Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Masqueraders Around Disaster: Clinical Features of Scrub Typhus in Fukushima, Japan.

Background: Scrub typhus (ST) is endemic in Fukushima, with the largest number of cases reported in Japan from 2009 to 2010. Although ST is highly treatable, its atypical clinical presentation impedes diagnosis, causing delays in treatment. Methods: We review the clinical features of ST in adults from 2008 to 2017 at Ohta Nishinouchi General Hospital in Fukushima, Japan. Results: Fifty-five cases (serotype Karp 24, Irie/Kawasaki 21, Hirano/Kuroki 10) of ST were confirmed via serology based on elevated immunoglobulin (Ig)M and IgG and polymerase chain reaction positivity of eschar samples. The mean age was 69 years, and 64% were female. The case fatality rate was 1.8% (1/55). Approximately 70% of cases (38/55) were not diagnosed as ST upon the initial clinic visit. Inappropriate use of antibiotics was identified in 22% of cases (12/55). In terms of atypical clinical features, 1 or more of the manifestations, fever, rash, and eschar, was absent in 31% of cases (17/55). Approximately 11% of cases presented without eschar (6/55; Karp 1, Irie/Kawasaki 1, Hirano/Kuroki 4). Moreover, severe complications were observed with shock and disseminated intravascular coagulation in 7% of cases (4/55), Thus, while 53% of cases presented with the typical triad (29/55), unusual complications and atypical features occurred in 40% (22/55). Conclusions: Diagnosis of ST becomes clinically challenging in the absence of typical features. In Fukushima, an endemic area of ST, an atypical presentation involving multisystem disease is common.

Driving Behavior during Left-Turn Maneuvers at Intersections on Left-Hand Traffic Roads.

Understanding left-turn vehicle-pedestrian accident mechanisms is critical for developing accident-prevention systems. This study aims to clarify the features of driver behavior focusing on drivers' gaze, vehicle speed, and time to collision (TTC) during left turns at intersections on left-hand traffic roads. Herein, experiments with a sedan and light-duty truck (< 7.5 tons GVW) are conducted under four conditions: no pedestrian dummy (No-P), near-side pedestrian dummy (Near-P), far-side pedestrian dummy (Far-P) and near-and-far side pedestrian dummies (NF-P). For NF-P, sedans have a significantly shorter gaze time for left-side mirrors compared with light-duty trucks. The light-duty truck's average speed at the initial line to the intersection (L1) and pedestrian crossing line (L0) is significantly lower than the sedan's under No-P, Near-P, and NF-P conditions, without any significant difference between any two conditions. The TTC for sedans is significantly shorter than that for trucks with near-side pedestrians (Near-P and NF-P) and far-side pedestrians in Far-P. These insights can contribute to the ongoing development of accident-prevention safety systems for left-turning maneuvers at intersections.

Senescence of human pancreatic beta cells enhances functional maturation through chromatin reorganization and promotes interferon responsiveness.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

Determining the Compaction State of Genes Using DNA FISH.

DNA fluorescence in situ hybridization (FISH) enables the visualization of chromatin architecture and the interactions between genomic loci at a single-cell level, complementary to genome-wide methods such as Hi-C. DNA FISH uses fluorescent-labeled DNA probes targeted to the loci of interest, allowing for the analysis of their spatial positioning and proximity with microscopy. Here, we describe an optimized experimental procedure for DNA FISH, from probe design and sample preparation through imaging and image quantification. This protocol can be readily applied to querying the spatial positioning of genomic loci of interest.

Text mining for contexts and relationships in cancer genomics literature.

MOTIVATION: Scientific advances build on the findings of existing research. The 2001 publication of the human genome has led to the production of huge volumes of literature exploring the context-specific functions and interactions of genes. Technology is needed to perform large-scale text mining of research papers to extract the reported actions of genes in specific experimental contexts and cell states, such as cancer, thereby facilitating the design of new therapeutic strategies. RESULTS: We present a new corpus and Text Mining methodology that can accurately identify and extract the most important details of cancer genomics experiments from biomedical texts. We build a Named Entity Recognition model that accurately extracts relevant experiment details from PubMed abstract text, and a second model that identifies the relationships between them. This system outperforms earlier models and enables the analysis of gene function in diverse and dynamically evolving experimental contexts. AVAILABILITY AND IMPLEMENTATION: Code and data are available here: https://github.com/cambridgeltl/functional-genomics-ie.

Infectious virus shedding duration reflects secretory IgA antibody response latency after SARS-CoV-2 infection.

