Polygala lewtonii, an amphicarpic, perennial species endemic to fire-prone Florida sandhill and scrub, where fire kills plants but subsequently increases recruitment and population sizes. Polygala lewtonii produces three flower types: above-ground chasmogamous flowers and above-ground and below-ground cleistogamous flowers, with previous research demonstrating chasmogamous flowers produce a much greater proportion of seeds than cleistogamous flowers. We quantified outcrossing in seeds produced by chasmogamous flowers to determine whether it differed from the 100 % self-fertilized below-ground seeds. Approximately 25 % of seeds from chasmogamous flowers showed evidence of cross-pollination. Assuming that chasmogamous flowers produce the majority of the above-ground seeds, as was shown previously, this indicates it is possible to differentiate between germination by above-ground versus below-ground seeds in post-fire colonization. We next compared genetic diversity, admixture, inbreeding and population genetic structure pre- and post-fire. If fire promoted germination of chasmogamous seeds, heterozygosity and admixture would increase, and genetic structure and inbreeding would decrease. Instead, inbreeding and genetic structure increased and admixture decreased, suggesting that the below-ground selfed seeds  increased their contribution to the population after fire, possibly because fire reduced above-ground seed viability. Additionally, new alleles not found previously in range-wide analyses emerged from the seed bank post-fire. These results suggest that amphicarpy is a powerful adaptation to preserve genetic variation, maintain adaptive potential and promote rapid post-fire colonization.Amphicarpic plants produce both above-ground and below-ground seeds. Because below-ground seeds are protected in the soil and may maintain viability when above-ground conditions are stressful, they were proposed as an adaptation to recolonize a site after disturbance. However, whether below-ground seeds are the main colonizers after a disturbance remains unknown. Our goal was to understand whether recolonization by an amphicarpic species after fire was accomplished primarily through germination of seeds produced above-ground or below-ground. We investigated  Polygala lewtonii, a species endemic to fire-prone Florida sandhill and scrub. Polygala lewtonii has three flower types: above-ground chasmogamous (open-pollinated) and cleistogamous  flowers and below-ground cleistogamous flowers, with fruits produced both above-ground and below-ground. We used a genetic approach to test whether post-fire recolonization was accomplished primarily through germination of above-ground or below-ground seed. The below-ground selfed seeds showed an increased contribution to the population and new alleles emerged from the seed bank post-fire. These results suggest that amphicarpy is an adaptation to promote rapid post-fire colonization and preserve genetic variation and adaptive potential.We examined the mating system’s response to fire in  Understanding how reproductive strategies evolve in disturbance-maintained ecosystems connects basic evolutionary theory to conservation applications. Episodic mortality from regular disturbance tends to reduce population genetic variation and increase inbreeding . Adaptivt ≤ 0.8  is a federally endangered , smoke stimulated seed germination  CH flowers, (ii) above-ground CL flowers and (iii) below-ground CL flowers. In P. lewtonii, open-pollinated CH flowers also have a delayed selfing mechanism  is a short-lived (2–10 years) perennial, reaching ~20 cm tall  in the spring of 2017 see . We alsoP. lewtonii within the site, yielding an estimated area of ~0.28 km2. Within this study area, four blocks were created, and within each block, eight collection plots were established that ranged from 1 to 4 m in radius containing a minimum of nine P. lewtonii individuals, with a minimum of 10 m separating plots in the same block. The closest plots in neighbouring blocks were separated by larger distances . By design, plots were placed at a very fine spatial scale to understand how genotypes and genetic clusters were distributed spatially, with the goal of providing insight into the patterns of reproduction and the dispersal of selfed versus outcrossed seeds. In the 2014 sampling, leaves were collected from nine individuals per plot in each of eight plots per block, totalling 72 individuals per block, or 288 individuals total across the four blocks. A prescribed fire the following year (2 May 2015) burned and killed all plants in the blocks and plots except block 1 . To analyse whether seeds produced by CH flowers were derived from inbreeding versus outcrossing, we counted the number of non-maternal alleles in the offspring, which were presumably contributed by the pollen donor. Seeds were placed into three categories: individuals having zero non-maternal alleles, those having only one non-maternal allele and those having more than one non-maternal allele. Individuals with zero non-maternal alleles were products of either selfing or biparental inbreeding involving highly genetically similar individuals. Individuals with one non-maternal allele were either the product of cross-pollination between close relatives or genotyping error. Individuals with multiple non-maternal alleles were likely products of outcrossing.For seed data, we compared the heterozygosity of seeds from CH flowers to that expected under pure selfing by conducting a simulation of pure inbreeding in R version 3.4.1 . For eacA) and number of private alleles per population (AP). We used FSTAT v. 2.94 (HO and HE); if HO increased post-fire, it would indicate that a greater proportion of outcrossed seeds from CH flowers preferentially germinated in response to fire. If HO decreased or remained at pre-fire levels , it would indicate that seeds that germinated were predominantly produced by selfing or biparental inbreeding.For each block both pre- and post-fire, we used GenAlEx version 6.503  to calcu v. 2.94  to calcuF). Because technical artefacts such as genotyping errors and null alleles  inflate inbreeding coefficients, we used INEST version 2 (FB). We used the Bayesian Markov chain Monte Carlo (MCMC) approach with 5 000 000 iterations, a burn-in of 500 000, sampling every 250th iteration. If F and FB increased post-fire, this would indicate that fire stimulated the germination of seeds produced by inbreeding, whereas a decrease would indicate that a greater proportion of outcrossed seed germinated post-fire.To detect whether fire stimulated germination of selfed versus outcrossed seeds, we also used GenAlEx to measure the inbreeding coefficient  analyses as implemented in GenAlEx version 6.5 . Analysea priori population designations, we utilized InStruct version 1.0  and the −ln likelihood values from InStruct and selected the value where they plateaued and showed clear patterns of genetic structure.To estimate genetic structure independently of To quantify how admixture and genetic structure changed after a fire, we identified the predominant genetic cluster to which individuals were assigned, then calculated the percentage of individuals in each plot both pre- and post-fire that showed their greatest assignment to the predominant cluster. We also calculated average admixture proportions at the predominant InStruct cluster across all individuals in a plot, both pre- and post-fire. If below-ground selfed seed predominantly germinated after a fire, then we would expect post-fire plots to have more individuals and greater average admixture proportions assigned to the predominant genetic cluster. If above-ground seeds preferentially germinated after a fire, then because these seeds are capable of dispersal, we would expect post-fire plots to show fewer individuals assigned to a predominant genetic cluster. Provided that above-ground seeds showed some outcrossing, we would also expect lower average admixture proportions assigned to the predominant genetic cluster.Finally, we repeated analyses described above with individuals grouped into the predominant genetic cluster assigned by INSTRUCT. Analyses included estimates of genetic diversity, AMOVA and average admixture proportions at the majority cluster.[see].Seeds produced by CH flowers showed evidence of limited outcrossing. After removing 10 seeds from the analysis because of genotyping failure or genotyping errors, the final data set contained 40 seeds from nine maternal individuals, of which 30 out of 40 (75 %) had only maternal alleles present, 4 of 40 (10 %) differed from their mother by only one allele and 6 of 40 (15 %) differed from their mother by three or more alleles. When comparing observed inbreeding metrics to those simulated under 100 % selfing, the IR among seeds from open-pollinated flowers (0.718) was well below the range of values simulated for pure selfing , observed heterozygosity (HO) and inbreeding coefficients increased slightly after fire. The mean pre-fire HE averaged across loci was 0.311, whereas it was 0.322 post-fire  reached values close to one. Inbreeding coefficients increased slightly from pre-fire values  had only maternal alleles present, indicative of self-fertilization, biparental inbreeding or geitonogamy. Given the low seed set previously observed in P. lewtonii when pollinators were excluded  would still be greater than that expected for the 100 % selfed below-ground seed, indicating that it would be possible to detect whether above-ground or below-ground seed predominantly germinated after a fire.F and FB), indicating high rates of selfing or inbreeding. After fire, both observed and expected heterozygosity increased slightly, likely due to a slight increase in allelic diversity post-fire reflecting new alleles emerging from the seed bank. However, post-fire expected heterozygosity increased slightly more than observed heterozygosity, resulting in greater inbreeding coefficients. If seeds produced by above-ground flowers preferentially germinated after a fire, then given the greater seed set of CH flowers , the AMOVA-derived FIS decreased after fire. The apparent discrepancy likely reflects differences in how different methods quantify the effects of genetic drift on inbreeding. GenAlex and INest measure inbreeding based on the probability of identity by descent ) curves for InStruct analysis of Polygala lewtonii as presented in K = 1–10.plab067_suppl_Supplementary_MaterialClick here for additional data file.


