IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Structural basis of aggregate binding by the AAA+ disaggregase ClpG.

Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a ß-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

A metabolite binding protein moonlights as a bile-responsive chaperone.

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.

Musculoskeletal Pain, Physical Activity, Muscle Mass, and Mortality in Older Adults: Results from the Korean Longitudinal Study on Health and Aging (KLoSHA).

Background and objectives: Musculoskeletal (MSK) pain significantly impacts physical activity and quality of life in older adults, potentially influencing mortality. This study explored the relationship between MSK pain, physical activity, muscle mass, and mortality among older adults. Material and Methods: We studied 1000 participants in the Korean Longitudinal Study on Health and Aging (KLoSHA), a prospective, population-based cohort study of people aged 65 years or older. Survival status was tracked over a 5-year period. Correlations between low back pain (LBP), knee pain, regular exercise, appendicular skeletal muscle mass (ASM), and other variables were analyzed. Logistic regression analyses were used to identify independent risk factors for mortality. Results: Of the total participants, 829 (82.9%) survived over a 5-year period. Survivors tended to be younger, had a higher BMI, and were more active in regular exercise. In contrast, non-survivors exhibited a higher prevalence of both LBP and knee pain, along with increased instances of multiple MSK pains. Lower ASM correlated moderately with LBP and knee pain, whereas higher ASM was associated with regular exercise. There was a moderate correlation between LBP and knee pain, both of which were associated with a lack of regular exercise. Age, sex, ASM, and regular exercise were significant predictors, even though MSK pain itself did not directly predict all-cause mortality. Conclusions: This study demonstrated the independent association between ASM, regular exercise, and mortality. Although MSK pain did not directly correlate with all-cause mortality, the non-survivor group had higher levels of both single and multiple MSK pains. Recognizing the interplay of MSK pain, physical activity, and muscle mass for older adults, the research underscores the need for holistic strategies to enhance health outcomes in older individuals with MSK pain.

Aging: All roads lead to mitochondria.

Mitochondria were described as early as 1890 as ubiquitous intracellular structures by Ernster and Schatz (1981) [1]. Since then, the accretion of knowledge in the past century has revealed much of the molecular details of mitochondria, ranging from mitochondrial origin, structure, metabolism, genetics, and signaling, and their implications in health and disease. We now know that mitochondria are remarkably multifunctional and deeply intertwined with many vital cellular processes. They are quasi-self organelles that still possess remnants of its bacterial ancestry, including an independent genome. The mitochondrial free radical theory of aging (MFRTA), which postulated that aging is a product of oxidative damage to mitochondrial DNA, provided a conceptual framework that put mitochondria on the map of aging research. However, several studies have more recently challenged the general validity of the theory, favoring novel ideas based on emerging evidence to understand how mitochondria contribute to aging and age-related diseases. One prominent topic of investigation lies on the fact that mitochondria are not only production sites for bioenergetics and macromolecules, but also regulatory hubs that communicate and coordinate many vital physiological processes at the cellular and organismal level. The bi-directional communication and coordination between the co-evolved mitochondrial and nuclear genomes is especially interesting in terms of cellular regulation. Mitochondria are dynamic and adaptive, rendering their function sensitive to cellular context. Tissues with high energy demands, such as the brain, seem to be uniquely affected by age-dependent mitochondrial dysfunction, providing a foundation for the development of novel mitochondrial-based therapeutics and diagnostics.

Comparable humoral and cellular immunity against Omicron variant BA.4/5 of once-boosted BA.1/2 convalescents and twice-boosted COVID-19-naïve individuals.

The fourth vaccination dose confers additional protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with no prior coronavirus disease-19 (COVID-19). However, its immunological benefit against currently circulating BA.4/5 is unclear in individuals who have received a booster shot and been infected with Omicron variant BA.1/2. We analyzed immune responses in whom had been boosted once and did not have COVID-19 (n = 16), boosted once and had COVID-19 when BA.1/2 was dominant in Korea (Hybrid-6M group, n = 27), and boosted twice and did not have COVID-19 (Vx4 group, n = 15). Antibody binding activities against RBDo BA.1 and RBDo BA.4/5 , antigen-specific memory CD4+ and CD8+ T-cell responses against BA.4/5, and B-cell responses against SARS-CoV-2 wild-type did not differ statistically between the Hybrid-6M and Vx4 groups. The humoral and cellular immune responses of the Hybrid-6M group against BA.4/5 were comparable to those of the Vx4 group. Individuals who had been boosted and had an Omicron infection in early 2022 may not have high priority for an additional vaccination.