Infectious virus shedding from individuals infected with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is used to estimate human-to-human transmission risk. Control of SARS-CoV-2 transmission requires identifying the immune correlates that protect infectious virus shedding. Mucosal immunity prevents infection by SARS-CoV-2, which replicates in the respiratory epithelium and spreads rapidly to other hosts. However, whether mucosal immunity prevents the shedding of the infectious virus in SARS-CoV-2-infected individuals is unknown. We examined the relationship between viral RNA shedding dynamics, duration of infectious virus shedding, and mucosal antibody responses during SARS-CoV-2 infection. Anti-spike secretory IgA antibodies (S-IgA) reduced viral RNA load and infectivity more than anti-spike IgG/IgA antibodies in infected nasopharyngeal samples. Compared with the IgG/IgA response, the anti-spike S-IgA post-infection responses affected the viral RNA shedding dynamics and predicted the duration of infectious virus shedding regardless of the immune history. These findings highlight the importance of anti-spike S-IgA responses in individuals infected with SARS-CoV-2 for preventing infectious virus shedding and SARS-CoV-2 transmission. Developing medical countermeasures to shorten S-IgA response time may help control human-to-human transmission of SARS-CoV-2 infection and prevent future respiratory virus pandemics.

Beyond SAHF: An integrative view of chromatin compartmentalization during senescence.

Cellular senescence, a persistent form of cell cycle arrest, has been linked to the formation of heterochromatic foci, accompanied by additional concentric epigenetic layers. However, senescence is a highly heterogeneous phenotype, and the formation of these structures is context dependent. Recent developments in the understanding of the high-order chromatin organization have opened new avenues for contextualizing the nuclear and chromatin phenotypes of senescence. Oncogene-induced senescence displays prominent foci and typically exhibits increased chromatin compartmentalization, based on the chromosome conformation assays, as marked by increased transcompaction and segregation of the heterochromatin and euchromatin. However, other types of senescence (e.g., replicative senescence) exhibit comparatively lower levels of compartmentalization. Thus, a more integrative view of the global rearrangement of the chromatin architecture that occurs during senescence is emerging, with potential functional implications for the heterogeneity of the senescence phenotype.

Cellular senescence: beneficial, harmful, and highly complex.

The contribution of cellular senescence to a diverse range of biological processes, including normal physiology, ageing, and pathology were long overlooked but have now taken centre stage. In this Editorial, we will briefly outline the review and original work articles contained in The FEBS Journal's Special Issue on Senescence in Ageing and Disease. It is beginning to be appreciated that senescent cells can exert both beneficial and adverse effects following tissue injury. Additionally, while these cells play critical roles for maintaining a healthy physiology, they also promote ageing and certain pathological conditions (including developmental disorders). Progress has been made in re-defining and identifying senescent cells, especially in slow-proliferating or terminally differentiated tissues, such as the brain and cardiovascular system. Novel approaches and techniques for isolating senescent cells will greatly increase our appreciation for senescent properties in tissues. The inter-organ communication between senescent cells and other residents of the tissue microenvironment, via the senescence-associated secretory phenotype (SASP), is a focus of several reviews in this Special Issue. The importance of the SASP in promoting tumour development and the evolution of SARS CoV-2 variants is also highlighted. In one of the two original articles included in the issue, the impact of dietary macronutrients and the presence of senescent cells in mice is investigated. Lastly, we continue to deepen our understanding on the use of senolytics and senomorphics to specifically target senescent cells or their secreted components, respectively, which is discussed in several of the reviews included here.

Contamination of Blood Cultures From Arterial Catheters and Peripheral Venipuncture in Critically Ill Patients: A Prospective Multicenter Diagnostic Study.