B-cells undergo somatic hypermutation and affinity maturation in germinal centers. Somatic hypermutated germinal center B-cells (GCBs) compete to engage with and capture antigens on follicular dendritic cells. Recent studies show that when encountering membrane antigens, GCBs generate actin-rich pod-like structures with B-cell receptor (BCR) microclusters to facilitate affinity discrimination. While deficiencies in actin regulators, including the Wiskott-Aldrich syndrome protein (WASp), cause B-cell affinity maturation defects, the mechanism by which actin regulates BCR signaling in GBCs is not fully understood. Using WASp knockout (WKO) mice that express Lifeact-GFP and live-cell total internal reflection fluorescence imaging, this study examined the role of WASp-mediated branched actin polymerization in the GCB immunological synapse. After rapid spreading on antigen-coated planar lipid bilayers, GCBs formed microclusters of phosphorylated BCRs and proximal signaling molecules at the center and the outer edge of the contact zone. The centralized signaling clusters localized at actin-rich GCB membrane protrusions. WKO reduced the centralized micro-signaling clusters by decreasing the number and stability of F-actin foci supporting GCB membrane protrusions. The actin structures that support the spreading membrane also appeared less frequently and regularly in WKO than in WT GCBs, which led to reductions in both the level and rate of GCB spreading and antigen gathering. Our results reveal essential roles for WASp in the generation and maintenance of unique structures for GCB immunological synapses. B-cell-mediated antibody responses provide essential immune protection against infectious diseases. Antibodies and antibody responses have been explored as immune therapies and preventatives for cancer, immune disorders, and infectious diseases. Upon encountering cognate antigen, mature B-cells in B-cell follicles of the secondary lymphoid organs are activated through the B-cell antigen receptor (BCR) to proliferate, leading to the formation of germinal centers (GCs). B-cells undergo somatic hypermutation in immunoglobulin genes of the BCR in the dark zone (DZ) of GCs. Germinal center B-cells (GCBs) with mutated BCRs then migrate to the light zone (LZ), where GCBs compete with each other to engage and capture antigen through their clonal-specific BCRs . BCR’s aDeficiencies of actin regulators responsible for generating branched actin structures cause concurrent immune deficiencies and autoimmune diseases in humans. These actin regulators include ARPC1B, a hematopoietic-specific ARPC1 isoform of Arp2/3 complex that nucleates branched actin , and a hThe activation of B-cells from all subsets is initiated by the binding of the BCR to antigens. In GCs, B-cells encounter antigens on the surface of follicular dendritic cells or soluble antigen diffusing into GCs . BindingB-cell affinity maturation in GCs is antigen-driven, indicating the crucial role of BCR-antigen engagement and subsequent signaling . CompareThe role of the actin cytoskeleton in BCR signaling of naïve B-cells has been extensively studied. Upon encountering cognate antigens with multi-valency or those associated with membranes, the earliest signaling triggers transient actin depolymerization, releasing surface BCRs from restraints to lateral mobility imposed by the cortical actin network . FollowiWhile deficiencies in actin regulators significantly impact GCBs, the exact role of the actin cytoskeleton in GCB BCR signaling has not been fully understood. Recent studies have shown a unique architecture of the GCB IS formed on antigen-presenting membranes. Distinct from the relatively smooth membrane contact of naive B-cells, GCBs generate actin- and ezrin-rich pod-like structures to contact antigen-presenting surfaces, which leads to the formation of less centralized BCR microclusters . The staThis study examined the mechanism by which WASp promoted polymerization of branched actin in the formation of GCB ISs, using WASp knockout mice expressing Lifeact-GFP that binds to F-actin and total internal reflection fluorescence microscopy. Here, we show that when interacting with planar lipid bilayers coated with a high density of Fab’ fragment of anti-BCR antibody, WT GCBs form centralized BCR signaling microclusters at plasma membrane protrusions. These centralized signaling microclusters are surrounded by regions of low signaling, in contact with the antigen-presenting membrane. Branched actin generated by WASp is required for the formation and stabilization of the unique membrane structure of the GCB ISs by generating and sustaining actin networks that support plasma membrane protrusions as well as interactions with antigen-presenting membranes in the periphery of the IS.Wild type  and WASp knockout (WKO) mice on a C57BL/6 background were purchased from Jackson Laboratories (Cat# 000664 and 019458). A lack of WASp expression in WKO mice was verified using western blotting . LifeactWT and WKO mice with or without expressing Lifeact-GFP were immunized intraperitoneally with sheep red blood cells (SRBC) (Innovative Research Cat# ISHRBC10P) twice 7 days apart and euthanized at 7 days following the second immunization. Splenocytes were released from the spleens using frosted glass slides and filtered through 40 μm cell strainer . Red blood cells were lysed using ACK lysing buffer (Gibico Cat# 10492-01). GCBs were enriched using a negative selection method based on a published protocol . BrieflyWestern blotting was used to verify WASp-deficiency in WKO mice. Splenocytes from WT and WKO mice were lysed, and cell lysates were analyzed by western blotting, probing for WASp (Santa Cruz Cat# 365859). The blots were stripped and probed for GAPDH (Proteintech Cat# HRP-60004) as loading controls.To determine the purity of enriched GCBs and compare the sizes and the surface BCR levels of GCBs from WT and WKO mice, splenocytes and isolated GCBs were stained with antibodies specific to B220 (Biolegend Cat# 103236), GL7 (Invitrogen Cat# 12-5902-82), CD95 (BD Biosciences Cat# 557653), and IgG (Biolegend Cat# 405315) and analyzed by a BD FACS Canto II flow cytometer (BD Sciences) and Flowjo software.2 fragment of anti-mouse IgM + G antibody (Jackson ImmunoResearch Cat# 115-006-068) using a previously published protocol (Mono-biotinylated Fab’ (mB-Fab’ or Fab’) was generated from the F(ab’)protocol . mB-Fab’protocol . CoverslImages were acquired using a TIRF microscope . Interference reflection images (IRM) and AF488 and AF546 images were acquired sequentially. To identify light-zone and dark-zone GCBs, enriched GCBs were stained with AF488-anti-CD86 (Invitrogen Cat# 53-0869-42) and PE-anti-CXCR4 (Invitrogen Cat# 12-9991-82) and then incubated with Fab’-PLBs for 5 min at 37°C before fixation with 4% paraformaldehyde. Cells with high levels of CD86 staining and low levels of CXCR4 staining were identified as light-zone GCB. After initial characterization, enriched GCBs were pre-stained with anti-CXCR4 antibodies before incubating with Fab’-PLB for all the experiments, and cells with no or low level of CXCR4 staining were identified as light-zone GCBs.To analyze