Tracking antigen-specific TCR clonotypes in SARS-CoV-2 infection reveals distinct severity trajectories.

Despite the importance of antigen-specific T cells in infectious disease, characterizing and tracking clonally amplified T cells during the progression of a patient's symptoms remain unclear. Here, we performed a longitudinal, in-depth single-cell multiomics analysis of samples from asymptomatic, mild, usual severe, and delayed severe patients of SARS-CoV-2 infection. Our in-depth analysis revealed that hyperactive or improper T-cell responses were more aggressive in delayed severe patients. Interestingly, tracking of antigen-specific T-cell receptor (TCR) clonotypes along the developmental trajectory indicated an attenuation in functional T cells upon severity. In addition, increased glycolysis and interleukin-6 signaling in the cytotoxic T cells were markedly distinct in delayed severe patients compared to usual severe patients, particularly in the middle and late stages of infection. Tracking B-cell receptor clonotypes also revealed distinct transitions and somatic hypermutations within B cells across different levels of disease severity. Our results suggest that single-cell TCR clonotype tracking can distinguish the severity of patients through immunological hallmarks, leading to a better understanding of the severity differences in and improving the management of infectious diseases by analyzing the dynamics of immune responses over time.

A recently isolated human commensal Escherichia coli ST10 clone member mediates enhanced thermotolerance and tetrathionate respiration on a P1 phage-derived IncY plasmid.

The ubiquitous human commensal Escherichia coli has been well investigated through its model representative E. coli K-12. In this work, we initially characterized E. coli Fec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared to E. coli K-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition, E. coli Fec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpGGI of TLPQC constitutes a major determinant of the thermotolerance of E. coli Fec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpGGI-Fec10 compared to the nearly identical Pseudomonas aeruginosa ClpGGI-SG17M. Furthermore, we noted a unique contribution of ClpGGI-Fec10 to the exquisite thermotolerance of E. coli Fec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensal E. coli isolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.

Broad humoral and cellular immunity elicited by one-dose mRNA vaccination 18 months after SARS-CoV-2 infection.

BACKGROUND: Practical guidance is needed regarding the vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals in resource-limited countries. It includes the number of vaccine doses that should be given to unvaccinated patients who experienced COVID-19 early in the pandemic. METHODS: We recruited COVID-19 convalescent individuals who received one or two doses of an mRNA vaccine within 6 or around 18 months after a diagnosis of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. Their samples were assessed for IgG-binding or neutralizing activity and cell-mediated immune responses against SARS-CoV-2 wild-type and variants of concern. RESULTS: A total of 43 COVID-19 convalescent individuals were analyzed in the present study. The results showed that humoral and cellular immune responses against SARS-CoV-2 wild-type and variants of concern, including the Omicron variant, were comparable among patients vaccinated within 6 versus around 18 months. A second dose of vaccine did not significantly increase immune responses. CONCLUSION: One dose of mRNA vaccine should be considered sufficient to elicit a broad immune response even around 18 months after a COVID-19 diagnosis.

Tumor-associated myeloid cells provide critical support for T-ALL.

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells, from the thymic tumor microenvironment support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in 2 distinct mouse models of T-ALL and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of antitumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid-depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

Global spliceosome activity regulates entry into cellular senescence.

Cellular senescence is a state of permanent growth arrest that can ultimately contribute to aging. Senescence can be induced by various stressors and is associated with a myriad of cellular functions and phenotypic markers. Alternative splicing is emerging as a critical contributor to senescence and aging. However, it is unclear how the composition and function of the spliceosome are involved in senescence. Here, using replicative and oxidative stress-induced senescence models in primary human fibroblasts, we report a common shift in the expression of 58 spliceosomal genes at the pre-senescence stage, prior to the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity. Spliceosomal perturbation, induced by pharmacologic and genetic inhibition of splicesomal genes, triggered cells to enter senescence, suggesting a key role as a gatekeeper. Association analysis of transcription factors based on the 58 splicesomal genes revealed Sp1 as a key regulator of senescence entry. Indeed, Sp1 depletion suppressed the expression of downstream spliceosomal genes (HNRNPA3, SRSF7, and SRSF4) and effectively induced senescence. These results indicate that spliceosomal gene sets, rather than a single spliceosomal gene, regulate the early transition into senescence prior to SA-ß-gal expression. Furthermore, our study provides a spliceosome signature that may be used as an early senescence marker.

