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Background During the ongoing COVID-19 pandemic, healthcare-associated transmission of respiratory viral infections (RVI) is a concern. To reduce the impact of SARS-CoV-2 and other respiratory viruses on patients and healthcare workers (HCWs) we devised and evaluated a multi-tiered infection control strategy with the goal of preventing nosocomial transmission of SARS-CoV2 and other RVIs across a large healthcare campus. Methodology From January-June 2020, a multi-tiered infection control strategy was implemented across a healthcare campus in Singapore, comprising the largest acute tertiary hospital as well as four other subspecialty centres, with more than 10,000 HCWs. Drawing on our institution’s experience with an outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003, this strategy included improved patient segregation and distancing, and heightened infection prevention and control (IPC) measures including universal masking. All symptomatic patients were tested for COVID-19 and common RVIs. Results A total of 16,162 admissions campus-wide were screened; 7.% (1155/16162) tested positive for COVID-19. Less than 5% of COVID-19 cases (39/1155) were initially detected outside of isolation wards in multi-bedded cohorted wards. Improved distancing and enhanced IPC measures successfully mitigated onward spread even amongst COVID-19 cases detected outside of isolation. COVID-19 rates amongst HCWs were kept low (0.13%, 17/13066) and reflected community acquisition rather than nosocomial spread. Rates of healthcare-associated-RVI amongst inpatients fell to zero and this decrease was sustained even after the lifting of visitor restrictions. Conclusion This multi-tiered infection control strategies can be implemented at-scale to successfully mitigate healthcare-associated transmission of respiratory viral pathogens., Highlights • A multi-tiered infection control strategy successfully contained both COVID-19 and common respiratory viral infections throughout a large healthcare system over six months of surveillance. • Despite caring for >1500 COVID-19 cases, no patient-staff transmission occurred. • Rates of healthcare-associated respiratory viral infections fell to zero over a six-month period. • Key components of the strategy included improved patient segregation and distancing, and universal masking.

Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body.

Highlights • Common viruses caused two-fifths of respiratory-illness-related hospitalizations, amidst a COVID-19 outbreak. • The co-infection rate between SARS-CoV-2 and other respiratory viruses was low, at 1.4%. • No increased morbidity or mortality with COVID-19 co-infections. • In-hospital mortality and intubation lower for COVID-19 compared with other respiratory viruses., Aims During the ongoing COVID-19 outbreak, co-circulation of other common respiratory viruses can potentially result in co-infections; however, reported rates of co-infections for SARS-CoV-2 vary. We sought to evaluate the prevalence and etiology of all community acquired viral respiratory infections requiring hospitalization during an ongoing COVID-19 outbreak, with a focus on co-infection rates and clinical outcomes. Methods Over a 10-week period, all admissions to our institution, the largest tertiary hospital in Singapore, were screened for respiratory symptoms, and COVID-19 as well as a panel of common respiratory viral pathogens were systematically tested for. Information was collated on clinical outcomes, including requirement for mechanical ventilation and in hospital mortality. Results One-fifth (19.3%, 736/3807) of hospitalized inpatients with respiratory symptoms had a PCR-proven viral respiratory infection; of which 58.5% (431/736) tested positive for SARS-CoV-2 and 42.2% (311/736) tested positive for other common respiratory viruses. The rate of co-infection with SARS-CoV-2 was 1.4% (8/431); all patients with co-infection had mild disease and stayed in communal settings. The in-hospital mortality rate and proportion of COVID-19 patients requiring invasive ventilation was low, at around 1% of patients; these rates were lower than patients with other community-acquired respiratory viruses admitted over the same period (p

The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. We used a novel IL-21eGFP reporter mouse strain to analyze the diversity and role of Tfh cells. Through the analysis of GFP expression in lymphoid organs of IL-21eGFP mice, we identified a subpopulation of GFP+, high IL-21 producing Tfh cells present only in Peyer’s Patches. GFP+Tfh cells were found to be polyclonal and related to GFP−Tfh cells of Peyer’s Patches in TCR repertoire composition and overall gene expression. Studies on the mechanisms of induction of GFP+Tfh cells demonstrated that they required the intestinal microbiota and a diverse repertoire of CD4+ T cells and B cells. Importantly, ablation of GFP+ cells resulted in a reduced frequency of Peyer’s Patches IgG1 and germinal center B cells in addition to small but significant shifts in gut microbiome composition. Our work highlights the diversity among IL-21 producing CD4+ Tfh cells, and the interrelationship between the intestinal bacteria and Tfh cell responses in the gut.