signaling and F-actin in GCBs, enriched GCBs were incubated with Fab’-PLBs or Tf-PLBs at 37°C for varying lengths of time, fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin, and stained with antibodies specific for phospho-CD79a Y182 , phospho-SHIP1 Y1020 , phospho-SHP1 Y536 (Abcam Cat# ab51171), phospho-Syk (Y525/Y526) , phospho-Akt (S473) , or AF488-phalloidin (Cytoskeleton Cat# PHDG1). Cell contact area, the total (TFI) and mean fluorescence intensity (MFI) in the cell contact zone, and fluorescence intensity (FI) along lines across cells were determined based on IRM and TIRF images using NIH ImageJ.2, and imaged by TIRF, acquiring one frame every 2 s. Cell contact area, the TFI, and the MFI in the cell contact zone were determined based on IRM and TIRF images analyzed with custom-made codes using MATLAB software . Increasing rates of the cell contact area and the MFI of AF546-Fab’ or Lifeact-GFP in the contact zone were determined using the slopes of the contact area or MFI vs. time plots and linear regression. Kymographs of time-lapse images were generated using NIH ImageJ.For live-cell imaging, GCBs expressing Lifeact-GFP were incubated with Fab’-PLBs at 37°C, 5% COFor the analysis of BCR-Fab’ cluster growth, one randomly selected kymograph was generated for each cell from time-lapse images using NIH ImageJ. AF546-Fab’ clusters, which were visually distinguishable and trackable for at least 7 min, were identified by visual inspection. Five such AF546-Fab’ clusters were randomly selected from each kymograph. The FI along each cluster track in kymographs was measured.Phalloidin and Lifeact-GFP foci in individual cells were identified visually and manually, aided by intensity maps generated using NIH ImageJ. The MFI of all identifiable foci and the MFI of the entire contact zone of individual cells were determined using NIH ImageJ manually. F-actin foci were identified when the MFI of phalloidin foci was 2-folds or the MFI of Lifeact-GFP foci was 1.5-fold of their MFI in the cell contact zone. The relative lifetime of actin foci was determined by the duration that individual actin foci can be detected in a kymograph. The width ratio of the adherent regions, the wider side relative to the narrower side, in individual cells was determined using a randomly selected kymograph per cell generated using time-lapse IRM images and an average of four time points: 3, 5, 7, and 10 min.t-tests (Prism-GraphPad software) when only two groups were compared, and one-way ANOVA when 3 or more groups were compared. All data were presented as the mean ± SD (standard deviation).Statistical significance was assessed using unpaired, two-tailed Student’s To examine germinal center B-cell (GCB) immunological synapses (ISs), we isolated GCBs from sheep red blood cell (SRBC)-immunized mice . GCBs weIn the contact zone of WT LZ GCBs, pCD79a and pSyk were primarily detected as numerous puncta in the central area . StaininB-cells undergo actin-driven spreading when interacting with antigen-presenting surfaces, increasing the contact area . We utilUpon interacting with antigen on presenting surfaces, antigen-engaged BCRs aggregate into microclusters, leading to signaling activation . To examWASp is an actin nucleation-promoting factor that activates Arp2/3-mediated polymerization of branched actin . We evalPrevious studies have shown that the GCB IS consists of actin-rich protrusions engaging antigen on presenting surfaces . ConsistIn addition to the centralized F-actin foci, we observed actin structures at the outer edge of the contact zone, supporting the spreading membrane . We deteGerminal centers are critical for selecting high-affinity antigen-specific B-cells and eliminating non-specific B-cells generated during somatic hypermutation . A lack Recent studies have shown that GCBs from human tonsils and mouse spleens engage membrane antigen through actin-rich pod-like membrane protrusions and form BCR microclusters at the tips of these protrusions . This orThis study further shows that activated signaling molecules, including both positive  and negative (SHP1) signaling molecules, appear at the protrusions at similar times. The peak levels of phosphorylated CD79a and Syk persist at least for 20 min. In contrast, stimulatory kinases in naïve B-cells interacting with the same type of Fab’-PLB are first activated and then down-regulated by activated phosphatases . Our finWASp has been shown to have B-cell intrinsic roles in the germinal center, causing GCB hyper-proliferation and plasma cell differentiation in WASp-deficient mouse models . HoweverIn addition to its role in GCB spreading, the new finding of this study is that WASp is essential for the formation and stabilization of pod-like membrane protrusions, the unique membrane structures of the GCB IS. WASp is responsible for the generation and/or extended lifetime of actin foci that support the membrane protrusions at the center of the GCB contact zone, as both the number of actin foci and their relative lifetime are reduced in WKO GCBs. This finding suggests that actin structures supporting pod-like membrane protrusions are based on branched actin networks. Furthermore, WASp is also required for the stability and regularity of branched actin structures at the outer edge of the GCB contact membrane after rapid spreading. These actin structures, which appear to be tread milling, function to maintain the GCB contact area and the GCB adherence to antigen-presenting surfaces surrounding the centralized membrane protrusions. The role of these actin structures in GCBs is opposite to what was observed in naïve B-cells, where actin at the outer edge of the contact zone drives B-cell contraction following spreading, which promotes signaling attenuation . This fuThe reductions in BCR phosphorylation, BCR clustering, and cell spreading in WKO GCBs compared to WT GCBs suggest that WASp promotes BCR activation by increasing cell spreading and BCR clustering at the contact zone. These findings are similar to what we observed in WKO native B-cells , suggestThe moderate reduction in cell spreading, BCR clustering, and signaling in WKO GCBs indicates that WASp is not essential for BCR signaling in GCBs. The WASp family has additional members, including N-WASp and WAVE, which can all activate Arp2/3-mediated branched actin polymerization . We haveIn summary, this study has identified a role for WASp and WASp-activated branched actin polymerization in the generation and maintenance of the unique architecture of the GCB IS. This unique architecture is essential for GCBs to discriminate antigen-binding affinity for the selection of high-affinity antigen-specific B-cells to mount antibody responses. The defective GCB IS architecture provides a cellular mechanism by which WASp deficiency causes immune disorders.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland  and Chongqing Medical University .YL, WS, AU, and XZ established the study design and provided the research strategy. YL and AB performed the experiments and the data analysis. YL and WS prepared and wrote the manuscript. All authors read and approved the manuscript.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.