BACKGROUND: Collecting blood cultures from indwelling arterial catheters is an attractive option in critically ill adult patients when peripheral venipuncture is difficult. However, whether the contamination proportion of blood cultures from arterial catheters is acceptable compared with that from venipuncture is inconclusive. RESEARCH QUESTION: Is contamination of blood cultures from arterial catheters noninferior to that from venipuncture in critically ill adult patients with suspected bloodstream infection? STUDY DESIGN AND METHODS: In this multicenter prospective diagnostic study conducted at five hospitals, we enrolled episodes of paired blood culture collection, each set consisting of blood drawn from an arterial catheter and another by venipuncture, were obtained from critically ill adult patients with cilinical indication. The primary measure was the proportion of contamination, defined as the number of false-positive results relative to the total number of procedures done. The reference standard for true bloodstream infection was blinded assessment by infectious disease specialists. We examined the noninferiority hypothesis that the contamination proportion of blood cultures from arterial catheters did not exceed that from venipuncture by 2.0%. RESULTS: Of 1,655 episodes of blood culture from December 2018 to July 2021, 590 paired blood culture episodes were enrolled, and 41 of the 590 episodes (6.9%) produced a true bloodstream infection. In blood cultures from arterial catheters, 33 of 590 (6.0%) were positive, and two of 590 (0.3%) were contaminated; in venipuncture, 36 of 590 (6.1%) were positive, and four of 590 (0.7%) were contaminated. The estimated difference in contamination proportion (arterial catheter - venipuncture) was -0.3% (upper limit of one-sided 95% CI, +0.3%). The upper limit of the 95% CI did not exceed the predefined margin of +2.0%, establishing noninferiority (P for noninferiority < .001). INTERPRETATION: Obtaining blood cultures from arterial catheters is an acceptable alternative to venipuncture in critically ill patients. CLINICAL TRIAL REGISTRATION: University Hospital Medical Information Network Center (UMIN-CTR); No.: UMIN000035392; URL: https://center6.umin.ac.jp/.

Association between hospital internal medicine physician workforce and patient admissions during the COVID-19 pandemic in Japan.

BACKGROUND: Hospital physician workforce in Japan is the lowest among developed countries. Many patients with novel coronavirus disease 2019 (COVID-19) with high risk of mortality could not be hospitalized during case surges in Japan and only about 5% of total acute care beds were used as COVID-19 beds nationwide. However, the relationship between the number of hospital physicians and patient admissions remains unclear. Thus, we aimed to evaluate this relationship in areas with the highest incidences during the surges. METHODS: Data collection was performed for teaching hospitals accredited with the specialty of internal medicine in three prefectures which experienced the highest COVID-19 incidences in Japan (Tokyo, Osaka, Okinawa). Association was examined between the number of full-time physicians (internal medicine staff physicians and residents) and admissions of internal medicine patients through ambulance transport from April 2020 to March 2021. Analysis was conducted separately for community hospitals and university hospitals because the latter have roles as research institutions in Japan. Community hospitals included private, public, and semi-public hospitals. RESULTS: Of 117 teaching hospitals in three prefectures, data from 108 teaching hospitals (83 community hospitals and 25 university hospitals) were available. A total of 102,400 internal medicine patients were admitted to these hospitals during the one-year period. Private hospitals received the greatest mean number of patient admissions (290 per 100 beds), followed by public hospitals (227) and semi-public hospitals (201), and university hospitals (94). Among community hospitals, a higher number of resident physicians per 100 beds was significantly associated with a greater number of patient admissions per 100 beds with beta coefficient of 11.6 (95% CI, 1.5 to 21.2, p = 0.025) admissions by one physician increase per 100 beds. There was no such association among university hospitals. CONCLUSIONS: Community hospitals with many resident physicians accepted more internal medicine admissions through ambulance transport during the COVID-19 pandemic. An effective policy to counter physician shortage in hospitals in Japan may be to increase internal medicine resident physicians among community hospitals to save more lives.

Bioprinting microporous functional living materials from protein-based core-shell microgels.

Living materials bring together material science and biology to allow the engineering and augmenting of living systems with novel functionalities. Bioprinting promises accurate control over the formation of such complex materials through programmable deposition of cells in soft materials, but current approaches had limited success in fine-tuning cell microenvironments while generating robust macroscopic morphologies. Here, we address this challenge through the use of core-shell microgel ink to decouple cell microenvironments from the structural shell for further processing. Cells are microfluidically immobilized in the viscous core that can promote the formation of both microbial populations and mammalian cellular spheroids, followed by interparticle annealing to give covalently stabilized functional scaffolds with controlled microporosity. The results show that the core-shell strategy mitigates cell leakage while affording a favorable environment for cell culture. Furthermore, we demonstrate that different microbial consortia can be printed into scaffolds for a range of applications. By compartmentalizing microbial consortia in separate microgels, the collective bioprocessing capability of the scaffold is significantly enhanced, shedding light on strategies to augment living materials with bioprocessing capabilities.

p53 in senescence - it's a marathon, not a sprint.