The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. We used a novel IL-21eGFP reporter mouse strain to analyze the diversity and role of Tfh cells. Through the analysis of GFP expression in lymphoid organs of IL-21eGFP mice, we identified a subpopulation of GFP+, high IL-21 producing Tfh cells present only in Peyer’s Patches. GFP+Tfh cells were found to be polyclonal and related to GFP−Tfh cells of Peyer’s Patches in TCR repertoire composition and overall gene expression. Studies on the mechanisms of induction of GFP+Tfh cells demonstrated that they required the intestinal microbiota and a diverse repertoire of CD4+ T cells and B cells. Importantly, ablation of GFP+ cells resulted in a reduced frequency of Peyer’s Patches IgG1 and germinal center B cells in addition to small but significant shifts in gut microbiome composition. Our work highlights the diversity among IL-21 producing CD4+ Tfh cells, and the interrelationship between the intestinal bacteria and Tfh cell responses in the gut.

Signaling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs) by opposing MAP kinase signaling. This contrasts with most cell types where STAT3signaling induces differentiation. We find that STAT3binding across the pluripotent genome is dependent upon Brg, the ATPase subunit of a specialized chromatin remodeling complex (esBAF) found in ESCs. Brg is required to establish chromatin accessibility at STAT3 binding targets, in essence preparing these sites to respond to LIF signaling. Moreover, Brg deletion leads to rapid Polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg-activated targets genome-wide, including the target genes of the LIF signaling pathway. Hence, one crucial role of Brg in ESCs involves its ability to potentiate LIF signaling by opposing PcG. Contrary to expectations, Brg also facilitates PcG function at classical PcG target including all four Hox loci, reinforcing their repression in ESCs. These findings reveal that esBAF does not simply antagonize PcG, but rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency.

The generation of long-lived plasma cells and memory B cells producing high-affinity antibodies depends on the maturation of B cell responses in germinal centers. These processes are essential for long-lasting antibody-mediated protection against infections. IgE antibodies are important for defense against parasites and toxins and can also mediate anti-tumor immunity. However, high-affinity IgE is also the main culprit responsible for the manifestations of allergic disease, including life-threatening anaphylaxisAnaphylaxis . Thus, generation of high-affinity IgE must be tightly regulated. Recent studies of IgE B cell biology have unveiled two mechanisms that limit high-affinity IgE memory responses: First, B cells that have recently switched to IgE production are programmed to rapidly differentiate into plasma cells,Plasma cells and second, IgE germinal centerGerminal center cells are transient and highly apoptotic. Opposing these processes, we now know that germinal center-derived IgG B cells can switch to IgE production, effectively becoming IgE-producing plasma cells. In this chapter, we will discuss the unique molecular and cellular pathways involved in the generation of IgE antibodies.

The generation of long-lived plasma cells and memory B cells producing high-affinity antibodies depends on the maturation of B cell responses in germinal centers. These processes are essential for long-lasting antibody-mediated protection against infections. IgE antibodies are important for defense against parasites and toxins and can also mediate anti-tumor immunity. However, high-affinity IgE is also the main culprit responsible for the manifestations of allergic disease, including life-threatening anaphylaxisAnaphylaxis . Thus, generation of high-affinity IgE must be tightly regulated. Recent studies of IgE B cell biology have unveiled two mechanisms that limit high-affinity IgE memory responses: First, B cells that have recently switched to IgE production are programmed to rapidly differentiate into plasma cells,Plasma cells and second, IgE germinal centerGerminal center cells are transient and highly apoptotic. Opposing these processes, we now know that germinal center-derived IgG B cells can switch to IgE production, effectively becoming IgE-producing plasma cells. In this chapter, we will discuss the unique molecular and cellular pathways involved in the generation of IgE antibodies.