Hematopoietic stem cell transplants (HSCT) are becoming more widespread as a result of optimization of conditioning regimens and prevention of short-term complications with prophylactic antibiotics and antifungals. However, pulmonary complications post-HSCT remain a leading cause of morbidity and mortality and are a challenge to clinicians in both diagnosis and treatment. This comprehensive review provides a primer for non-pulmonary healthcare providers, synthesizing the current evidence behind common infectious and non-infectious post-transplant pulmonary complications based on time . Utilizing the combination of timing of presentation, clinical symptoms, histopathology, and radiographic findings should increase rates of early diagnosis, treatment, and prognostication of these severe illness states. Hematopoietic stem cell transplant (HSCT) involves replacing a patient’s bone marrow with hematopoietic stem or progenitor cells from peripheral blood, bone marrow, or umbilical cord from another person or the same individual to restore immune–hematopoietic function after the underlying disease is eliminated . PulmonaHematopoietic stem cell transplant (HSCT) is a multi-step process with a high risk for complications during marrow ablation, during engraftment, or afterwards ,6,7. SucHematopoietic stem cells can be obtained from self (autologous or auto-HSCT) or from a donor . Deciding whether to use autologous versus allogeneic transplant is dependent on multiple patient-specific factors and donor availability . AutologAdvantages of allogeneic transplant include the ability to correct congenital or acquired defects and the immunologic, anti-tumor effects that can occur in response to persistent disease referred to as the graft versus tumor (GVT) effect . AllogenAfter selecting the source of stem cells, the host is prepared via high dose myeloablation therapy, reduced intensity myeloablation, or non-myeloablative conditioning . Once a 3, a platelet count greater than 20,000/microliter without any transfusions for one week, and a hematocrit greater than 25% for at least 20 days without any transfusions [While there are several definitions of successful engraftment, patients typically develop a neutrophil count greater than 500/mmsfusions . For autsfusions . For allsfusions . Neutropsfusions .The diagnosis and management of post-HSCT pulmonary manifestations requires a multidisciplinary approach. Diagnosis can be challenging as post-HSCT syndromes often have nonspecific clinical presentations. Utilizing common timelines of illness presentation post-HSCT  in combiInfectious and non-infectious pulmonary complications are classified by etiology and temporality in reference to HSCT, as it reflects the immunologic state of the patient. The often-cited time course for non-infectious entities consists of the pre/peri-engraftment phase (first 30 days), early post-transplantation phase (30–100 days), and the late post-transplantation phase (after 100 days). Infectious complications will be discussed separately.Conditions in the peri-engraftment period occur secondary to neutropenia, transfusion-related events, and toxicity of conditioning regimens. Non-infectious and infectious etiologies are equally likely to develop and often co-occur. An attempt should be made to identify and treat infection prior to initiation of immunosuppressive treatment for non-infectious complications; however, in critically ill patients, infectious and non-infectious complications may require concurrent treatment. Cardiogenic causes of acute lung injury include volume overload resulting from large fluid volumes administered during chemotherapy, cardiac toxicity due to prior chemotherapy or radiation therapy, and transfusion-associated circulatory overload (TACO) after multiple blood products are transfused. Non-cardiogenic causes include chemotherapy pneumonitis and transfusion-related acute lung injury (TRALI). Chemotherapy-associated pneumonitis can occur due to a number of different chemotherapeutic agents including melphalan, busulfan, and bis(chloroethyl)nitrosourea (BCNU) . Drug asPeri-engraftment respiratory distress syndrome (PERDS) has a wide range of reported incidence with a propensity to occur more frequently in allogeneic HSCT . It is oWhile most patients with PERDS respond favorably to treatment, some demonstrate progressive acute lung injury with lung consolidation and increasing alveolar collapse requiring intubation b,c. Up tDiffuse alveolar hemorrhage (DAH) has similar rates in both allogeneic (3–7%) and autologous (1–5%) HSCT recipients ,26,27,28DAH presents with fever, dyspnea, non-productive cough, and diffuse pulmonary infiltrates. Hemoptysis is relatively rare, making it a non-reliable sign for diagnosis. Computed tomography (CT) of the chest usually demonstrates patchy multifocal or diffuse ground-glass opacity with superimposed interlobular and intralobular septal thickening, creating a “crazy paving” pattern d. BronchThis period is characterized by impaired cellular and humoral immunity and delayed lung injury from conditioning/preparative regimens. During this phase, the neutrophil count is generally normal, and the frequency of infectious complications typically decreases. Delayed complications of radiation and chemotherapy can produce manifestations similar to other HSCT-related complications during this period. Idiopathic pneumonia syndrome (IPS) represents a spectrum of diseases occurring between 20 and 42 days post-transplant, with the range extending up to 100+ days post-transplant. It is more common in allogeneic HSCT recipients . MyeloabWhile both PERDS and DAH technically meet the diagnostic criteria of IPS, the three disease entities behave differently. High-dose corticosteroids continue to be the treatment of choice with variable response. Up to 85% of IPS cases are non-responsive to corticosteroids and progress to respiratory failure with up to 90% requiring mechanical ventilation ,28. OverPulmonary cytolytic thrombi are intravascular thromboemboli accompanied by the infiltration of monocytes into small and medium pulmonary vessels surrounded by areas of hemorrhage or infarct . This phPost-transplant lymphoproliferative disease (PTLD) is a heterogenous group of lymphoid disorders that can complicate solid organ transplants as well as HSCTs. While in solid organ transplants, most PTLD is host-derived, the majority of PTLD (55–65%) in HSCT is due to reactivation and proliferation of Epstein–Barr virus (EBV)–infected donor derived B-lymphocytes due to weakened cytotoxic T-cell immunity . HoweverThe term late-onset non-infectious pulmonary complications (LONIPCS) has been used to describe complications that occur 3 months after transplant . These aChronic graft-versus-host disease (cGVHD) is currently the leading cause of long-term morbidity and mortality post-allogeneic HSCT . ClinicaBOS is a clinical rather than a histopathological diagnosis . BOS is BOS is irreversible with variable progression and mortality rates of up to 60% . ManagemOrganizing pneumonia (OP) has an incidence of approximately 10% after HSCT . It occuMore recently, studies have focused on the prevalence of interstitial lung diseases (ILD) after HSCT . While tOne example of post-HSCT ILD is pleuroparenchymal fibroelastosis (PPFE), a rare form of interstitial pneumonia. PPFE can be idiopathic or secondary, the latter of which has been associated with HSCT. It occurs in <0.5% of patients post-HSCT and typically presents many years post-transplantation ,64,65. TPulmonary veno-occlusive disease (PVOD) is a rare variant of pulmonary arterial hypertension (PAH) in which intimal proliferation occurs in the small pulmonary venules. A recent autopsy series suggests that it may be under-recognized with pathologic evidence of PVOD in 12 of the 35 autopsies in allo-HSCT recipients . The etiThe peri-engraftment phase is hallmarked by mucositis due to conditioning therapy and prolonged neutropenia due to non-functional transplanted marrow . The risBacterial infections account for approximately 90% of infections during the peri-engraftment period after allo-HSCT  and inclAspergillus and even Mucor and generally includes a vessel occlusion sign, which was