either inclusive fitness or reciprocal altruism. Future studies could investigate costly signaling and adherence to social norms to avoid negative consequences as ultimate explanations for advantageous inequity aversion.

Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from patients with severe COVID-19 every third to seventh day during hospitalization and every third month after recovery. We profiled their antigen-specific immune cell dynamics by combining single-cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and B cell receptor-Seq (BCR-Seq) with oligo-tagged antigen baits. While the proportion of Spike receptor binding domain-specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen-specific cells, and affinity maturation was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to 1 year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen-specific B cell responses in longitudinally sampled patients infected with COVID-19.

Background: The risk of SARS-CoV-2 infection among health care workers (HCWs) is a concern, but studies that conclusively determine whether HCWs are over-represented remain limited. Furthermore, methods used to confirm past infection vary and the immunological response after mild COVID-19 is still not well defined., Method: 314 HCWs were recruited from a Swedish Infectious Diseases clinic caring for COVID-19 patients. IgG antibodies were measured using two commercial assays (Abbot Architect nucleocapsid (N)-assay and YHLO iFlash-1800 N and spike (S)-assays) at five time-points, from March 2020 to January 2021, covering two pandemic waves. Seroprevalence was assessed in matched blood donors at three time-points. More extensive analyses were performed in 190 HCWs in September/October 2020, including two additional IgG-assays (DiaSorin LiaisonXL S1/S2 and Abbot Architect receptor-binding domain (RBD)-assays), neutralizing antibodies (NAbs), and CD4 + T-cell reactivity using an in-house developed in vitro whole-blood assay based on flow cytometric detection of activated cells after stimulation with Spike S1-subunit or Spike, Membrane and Nucleocapsid (SMN) overlapping peptide pools., Findings: Seroprevalence was higher among HCWs compared to sex and age-matched blood donors at all time-points. Seropositivity increased from 6.4% to 16.3% among HCWs between May 2020 and January 2021, compared to 3.6% to 11.9% among blood donors. We found significant correlations and high levels of agreement between NAbs and all four commercial IgG-assays. At 200-300 days post PCR-verified infection, there was a wide variation in sensitivity between the commercial IgG-assays, ranging from <30% in the N-assay to >90% in the RBD-assay. There was only moderate agreement between NAbs and CD4 + T-cell reactivity to S1 or SMN. Pre-existing CD4 + T-cell reactivity was present in similar proportions among HCW who subsequently became infected and those that did not., Conclusions: HCWs in COVID-19 patient care in Sweden have been infected with SARS-CoV-2 at a higher rate compared to blood donors. We demonstrate substantial variation between different IgG-assays and propose that multiple serological targets should be used to verify past infection. Our data suggest that CD4 + T-cell reactivity is not a suitable measure of past infection and does not reliably indicate protection from infection in naive individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marklund, Leach, Nyström, Lundgren, Liljeqvist, Nilsson, Yilmaz, Andersson, Bemark and Gisslén.)

When speaking to infants, parents typically use infant-directed speech, a speech register that in several aspects differs from that directed to adults. Vowel hyperarticulation, that is, extreme articulation of vowels, is one characteristic sometimes found in infant-directed speech, and it has been suggested that there exists a relationship between how much vowel hyperarticulation parents use when speaking to their infant and infant language development. In this study, the relationship between parent vowel hyperarticulation and phonetic complexity of infant vocalizations is investigated. Previous research has shown that on the level of subject means, a positive correlational relationship exists. However, the previous findings do not provide information about the directionality of that relationship. In this study the relationship is investigated on a conversational turn level, which makes it possible to draw conclusions on whether the behavior of the infant is impacting the parent, the behavior of the parent is impacting the infant, or both. Parent vowel hyperarticulation was quantified using the vhh-index, a measure that allows vowel hyperarticulation to be estimated for individual vowel tokens. Phonetic complexity of infant vocalizations was calculated using the Word Complexity Measure for Swedish. Findings were unexpected in that a negative relationship was found between parent vowel hyperarticulation and phonetic complexity of the immediately following infant vocalization. Directionality was suggested by the fact that no such relationship was found between infant phonetic complexity and vowel hyperarticulation of the immediately following parent utterance. A potential explanation for these results is that high degrees of vowel hyperarticulation either provide, or co-occur with, large amounts of phonetic and/or linguistic information, which may occupy processing resources to an extent that affects production of the next vocalization., (Copyright © 2021 Marklund, Marklund and Gustavsson.)