Longitudinal Analysis of Human Memory T-Cell Response According to the Severity of Illness up to 8 Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: Understanding the memory T-cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for assessing the longevity of protective immunity after SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination. However, the longitudinal memory T-cell response up to 8 months post-symptom onset (PSO) according to the severity of illness is unknown. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with COVID-19 who experienced asymptomatic, mild, or severe illness at 2, 5, and 8 months PSO. SARS-CoV-2 spike, nucleocapsid, and membrane protein-stimulated PBMCs were subjected to flow cytometry analysis. RESULTS: A total of 24 patients (7 asymptomatic, 9 with mild disease, and 8 with severe disease) and 6 healthy volunteers were analyzed. SARS-CoV-2-specific OX40+CD137+CD4+ T cells and CD69+CD137+CD8+ T cells persisted at 8 months PSO. Also, antigen-specific cytokine-producing or polyfunctional CD4+ T cells were maintained for up to 8 months PSO. Memory CD4+ T-cell responses tended to be greater in patients who had severe illness than in those with mild or asymptomatic disease. CONCLUSIONS: Memory response to SARS-CoV-2, based on the frequency and functionality, persists for 8 months PSO. Further investigations involving its longevity and protective effect from reinfection are warranted.

MOTS-c: A Mitochondrial-Encoded Regulator of the Nucleus.

Mitochondria are increasingly being recognized as information hubs that sense cellular changes and transmit messages to other cellular components, such as the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes. Nonetheless, the interaction between mitochondria and the nucleus is of special interest because they both host part of the cellular genome. Thus, the communication between genome-bearing organelles would likely include gene expression regulation. Multiple nuclear-encoded proteins have been known to regulate mitochondrial gene expression. On the contrary, no mitochondrial-encoded factors are known to actively regulate nuclear gene expression. MOTS-c (mitochondrial open reading frame of the 12S ribosomal RNA type-c) is a recently identified peptide encoded within the mitochondrial 12S ribosomal RNA gene that has metabolic functions. Notably, MOTS-c can translocate to the nucleus upon metabolic stress (e.g., glucose restriction and oxidative stress) and directly regulate adaptive nuclear gene expression to promote cellular homeostasis. It is hypothesized that cellular fitness requires the coevolved mitonuclear genomes to coordinate adaptive responses using gene-encoded factors that cross-regulate the opposite genome. This suggests that cellular gene expression requires the bipartite split genomes to operate as a unified system, rather than the nucleus being the sole master regulator.

Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria.

AAA+ disaggregases solubilize aggregated proteins and confer heat tolerance to cells. Their disaggregation activities crucially depend on partner proteins, which target the AAA+ disaggregases to protein aggregates while concurrently stimulating their ATPase activities. Here, we report on two potent ClpG disaggregase homologs acquired through horizontal gene transfer by the species Pseudomonas aeruginosa and subsequently abundant P. aeruginosa clone C. ClpG exhibits high, stand-alone disaggregation potential without involving any partner cooperation. Specific molecular features, including high basal ATPase activity, a unique aggregate binding domain, and almost exclusive expression in stationary phase distinguish ClpG from other AAA+ disaggregases. Consequently, ClpG largely contributes to heat tolerance of P. aeruginosa primarily in stationary phase and boosts heat resistance 100-fold when expressed in Escherichia coli This qualifies ClpG as a potential persistence and virulence factor in P. aeruginosa.

Investigation on the Printed CNT-Film-Based Electrochemical Sensor for Detection of Liquid Chemicals.