noted to have a sensitivity of 0.78 and specificity of 0.91 for angioinvasive disease in one study [Aspergillus strains resistant to more commonly used azoles [Invasive fungal infections (IFI) can affect patients early in their post-transplant course. Invasive aspergillosis is the most common invasive fungal infection HSCT recipients ,83. The ne study . The diane study . Posacond azoles . VoriconZygomycosis is the second most common mold infection in HSCT with an increasing incidence over the past few decades . Rare inCandida glabrata and Candida krusei, especially in those with cGVHD [Pneumocystis jiroveci pneumonia (PJP) has a low incidence due to use of prophylaxis. Without adequate prophylaxis, it has an incidence of 5–16% [Candidal infections occur less frequently as a result of universal prophylaxis with fluconazole. In high-risk patients, posaconazole may be the preferred prophylactic therapy due to the emergence of resistant non-albicans candida species including th cGVHD . Similarof 5–16%  and ofteof 5–16% . CT findof 5–16% . TMP-SMXof 5–16% .Viral pneumonia from reactivation is common during the peri-engraftment phase and should be closely monitored while patients are immunosuppressed. Viral infection from herpes simplex virus (HSV)-1 and -2 occur in peri-engraftment and early post-transplantation phase, those from cytomegalovirus (CMV) and human herpes virus-6 (HHV-6) are often present in early post-transplantation phase, and infection from varicella zoster virus (VZV) is more common in the late post-transplantation phase . The incCMV pneumonia has an incidence as high as 20% in allo-HSCT recipients . In contHSV can have pulmonary manifestations including tracheobronchitis and pneumonia . The brePatients continue to be at high risk for seasonal community-acquired respiratory viruses (CARV). Respiratory syncytial virus (RSV), influenza A and B, parainfluenza, and rhinovirus cause the majority of non-CMV viral respiratory infections ,109, espRSV is one of the more common CARV that is associated with high mortality rate. In a single center retrospective study of 280 allo-HSCT recipients with RSV infection, 29% had progression to lower respiratory tract infection . Older aIn 2019, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a coronavirus disease 2019 (COVID-19) global pandemic. The majority of HSCT patients who contract COVID-19 have mild or moderate disease . StudiesUpdated guidelines have been published by European Society for Blood and Marrow Transplantation (EBMT) and American Society for Transplantation and Cellular Therapy (ASTCT) for the management of HSCT patients in regard to COVID-19 ,125. PatOpen communication between these primary services and these subspecialists is necessary for the initial evaluation of these patients. There are multiple diagnostic tools available if non-invasive methods do not yield a diagnosis. The most frequently utilized and widely considered safest diagnostic tool is flexible bronchoscopy. Bronchoscopic findings result in identification of infectious pathogens in 31–71% cases and change in therapy in 24–52% of patients ,127. AltWhile prior studies have shown that transbronchial biopsies (TBBx) can increase the diagnostic yield compared to bronchoscopy alone , more reThe use of other more invasive modalities is controversial . PercutaCollectively, the risk of both infectious and non-infectious pulmonary complications post-HSCT are high, but careful consideration of the timeline post-HSCT, an assessment of individual and local risk factors, and early involvement of a multidisciplinary team in the care of these individuals can help to identify and treat these complications in a timely manner.


Lineage tracing studies have become a well-suited approach to reveal cellular hierarchies and tumor heterogeneity. Cellular heterogeneity, particularly in breast cancer, is still one of the main concerns regarding tumor progression and resistance to anti-cancer therapies. Here, we review the current knowledge about lineage tracing analyses that have contributed to an improved comprehension of the complexity of mammary tumors, highlighting how targeting different mammary epithelial cells and tracing their progeny can be useful to explore the intra- and inter-heterogeneity observed in breast cancer. In addition, we examine the strategies used to identify the cell of origin in different breast cancer subtypes and summarize how cellular plasticity plays an important role during tumorigenesis. Finally, we evaluate the clinical implications of lineage tracing studies and the challenges remaining to address tumor heterogeneity in breast cancer. Breast cancer is the most common cancer in women worldwide and is the second leading cause of cancer death in women, exceeded only by lung cancer . This type of cancer originates in the mammary gland, which is a ductal tree composed of two epithelial compartments: cells facing the ductal lumen called luminal cells (LCs), and basal cells (BCs) found in the outer layer with a capacity to contract, which includes basal progenitor cells and terminal differentiated myoepithelial cells. Luminal cells can be further subdivided into two independent subpopulations based on the expression of the hormone receptor, estrogen receptor alpha (ERα) [ERBB2/HER2 amplifications and loss of ERα expression; and triple-negative breast cancer (TNBC), characterized by the lack of expression of the aforementioned molecular markers [In clinical practice, breast tumors are classified based on the histological expression of ERα, progesterone receptor (PR), receptor tyrosine-protein kinase ErbB-2 (HER2), and the proliferation marker Ki-67. They are divided into three main groups: hormone receptor-positive subtype, which includes tumors expressing ERα and/or PR, which are subclassified as luminal A or B depending on the percentage of Ki-67; HER2-positive tumors, defined by the presence of At the cellular level, breast cancer is a heterogeneous disease , therebyDuring the last decade, lineage tracing studies have provided deeper insights into the cellular heterogeneity and molecular mechanisms underlying cellular plasticity in different subtypes of breast cancer. Accordingly, this review outlines the main milestones concerning this technology in the breast cancer context, as well as underlining unsolved questions and future prospects in the field.loxP sequences, allowing the expression of the reporter  ,20,21, wreporter A. The usransgene ,33  system was developed to specifically study the mammary gland [MMTV) promoter, which recombines Frt sequences exclusively in mammary epithelial cells. Similar to the doxycycline-dependent system, this in vivo strategy does not require the use of tamoxifen for activating Flp enzyme. Importantly, this model consists of a Cre alternative recombinase (Flp), which allows the combination of multiple site-specific recombination systems, such as Cre/loxP and Flp/Frt [A new genetically engineered mouse model using the Flippase (Flp)/flippase recognition target  with transcriptional profiles of different tumor subtypes ,40,41. ILgr6), which is expressed in LCs and BCs during the early stages of tumorigenesis, contributed to mammary tumor progression [pos cells were genetically labeled at the hyperplasia stage  in a mouse mammary tumor virus promoter-driven Polyomavirus middle T antigen breast cancer mouse model (MMTV-PyMT), which mostly generates luminal tumors, these cells clonally expanded, contributing to the formation of carcinomas [pos cells were involved in the formation of basal-like tumors. These results suggest that Lgr6pos cells could be the cell of origin in the luminal breast cancer subtype exclusively; however, the fact that the Lgr6 promoter is activated in LCs and BCs at the start point of the lineage tracing makes it complicated to draw strong conclusions regarding the true cell of origin in each breast cancer subtype. Remarkably, when Lgr5-expressing cells  were trd in BCs ,45, the of TNBCs ; neverthEsr1-Cre/MMTV-TVA/Rosa26-tdRFP mice infected with RCAS-Erbb2 (to generate HER2-positive tumors), the authors could demonstrate that ERαpos tumor cells with an overexpression of HER2 have to progressively lose their ERα