The tumour suppressor p53, a stress-responsive transcription factor, plays a central role in cellular senescence. The role of p53 in senescence-associated stable proliferative arrest has been extensively studied. However, increasing evidence indicates that p53 also modulates the ability of senescent cells to produce and secrete diverse bioactive factors (collectively called the senescence-associated secretory phenotype, SASP). Senescence has been linked with both physiological and pathological conditions, the latter including ageing, cancer and other age-related disorders, in part through the SASP. Cellular functions are generally dictated by the expression profile of lineage-specific genes. Indeed, expression of SASP factors and their regulators are often biased by cell type. In addition, emerging evidence suggests that p53 contributes to deregulation of more stringent lineage-specific genes during senescence. P53 itself is also tightly regulated at the protein level. In contrast to the rapid and transient activity of p53 upon stress ('acute-p53'), during senescence and other prolonged pathological conditions, p53 activities are sustained and fine-tuned through a combination of different inputs and outputs ('chronic-p53').

The Chromatin Regulator HMGA1a Undergoes Phase Separation in the Nucleus.

The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.

Inpatient antibiotic prescribing patterns using the World Health Organization (WHO) Access Watch and Reserve (AWaRe) classification in Okinawa, Japan: A point-prevalence survey.

Using point-prevalence methodology and the World Health Organization (WHO) Access, Watch, and Reserve Classification, we measured antibiotic use in 5 hospitals in Okinawa, Japan, on October 1, 2020. Overall, 29% of patients were prescribed an antibiotic on the survey date and the 3 most used antibiotics in the "Watch" category were cefazolin, ampicillin-sulbactam, and ampicillin.

Prevalence and characteristics of antibiotic prescription for acute COVID-19 patients in Japan.

COVID-19 is a viral infection and does not require antibiotics. The study aimed to elucidate a prescribing pattern of antibiotics for COVID-19. A nationwide cross-sectional study was conducted in Japan. The Diagnosis and Procedure Combinations (DPC) data was used to collect information, covering 25% of all acute care hospitals in the country. In 140,439 COVID-19 patients, 18,550 (13.21%) patients received antibiotics. Antibiotics were prescribed more often in inpatients (10,809 out of 66,912, 16.15%) than outpatients (7741 out of 73,527, 10.53%) (p < 0.001). Outpatient prescription was significantly associated with older patients (odds ratio [OR], 4.66; 95% confidence interval [CI] 4.41-4.93) and a greater Charlson index (OR with one-point index increase, 1.22; 95% CI 1.21-1.23). Inpatient prescription was significantly associated with older patients (OR 2.10; 95% CI 2.01-2.21), male gender (OR 1.12, 95% CI 1.07-1.18), a greater Charlson index (OR with one-point increase, 1.06; 95% CI 1.05-1.07), requirement of oxygen therapy (OR 3.44; 95% CI 3.28-3.60) and mechanical ventilation (OR 15.09; 95% CI 13.60-16.74). The most frequently prescribed antibiotic among outpatients was cefazolin, while that among inpatients was ceftriaxone. Antibiotic prescription is relatively low for acute COVID-19 in Japan. Antibiotic prescription was associated with older age, multi-morbidity, severe disease, and winter season.

Syphilitic osteomyelitis in a patient with HIV and cognitive biases in clinical reasoning: A case report.

RATIONALE: Diagnosing multifactorial, multidimensional symptoms unexplained by presumptive diagnosis is often challenging for infectious disease specialists. PATIENT CONCERNS: We report a rare case of a 30-year-old Japanese bisexual man with a history of virally suppressed human immunodeficiency virus and syphilis infections who developed chest pain and an erosive lesion under the lower midline jaw. DIAGNOSIS: Imaging examinations revealed erosive lesions on the sternum and left the ninth rib. Biopsy and polymerase chain reaction testing of sternal tissue specimens were noncontributory. However, due to elevated rapid plasma regain levels, a diagnosis of syphilitic osteomyelitis and gumma of the jaw was made. INTERVENTIONS: The patient was treated with 5 weeks of intravenous ceftriaxone and then with 8 weeks of oral amoxicillin. OUTCOME: After the antibiotic treatment, bone pain disappeared. We conducted a literature review on syphilitic osteomyelitis, and all of the articles included were case reports. Approximately half of the 46 patients with syphilitic osteomyelitis had HIV coinfection, and 10 (22%) patients lacked signs of early syphilis. Given its rarity, clinical data to establish appropriate guidelines for diagnosing and treating syphilitic osteomyelitis are still lacking. Cognitive biases, such as anchoring, cognitive overload bias, and premature closure, may contribute to diagnostic delays. LESSONS: In cases of idiopathic multiple bone lesions, syphilis must always be ruled out, and clinicians should guard against cognitive pitfalls when diagnosing rare diseases.

Transient Systemic Autophagy Inhibition Is Selectively and Irreversibly Deleterious to Lung Cancer.