Extreme or exaggerated articulation of vowels, or vowel hyperarticulation, is a characteristic commonly found in infant-directed speech (IDS). High degrees of vowel hyperarticulation in parent IDS has been tied to better speech sound category development and bigger vocabulary size in infants. In the present study, the relationship between vowel hyperarticulation in Swedish IDS to 12-month-old and phonetic complexity of infant vocalizations is investigated. Articulatory adaptation toward hyperarticulation is quantified as difference in vowel space area between IDS and adult-directed speech (ADS). Phonetic complexity is estimated using the Word Complexity Measure for Swedish (WCM-SE). The results show that vowels in IDS was more hyperarticulated than vowels in ADS, and that parents' articulatory adaptation in terms of hyperarticulation correlates with phonetic complexity of infant vocalizations. This can be explained either by the parents' articulatory behavior impacting the infants' vocalization behavior, the infants' social and communicative cues eliciting hyperarticulation in the parents' speech, or the two variables being impacted by a third, underlying variable such as parents' general communicative adaptiveness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marklund, Marklund and Gustavsson.)

The amplitude of the event-related N1 wave decreases with repeated stimulation. This repetition-attenuation has not previously been investigated in response to variable auditory stimuli, nor has the relative impact of acoustic vs. perceptual category repetition been studied. In the present study, N1 repetition-attenuation was investigated for speech and spectrally rotated speech with varying degrees of acoustic and perceptual category variation. In the speech condition, participants ( n = 19) listened to stimulus trains consisting of either the same vowel exemplar (no variability condition), different exemplars of the same vowel (low variability condition), or different exemplars of two different vowels (high variability condition). In the rotated speech condition, the spectrally rotated counterparts of the vowels were presented. Findings show N1 repetition-attenuation in the face of acoustic and perceptual category variability, but no impact of the degree of variability on the degree of N1 attenuation. Speech stimuli resulted in less attenuation than the acoustically matched non-speech stimuli, which is in line with previous findings. It remains unclear if the attenuation of the N1 wave is reduced as a result of stimuli being perceived as belonging to perceptual categories or as a result of some other characteristic of speech., (Copyright © 2020 Marklund, Gustavsson, Kallioinen and Schwarz.)

Background: To accurately interpret COVID-19 seroprevalence surveys, knowledge of serum-IgG responses to SARS-CoV-2 with a better understanding of patients who do not seroconvert, is imperative. This study aimed to describe serum-IgG responses to SARS-CoV-2 in a cohort of patients with both severe and mild COVID-19, including extended studies of patients who remained seronegative more than 90 days post symptom onset., Methods: SARS-CoV-2-specific IgG antibody levels were quantified using two clinically validated and widely used commercial serological assays (Architect, Abbott Laboratories and iFlash 1800, YHLO), detecting antibodies against the spike and nucleocapsid proteins., Results: Forty-seven patients (mean age 49 years, 38% female) were included. All (15/15) patients with severe symptoms and 29/32 (90.6%) patients with mild symptoms of COVID-19 developed SARS-CoV-2-specific IgG antibodies in serum. Time to seroconversion was significantly shorter (median 11 vs. 22 days, P = 0.04) in patients with severe compared to mild symptoms. Of the three patients without detectable IgG-responses after >90 days, all had detectable virus-neutralizing antibodies and in two, spike-protein receptor binding domain-specific IgG was detected with an in-house assay. Antibody titers were preserved during follow-up and all patients who seroconverted, irrespective of the severity of symptoms, still had detectable IgG levels >75 days post symptom onset., Conclusions: Patients with severe COVID-19 both seroconvert earlier and develop higher concentrations of SARS-CoV-2-specific IgG than patients with mild symptoms. Of those patients who not develop detectable IgG antibodies, all have detectable virus-neutralizing antibodies, suggesting immunity. Our results showing that not all COVID-19 patients develop detectable IgG using two validated commercial clinical methods, even over time, are vital for the interpretation of COVID-19 seroprevalence surveys., Competing Interests: The authors have declared that no competing interests exist.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA 1-5 . Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 μs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