expression in order to clonally expand and metastasize [pos cells were more aggressive than those originated from ERαneg cells, suggesting that the cell of origin plays an important role in the clinical outcome of breast cancer patients.Fascinatingly, a recent work combined lineage tracing analysis and the RCAS-TVA system. This consists of expressing the TVA cell surface receptor under the control of a specific target promoter recognized by an avian leukosis virus-derived vector (RCAS)  to eluciastasize . ImportaUndoubtedly, more studies are required to figure out which mammary epithelial cell type is the cell of origin for each specific tumor type. The lack of good preclinical breast cancer mouse models that truly recapitulate human subtypes, and the selection of suitable inducible Cre lines, which exclusively label one mammary epithelial cell type at a time, are the major concerns that the research community currently face to address this important issue.The epithelial-to-mesenchymal transition (EMT) is a biologic process by which epithelial cells lose their cell polarity and cell–cell adhesion to undergo multiple biochemical changes that enable them to assume mesenchymal attributes, such as an elongated shape, fibroblast-like morphology, enhanced mobility, invasiveness, resistance to apoptotic stimuli, and production of extracellular matrix components . CrucialMMTV-PyMT and MMTV-Neu), being a biological process that can be observed in vivo; however, the metastases developed were neither FSP1- nor Vimentin-derived progenies [low cells and FSP1pos-cells  [low tumor cells were injected into the circulation, these cells generated metastases [In vivo monitorization of different mesenchymal markers (FSP1 and Vimentin) using different breast cancer models demonstrated that the EMT process does not contribute to lung metastasis development . These srogenies ,52,53. Nrogenies . The maiEMTracer, a triple transgenic mouse model carrying Kit-CreER, EMTgene-LSL-Dre, and NR1-reporter. Thus, this EMTracer was crossed with the MMTV-PyMT model to monitor EMT during tumor progression [Kitpos cells recombined LoxP sequences expressing ZsGreen fluorescent protein, inducing Dre expression exclusively in mesenchymal cells which were positive for Vim or Cdh2/N-cadherin, turning tdTomato positive [The fact that many different cells can undergo EMT and the lack of a universal marker encouraged the combinatorial design of Cre and Dre systems to study this biological process. Recently, Li and colleagues generated gression . With thpositive . In theipositive . In addipositive , indicatThe heterogeneity underlying the EMT process could be explained by the emergence of a partial rather than a full EMT , resultiRosa26-CreERT2), Zomer and colleagues observed that only a small subset of tumor cells clonally expanded during tumor progression, and the vast majority of cells within the primary tumor either disappear, grow slowly, or partially expand to finally regress [New microscopy technologies, such as intravital or 3D-whole mount imaging, have allowed the visualization of mammary tumor progression from adenoma to carcinoma in vivo and at large-scale single-cell resolution, respectively ,58. Comb regress . The comMMTV-PyMT model demonstrated that the metastatic process is produced by the collective dissemination of cancer cells forming cohesive clusters rather than the serial seeding of single tumor cells [Performing multicolor fluorescent lineage tracing in the or cells . Concretor cells . Moreoveor cells .MMTV-Cre mice with HER2-Crainbow (HER2BOW) mice, which encoded for the three HER2 variants, fluorescently barcoded and flanked by LoxP sites. Using fluorescence and RNA sequencing, they could characterize the tumor phenotype associated with each barcoded isoform [A powerful approach is combining lineage tracing with scRNA-seq by introducing genetic barcodes. Indeed, using this strategy, Ginzel and colleagues measured the tumorigenic capacity of different oncogenic HER2 isoforms  within the same mammary gland . Specifi isoform . Althoug isoform .Many groups have tried to recapitulate the wide diversity of breast cancer subtypes detected in the clinics, designing preclinical mouse models that overexpress oncogenes or silence tumor suppressor genes. Indeed, recent studies have relied on Cre/lox systems for defining novel drivers of mammary tumorigenesis and assessing their consequences in different cellular contexts.Vim and Snai1, they found the MMTV-Cre/Brca1Co/Co/p53+/− [MMTV-Lpa [121 [+/− irradiated [MMTV-Cre/Brca1Co/Co/p53+/− [MMTV-Myc [WAP-Myc [WAP-Tag [MMTV-Aib1 [MMTV-Wnt1 [MMTV-Neu [MMTV-PyMT [WAP-Myc [MMTV-Myc [MMTV-Aib1 [MMTV-Hras [MMTV-Neu [+/− [−/− [−/− [MMTV-Aib1 [WAP-Cre/Etv6 [WAP-T121 [MMTV-Fgf3 [It was reported that specific murine strains can spontaneously develop breast tumors, such as CH3, that produces adenocarcinomas with a latency of 6–10 months ; BALB/c o/p53+/− ; DMBA-ino/p53+/− , few C3-To/p53+/− , MMTV-MyMMTV-Myc , WAP-Myc[WAP-Myc , WAP-Tag[WAP-Tag , MMTV-AiMTV-Aib1 , and MMTMTV-Wnt1  mice. ReMMTV-Neu , MMTV-PyMTV-PyMT , WAP-Myc[WAP-Myc , MMTV-MyMMTV-Myc , MMTV-AiMTV-Aib1 , MMTV-HrMTV-Hras , and WAPMTV-Hras . AdditioMMTV-Neu , Bgr1+/−Neu [+/− , p18−/− +/− [−/− , Rb−/− [−/− [−/− , MMTV-AiMTV-Aib1 , WAP-CreCre/Etv6 , WAP-T12WAP-T121 , and MMTMTV-Fgf3 .  or BCs (Lgr5-expressing cells), the resulting tumors were basal-like, HER2-positive, and luminal B (Krt8-derived tumor), or mainly luminal A and B (Lgr5-derived tumors) [Krt5 promoter to target BCs [H1047R in BCs led to the formation of luminal B tumors    and luminal markers (Keratin-18 and/or ERα), with similar but not identical gene expression profiles, which closely resembled that of human basal-like tumors, regardless of the cell of origin  could be the key to designing a model able to recapitulate the tumor evolution observed in human breast cancer.Clinical decisions are made depending on the molecular subtype diagnosed, which includes anti-hormone therapies for those patients with ERαpos or ERαneg cellular origin will determine their aggressiveness and metastatic capacity [Remarkably, lineage tracing studies have disclosed that the cell of origin matters. Here, we have presented several good examples, such as the work of Ding and colleagues that proved that HER2-positive tumors with an ERαcapacity . Moreovecapacity ,94,95,96capacity ,89 or HEcapacity ) were abcapacity ,91) compG12D [Human cells contain somatic mutations that have served as genetic barcodes to perform retrospective lineage tracing analysis in healthy and diseased human tissues . For exaG12D .Notably, over the last years, single-cell RNA sequencing has emerged as a new tool to replace lineage tracing studies, since it provides a recapitulation of the clonal dynamics of different cell populations within a tumor in a retrospective manner; however, the question of the identity of the tumor-initiating cell still remains to be solved with this type of technology. Combining both approaches would allow us to genetically and phenotypically trace each individual cell, redefining the phylogenetic trees, cell trajectories, and cell–cell interactions. In keeping with recent breakthroughs, the development of barcode systems has enabled us to target individual cells with unique nucleic acid sequences . This te® and Oncotype DX®, both of which predict the risk of distant disease recurrence. Moreover, among some spatial transcriptomic technologies, fluorescence in situ hybridization (FISH) methods are commonly used in cancer diagnosis as they allow the detection and chromosomal location of specific genes which are aberrantly expressed or harbor rare mutations in tumors [Beyond lineage tracing, multi-omic and high-throughput methods will help us to understand the complex genomics that underlie the human cancer disease. Indeed, single-cell genomics has enabled us to associate specific genetic mutations with different molecular subtypes , and sinn tumors . On the n tumors . The int