Autophagy is a conserved catabolic process that maintains cellular homeostasis. Autophagy supports lung tumorigenesis and is a potential therapeutic target in lung cancer. A better understanding of the importance of tumor cell-autonomous versus systemic autophagy in lung cancer could facilitate clinical translation of autophagy inhibition. Here, we exploited inducible expression of Atg5 shRNA to temporally control Atg5 levels and to generate reversible tumor-specific and systemic autophagy loss mouse models of KrasG12D/+;p53-/- (KP) non-small cell lung cancer (NSCLC). Transient suppression of systemic but not tumor Atg5 expression significantly reduced established KP lung tumor growth without damaging normal tissues. In vivo13C isotope tracing and metabolic flux analyses demonstrated that systemic Atg5 knockdown specifically led to reduced glucose and lactate uptake. As a result, carbon flux from glucose and lactate to major metabolic pathways, including the tricarboxylic acid cycle, glycolysis, and serine biosynthesis, was significantly reduced in KP NSCLC following systemic autophagy loss. Furthermore, systemic Atg5 knockdown increased tumor T-cell infiltration, leading to T-cell-mediated tumor killing. Importantly, intermittent transient systemic Atg5 knockdown, which resembles what would occur during autophagy inhibition for cancer therapy, significantly prolonged lifespan of KP lung tumor-bearing mice, resulting in recovery of normal tissues but not tumors. Thus, systemic autophagy supports the growth of established lung tumors by promoting immune evasion and sustaining cancer cell metabolism for energy production and biosynthesis, and the inability of tumors to recover from loss of autophagy provides further proof of concept that inhibition of autophagy is a valid approach to cancer therapy. SIGNIFICANCE: Transient loss of systemic autophagy causes irreversible damage to tumors by suppressing cancer cell metabolism and promoting antitumor immunity, supporting autophagy inhibition as a rational strategy for treating lung cancer. See related commentary by Gan, p. 4322.

High Yield of Blood Cultures in the Etiologic Diagnosis of Cellulitis, Erysipelas, and Cutaneous Abscess in Elderly Patients.

Background: Cellulitis is a common disease in the elderly, and detecting etiologic organisms with blood cultures is difficult because of the low positive rate and occasional skin contamination. Therefore, routine blood cultures are not recommended for uncomplicated cellulitis. However, it is unclear whether blood culture collection for the diagnosis of cellulitis in elderly patients is useful. Methods: This single hospital-based observational study was performed between April 2012 and March 2015 in Okinawa, Japan. All enrolled patients were aged 15 years or older and admitted to the Division of Infectious Diseases with suspected cellulitis, erysipelas, and cutaneous abscess. Two routine sets of blood cultures were obtained. Results: Two hundred and twenty-one patients were enrolled. The median age was 77 years. The proportion of bacteremia was 21.7% for all patients (48/221), 8.5% (4/47) for those <65 years, and 25.3% (44/174) for those ≥65 years old (P = .013). The skin contamination rate was 0.9% (2/221). The most common pathogen was Streptococcus dysgalactiae (62.5%). Gram-negative bacteremia not susceptible to cefazolin was detected in 8.3%. Cefazolin and ampicillin were the first- and second-most commonly used therapies. Anti-methicillin-resistant Staphylococcus aureus therapy was required in 3.6% of patients. In addition to age and severe infection, shaking chills and white blood count ≥13 000 cells/µL were independent risk factors of bacteremia. Conclusions: Two routine sets of blood cultures are recommended for the precise diagnosis and appropriate treatment of cellulitis in elderly patients, especially in patients with shaking chills or leukocytosis.

Senescence: An Identity Crisis Originating from Deep Within the Nucleus.

Cellular senescence is implicated in a wide range of physiological and pathological conditions throughout an organism's entire lifetime. In particular, it has become evident that senescence plays a causative role in aging and age-associated disorders. This is not due simply to the loss of function of senescent cells. Instead, the substantial alterations of the cellular activities of senescent cells, especially the array of secretory factors, impact the surrounding tissues or even entire organisms. Such non-cell-autonomous functionality is largely coordinated by tissue-specific genes, constituting a cell fate-determining state. Senescence can be viewed as a gain-of-function phenotype or a process of cell identity shift. Cellular functionality or lineage-specific gene expression is tightly linked to the cell type-specific epigenetic landscape, reinforcing the heterogeneity of senescence across cell types. Here, we aim to define the senescence cellular functionality and epigenetic features that may contribute to the gain-of-function phenotype.