During the first year of life, infants shift their focus in speech perception from acoustic to linguistic information. This perceptual reorganization is related to exposure, and a direct relation has previously been demonstrated between amount of daily language exposure and mismatch response (MMR) amplitude to a native consonant contrast at around one year of age. The present study investigates the same relation between amount of speech exposure and MMR amplitude to a native vowel contrast at four to eight months of age. Importantly, the present study uses spectrally rotated speech in an effort to take general neural maturation into account. The amplitude of the part of the MMR that is tied specifically to speech processing correlates with amount of daily speech exposure, as estimated using the LENA system., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level., Competing Interests: The authors have declared that no competing interests exist.

Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: During the last decades, healthcare-associated genotypes of methicillin-resistant Staphylococcus epidermidis (HA-MRSE) have been established as important opportunistic pathogens. However, data on potential reservoirs on HA-MRSE is limited. The aim of the present study was to investigate the dynamics and to which extent HA-MRSE genotypes colonize patients, healthcare workers (HCWs) and the environment in an intensive care unit (ICU)., Methods: Over 12 months in 2006-2007, swab samples were obtained from patients admitted directly from the community to the ICU and patients transferred from a referral hospital, as well as from HCWs, and the ICU environment. Patients were sampled every third day during hospitalization. Antibiotic susceptibility testing was performed according to EUCAST guidelines. Pulsed-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relatedness of a subset of MRSE isolates., Results: We identified 620 MRSE isolates from 570 cultures obtained from 37 HCWs, 14 patients, and 14 environmental surfaces in the ICU. HA-MRSE genotypes were identified at admission in only one of the nine patients admitted directly from the community, of which the majority subsequently were colonized by HA-MRSE genotypes within 3 days during hospitalization. Almost all (89%) of HCWs were nasal carriers of HA-MRSE genotypes. Similarly, a significant proportion of patients transferred from the referral hospital and fomites in the ICU were widely colonized with HA-MRSE genotypes., Conclusions: Patients transferred from a referral hospital, HCWs, and the hospital environment serve as important reservoirs for HA-MRSE. These observations highlight the need for implementation of effective infection prevention and control measures aiming at reducing HA-MRSE transmission in the healthcare setting.

The extent to which protein structures are preserved on transfer from solution to gas phase is a central question for native mass spectrometry. Here we compare the collision cross sections (Ω) of a wide range of different proteins and protein complexes (15-500 kDa) with their corresponding Stokes radii (RS). Using these methods, we find that Ω and RS are well correlated, implying overall preservation of protein structure in the gas phase. Accounting for protein hydration, a scaling term is required to bring Ω and RS into parity. Interestingly, the magnitude of this scaling term agrees almost entirely with the drag factor proposed by Millikan. RS were then compared with various different predicted values of Ω taken from their atomic coordinates. We find that many of the approaches used to obtained Ω from atomic coordinates miscalculate the physical sizes of the proteins in solution by as much as 20%. Rescaling of Ω estimated from atomic coordinates may therefore seem appropriate as a general method to bring theoretical values in line with those observed in solution.

We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI-Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size-growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media.