Endothelial dysfunction with subsequent thrombosis and, less commonly, vasculitis has been implicated during the active phase of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infection. However, less has been described during the recovery phase or as late sequelae. Here, we report a case of acute anterior wall ST-elevation myocardial infarction in a female patient with no medical history of cardiovascular risk factors as a post-infectious complication of coronavirus disease 2019 (COVID-19). Coronary angiography revealed total occlusion of her left anterior descending, right coronary arteries, and tight stenosis in the left circumflex artery. Successful revascularization with a staged percutaneous coronary intervention was achieved. To date, there is not much data regarding the late cardiovascular sequelae of COVID-19 and its possible mechanisms. Prolonged follow-up, even for mild cases of COVID-19, is advised for early diagnosis and treatment of long-term complications of COVID-19. Coronavirus disease 2019 (COVID-19)-mediated vasculopathy has been increasingly recognized as a serious complication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The complex interplay between endothelial dysfunction from direct viral infection and systemic inflammation is associated with various thrombotic complications and vasculitides -6.We present the case of a 58-year-old female with an unremarkable medical history except for a resolved COVID-19 infection. Three months ago, she had experienced fever, shortness of breath, and labile blood pressure, and had tested positive for COVID-19 by real-time reverse transcriptase-polymerase chain reaction. She did not require supplemental oxygen or hospitalization and was managed conservatively at home. Afterward, she started to complain of progressive exertional chest pain and dyspnea grade II relieved by rest; nevertheless, she did not seek medical advice. She presented three months after her initial diagnosis with COVID-19 to our medical facility complaining of acute onset of retrosternal chest pain radiating to her left arm and associated with diaphoresis and sweating. Her physical examination was unremarkable apart from tachycardia (heart rate of 105 beats/minute). Electrocardiography revealed anterolateral ST-segment elevations. She was diagnosed with anterior STEMI and transferred immediately for coronary angiography (CA). CA revealed total thrombotic occlusion of the left anterior descending artery (LAD) and total occlusion of the right coronary artery (RCA) receiving retrograde collaterals from the left system, and a proximal tight lesion in the left circumflex artery (LCx) . She underwent successful primary LAD PCI Video .After uneventful observation in the cardiac care unit for 48 hours, she underwent successful PCI of LCx and RCA Video .She was discharged on dual antiplatelet therapy with close follow-up visits.The pathogenesis of SARS-COV-2-mediated vasculopathy has been proposed as follows: (1) Co-expression of the angiotensin-converting enzyme 2 receptor and transmembrane serine protease 2 in endothelial cells facilitates the viral entry, thereby promoting endothelial damage, inflammation, and prothrombotic state ,8. The iIn addition to thrombotic complications, there is scarce evidence showing that coronary artery vasculitis can also manifest as a post-infectious complication of COVID-19, despite having mild or no previous pulmonary infection, as a part of the multisystem inflammatory syndrome ,11 GivenTo date, the mechanisms responsible for the association between COVID-19 and coronary artery vasculopathy, after the hyperinflammatory state has subsided, have not been determined. Several questions remain unanswered, including “how can we stratify those who are at higher risk of developing vasculopathy after resolution of COVID-19, and how long will they be susceptible to those complications?.” Long-term close follow-up of even mild cases of COVID-19 is required to diagnose late cardiovascular complications. Two-dimensional speckle-tracking echocardiography with left ventricular global longitudinal strain can be used as a validated method to determine early or late myocardial dysfunction following COVID-19 infection .So far, data regarding late cardiovascular complications of COVID-19 and its possible mechanisms are not available. Follow-up is required for even mild cases of COVID-19 for the early diagnosis and treatment of long-term consequences of COVID-19.


Escherichia coli (ETEC) is the main infectious agent responsible for piglet post-weaning diarrhea with high mortality rates. Antimicrobials represent the current principal strategy for treating ETEC infections in pig farms, but the occurrence of multi-resistant bacterial strains has considerably increased in the last decades. Thus, finding non-antibiotic alternatives becomes a real emergency. In this context, we investigated the effect of a live yeast strain, Saccharomyces cerevisiae var boulardii CNCM I-1079 (SB) in an in vitro model of the weaning piglet colon implemented with a mucus phase (MPigut-IVM) inoculated with ETEC and coupled with an intestinal porcine cell line IPI-2I. We showed that SB was able to modulate the in vitro microbiota through an increase in Bacteroidiaceae and a decrease in Prevotellaceae families. Effluents collected from the SB treated bioreactors were able to mitigate the expression level of genes encoding non-gel forming mucins, tight junction proteins, innate immune pathway, and pro-inflammatory response in IPI-2I cells. Furthermore, SB exerted a significant protective effect against ETEC adhesion on porcine IPEC-J2 intestinal cells in a dose-dependent manner and showed a positive effect on ETEC-challenged IPEC-J2 by lowering expression of genes involved in pro-inflammatory immune responses. Our results showed that the strain SB CNCM I-1079 could prevent microbiota dysbiosis associated with weaning and protect porcine enterocytes from ETEC infections by reducing bacterial adhesion and modulating the inflammatory response.Enterotoxigenic  Escherichia coli (ETEC) is the main pathogen responsible of post-weaning diarrhea , 9., 9.® Eva


Innate immune responses are tightly regulated by various pathways to control infections and maintain homeostasis. One of these pathways, the inflammasome pathway, activates a family of cysteine proteases called inflammatory caspases. They orchestrate an immune response by cleaving specific cellular substrates. Canonical inflammasomes activate caspase-1, whereas non-canonical inflammasomes activate caspase-4 and -5 in humans and caspase-11 in mice. Caspases are highly specific enzymes that select their substrates through diverse mechanisms. During inflammation, caspase activity is responsible for the secretion of inflammatory cytokines and the execution of a form of lytic and inflammatory cell death called pyroptosis. This review aims to bring together our current knowledge of the biochemical processes behind inflammatory caspase activation, substrate specificity, and substrate signalling. Proteases are central enzymes that mediate numerous signalling roles to ensure cellular functions and organismal homeostasis . DiscoveInflammatory caspases are a caspase subset activated by cellular platforms called inflammasomes . Albeit cysteine-dependent aspartate-specific proteases. Caspases use a catalytic dyad composed of an histidine (H237 in caspase-1) and a cysteine (Cys285 in caspase-1) [Caenorhabditis elegans (C.elegans) [The term caspase ,9 is derelegans) .Caspases have a conserved modular organisation: a N-terminal domain (of variable length and function), a large catalytic subunit, and a small catalytic subunit  Figure . These dThe first caspase group, the apoptotic caspases, can be further subdivided into initiator and executioner caspases based on their role within the apoptotic pathway. The initiator caspases , are monomeric and contain a long homotypic N-terminal domain required for recruitment to their respective activation platform. The initiator caspases can then be further divided into whether they participate in the extrinsic or intrinsic apoptotic pathway. The extrinsic pathway involves caspase-8 and -10, which are activated by complexes like the death-inducing signalling complex (DISC) following binding of death ligands to their cognate receptors ,15. The Executioner caspases contain a short pro-domain (<30 residues) and are synthesised as inactive dimeric