We studied electrochemical sensors using printed carbon nanotubes (CNT) film on a polyethylene telephtalate (PET) substrate. The mechanical stability of the printed CNT film (PCF) was confirmed by using bending and Scotch tape tests. In order to determine the optimum sensor structure, a resistance-type PCF sensor (R-type PCF sensor) and a comb-type PCF sensor (C-type PCF sensor) were fabricated and compared using a diluted NH3 droplet with various concentrations. The magnitude of response, response time, sensitivity, linearity, and limit of detection (LOD) were compared, and it was concluded that C-type PCF sensor has superior performance. In addition, the feasibility of PCF electrochemical sensor was investigated using 12 kinds of hazardous and noxious substances (HNS). The detection mechanism and selectivity of the PCF sensor are discussed.

Development of Therapeutic Antibodies and Modulating the Characteristics of Therapeutic Antibodies to Maximize the Therapeutic Efficacy.

Monoclonal antibodies (mAb) have been used as therapeutic agents for various diseases, and immunoglobulin G (IgG) is mainly used among antibody isotypes due to its structural and functional properties. So far, regardless of the purpose of the therapeutic antibody, wildtype IgG has been mainly used, but recently, the engineered antibodies with various strategies according to the role of the therapeutic antibody have been used to maximize the therapeutic efficacy. In this review paper, first, the overall structural features and functional characteristics of antibody IgG, second, the old and new techniques for antibody discovery, and finally, several antibody engineering strategies for maximizing therapeutic efficacy according to the role of a therapeutic antibody will be introduced.

Mitochondrial-derived peptides in energy metabolism.

Mitochondrial-derived peptides (MDPs) are small bioactive peptides encoded by short open-reading frames (sORF) in mitochondrial DNA that do not necessarily have traditional hallmarks of protein-coding genes. To date, eight MDPs have been identified, all of which have been shown to have various cyto- or metaboloprotective properties. The 12S ribosomal RNA (MT-RNR1) gene harbors the sequence for MOTS-c, whereas the other seven MDPs [humanin and small humanin-like peptides (SHLP) 1-6] are encoded by the 16S ribosomal RNA gene. Here, we review the evidence that endogenous MDPs are sensitive to changes in metabolism, showing that metabolic conditions like obesity, diabetes, and aging are associated with lower circulating MDPs, whereas in humans muscle MDP expression is upregulated in response to stress that perturbs the mitochondria like exercise, some mtDNA mutation-associated diseases, and healthy aging, which potentially suggests a tissue-specific response aimed at restoring cellular or mitochondrial homeostasis. Consistent with this, treatment of rodents with humanin, MOTS-c, and SHLP2 can enhance insulin sensitivity and offer protection against a range of age-associated metabolic disorders. Furthermore, assessing how mtDNA variants alter the functions of MDPs is beginning to provide evidence that MDPs are metabolic signal transducers in humans. Taken together, MDPs appear to form an important aspect of a retrograde signaling network that communicates mitochondrial status with the wider cell and to distal tissues to modulate adaptative responses to metabolic stress. It remains to be fully determined whether the metaboloprotective properties of MDPs can be harnessed into therapies for metabolic disease.

Mitonuclear genomics and aging.

Our cells operate based on two distinct genomes that are enclosed in the nucleus and mitochondria. The mitochondrial genome presumably originates from endosymbiotic bacteria. With time, a large portion of the original genes in the bacterial genome is considered to have been lost or transferred to the nuclear genome, leaving a reduced 16.5 Kb circular mitochondrial DNA (mtDNA). Traditionally only 37 genes, including 13 proteins, were thought to be encoded within mtDNA, its genetic repertoire is expanding with the identification of mitochondrial-derived peptides (MDPs). The biology of aging has been largely unveiled to be regulated by genes that are encoded in the nuclear genome, whereas the mitochondrial genome remained more cryptic. However, recent studies position mitochondria and mtDNA as an important counterpart to the nuclear genome, whereby the two organelles constantly regulate each other. Thus, the genomic network that regulates lifespan and/or healthspan is likely constituted by two unique, yet co-evolved, genomes. Here, we will discuss aspects of mitochondrial biology, especially mitochondrial communication that may add substantial momentum to aging research by accounting for both mitonuclear genomes to more comprehensively and inclusively map the genetic and molecular networks that govern aging and age-related diseases.