The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity.

After rederivation of a mouse parvovirus (MPV)-contaminated transgenic mouse strain, serology and PCR testing of the surrogate dam showed it to be infected with mouse parvovirus strain 1 (MPV-1). The rederived pups (n = 3) also were MPV-positive, according to serology. Despite MPV seropositivity, fecal PCR tests of the pups were negative, as were serologic results from direct-contact sentinels. Only one rederived pup survived, and this male was bred successfully. None of its mates or progeny seroconverted to MPV. At 14.5 mo of age, the rederived male mouse was euthanized; tissues were collected and submitted for MPV testing; both serologic tests and PCR analysis of mesenteric lymph nodes were MPV-negative. One explanation for the rederived pups' MPV seropostivity is passive transfer of maternal antibodies or a nonproductive MPV infection. This case illustrates that although routine serological testing of surrogate mothers and pups is appropriate, any positive results should be further investigated by using transmissibility testing (fecal PCR or contact sentinels or both) prior to repeat rederivation.

IL-21 is the signature cytokine produced by T follicular helper (Tfh) cells and is vital in driving both the germinal centre (GC) reaction and formation of high affinity antibodies. However, the degree of Tfh cell heterogeneity and function is not fully understood. By utilising a novel IL-21eGFP reporter mouse carrying a diphtheria toxin receptor (DTR)-enhanced green fluorescent protein (eGFP) fusion gene, we identified a subpopulation of highly differentiated Tfh cells within Peyer’s Patches (PPs). This subpopulation demonstrated highest expression of the key Tfh molecules - Bcl6, ICOS and IL-21, and was found within germinal centres and surrounding B cell areas. TCRb repertoire analysis of GFP+Tfh cells revealed that these were polyclonal and closely related to GFP− Tfh cells, indicating selection by common antigens. In vitro stimulation of IL-21eGFP CD4+ cells in the presence of IL-6, TGFb and retinoic acid led to the induction of GFP+ cells that did not co-express IL-17 or Foxp3. Treatment of IL-21eGFP mice with antibiotics led to an ablation of PP GFP+CD4+ cells meaning that the presence of these cells relies on an intact bacterial microbiota. Furthermore, both polyclonal CD4+ T cell and B cell activation is necessary to support the differentiation of GFP+Tfh cells. Finally, GFP+ cell depletion resulted in a reduced frequency of PP GC B cells and IgG1+ cells and small but significant shifts in gut microbiome composition. Therefore, using a new IL-21-reporter mouse we have identified a subpopulation of Tfh cells induced by the gut microbiota and necessary for optimal PP GC and IgG1 responses.

Chemically induced proximity (CIP) systems use small molecules and engineered proteins to control and study biological processes. However, small molecule–based systems for controlling protein abundance or activities have been limited by toxicity, instability, cost, and slow clearance of the small molecules in vivo. To address these problems, we modified proteins of the plant abscisic acid (ABA) stress response pathway to control the proximity of cellular proteins and showed that the system could be used to regulate transcription, signal transduction, and subcellular localization of proteins in response to exogenously applied ABA. We also showed that the ABA CIP system can be combined with other CIP systems to simultaneously control multiple processes. We found that, when given to mice, ABA was orally available and had a 4-hour half-life. These properties, along with its lack of toxicity and low cost, suggest that ABA may be well suited for therapeutic applications and as an experimental tool to control diverse cellular activities in vivo.

A molecular beam of ferrocene has been photodissociated with 193 nm excimer laser radiation. Two channels were observed. The major channel involves the incoherent absorption of two photons by ferrocene, followed by sequential elimination of the two Cp ligands. A minor, single photon channel produces FeCp in an excited (ligand field?) state. The energy disposal in both cases can be predicted by statistical, microcanonical models. The two photon channel occurs despite the fact that a single photon has sufficient energy to remove both Cp ligands, because the electronic energy interconverts to the ground electronic state. The RRKM dissociation rate after a single photon absorption is slow compared to the incoherent photon absorption rate, so a second photon is absorbed.