zymogens. Cleavage of executioner caspases by the initiator caspases allows full activation and therefore the cleavage of specific substrates to execute apoptotic cell death .The second main caspase group is the inflammatory caspases . The infA few caspases fall out of the traditional classification due to their unique functions. An example of this is caspase-14, a caspase involved in keratinocyte differentiation . CaspaseThe remainder of this review will focus on the mechanisms that govern inflammatory caspase activity.Inflammatory caspases exist as monomers under the cellular resting state and require dimerisation to become active. This dimerisation step is tightly regulated and is mediated by large, multi-protein signalling platforms called inflammasomes . InflammPRR-activating inflammasomes are diverse in nature and recognise or respond to a multitude of chemically different ligands (summarised in Inflammasome-activating PRRs contain either a Pyrin domain (PYD) or Caspase activation and recruitment domain (CARD), both of which belong to the death-fold domain family . The preThe ASC speck can also recruit and activate caspase-8 to trigger apoptosis .Inflammatory caspase-4 and -5 in humans, and caspase-11 in mice, are activated by the non-canonical inflammasome. Caspase-5 is conserved only in a few species (humans and great apes) and is believed to be the consequence of a genetic duplication. Until recently, these caspases were believed to directly binding lipopolysaccharide to trigger direct caspase dimerisation and activation . HoweverPseudomonas aeruginosa) [The cellular context controlling caspase-5 activation remain elusive. Specific LPS structures (e.g. Outer membrane vesicles from uginosa)  and NLRPuginosa)  were sugInflammatory caspases are highly specific proteases that cleave defined protein substrates to orchestrate the innate immune responses. In the following section, we will discuss how caspases achieve this specificity through diverse mechanisms .Caspases natural substrate must be present in the same cellular compartment as the caspase and their cleavage site present specific features. First, the P1′ position is usually occupied by a small aliphatic amino acid. Secondly, the cleavage site is usually located in a solvent-exposed flexible structural element and is accessible to the protease active site. Finally, caspase substrate often displays an optimal primary cleavage sequence .The substrates primary cleavage sequence bind through the caspase substrate-binding pocket, which can generally accommodate four amino acids from the substrate. According to the Schechter-Berger nomenclature , caspaseRecently, caspases were shown to cleave artificial substrates containing glutamic acid and phosphorylated serine in position P1, expanding the potential sequence of substrates .Although the focus has been towards the primary tetrapeptide, multiple studies have highlighted the influence of extended subsites on caspase specificity. The Salvesen group identified extended subsites in caspase-11 and caspase-5 substrates that increase cleavage of selected substrates ,76. ExteThe ability of certain caspases to cleave these primary sequences can also be influenced by post-translational modifications of the substrates. For example, phosphorylation of the substrates primary tetrapeptide has been shown to influence the ability of apoptotic caspases to cleave certain substrates ,77. HoweCaspase specificity to small peptides led to the development of caspase inhibitors. However, these inhibitors display relative specificity as they can target other caspases . For exaDeterminants outside the substrate-binding pocket have also been shown to influence caspase specificity .Exosites are structural motifs that allow binding of substrates independently of the primary substrate binding pocket. Although insufficient to allow substrate cleavage on their own, exosites enhance the cleavage efficiency of specific substrates and are used by various proteases to achieve protease specificity.Exosites were first observed in apoptotic caspases. Caspase-7 harbours an exosite in its N-terminal domain that consist of four lysines . RecentlUntil recently, no structure of caspases with their protein substrates were available. However, two recent papers outline unprecedent details on how exosites allow substrate specificity by inflammatory caspases.Shao's and Tsiao's lab reveal the structure of caspase-1, -4 and -11 bound to the C-terminal fragment of GSDMD  or the fSo far, GSDMD is the only inflammatory caspases substrate that has been clearly shown to bind on an inflammatory caspase exosite. However, recent evidence suggests that similar sites are also present for IL-1β .Developing inhibitors that target exosites instead of the primary substrate-binding pocket bear the promise of more specific inhibitors.Caspase substrate cleavage has three functional consequences: a loss-of-functions, a gain of functions or a no-consequence effect. Caspase cleavage may also affect protein stability and target specific substrates to proteasomal degradation ,86.Proteomic studies have identified substrates for caspase-1 in mice ,88 and hCaspase-1 was originally identified as an Interleukine-1β (IL-1β)-converting enzyme and was originally characterised to cleave and mature this cytokine. Caspase-1 also processes the IL-18 pro-form into its mature form. The cytokines IL-1β and IL-18 are unconventionally secreted through GSDMD pores or following cell lysis ,91. CaspCaspase-1 also cleaves IL-37, an anti-inflammatory cytokine, to promote IL-37 nuclear translocation and genetic repression of anti-inflammatory cytokines .Caspase-5 has been reported to cleave IL-1α in senescent cells, a process that may contribute to aging-associated inflammation .Gasdermin D (GSDMD) is a central executor of pyroptosis . FollowiCaspase-1 can cleave proapoptotic proteins like Bid and caspase-3 and -7. It has been suggested to control infection by specific pathogens and stands as a backup cell death mechanism if pyroptosis is counteracted by pathogens .Caspase-1, -4, -5, and -11 can cleave and inactivate cGAS to control type I IFN response and modulate antiviral responses .Caspase-1 has been shown to cleave other substrates however, the functional relevance of these substrates remains unclear. For example, caspase-1 may contribute to cell demise by cleaving many structural proteins . CaspasKnown caspase-4 and -5 substrates are minimal, and efforts to identify them thus far have been limited. Outside the substrates mentioned above, caspase-5 cleaves the transcription factor Max after glutamic acid , and SYADanio rerio), M (Mus musculus), H (Homo sapiens)). Human inflammatory caspases share high similarities with higher primates caspases.Numerous caspase substrates are conserved throughout evolution. Inflammatory caspases are present in vertebrates, from the zebrafish ,121 to hMultiple substrates seem to be conserved during evolution and many of their cleavage site position  is highlWhereas apoptotic caspase activity is controlled by the inhibitors of apoptosis proteins (IAP) , endogenSerpinB1 was suggested to be an endogenous inhibitor of inflammatory caspases in some cell types, such as neutrophils .The CARD-only proteins (COPs) and PYD-only proteins (POPs) can inhibit inflammatory caspases indirectly by regulating inflammasome formation and/or caspase recruitment to their activating platform .The involvement of inflammatory caspases in a range of diseases containing an inflammatory component  supVertex developed a caspase-1/4 inhibitor, VX765, that was taken to clinical trials . HoweverThe development of caspase inhibitors that target exosites highlights the possibility of more specific inhibitors.In addition, the development of exosite inhibitors has the potential to generate inhibitors that target deleterious functions of caspases, without affecting the beneficial ones, by modulating the cleavage of selected substrates.Importance in the field: Inflammatory caspases are crucial for regulated immune responses and linked to diverse pathologies from sepsis to cancer.Summary of the current thinking: Inflammatory caspases are activated either by canonical or non-canonical inflammasomes. GBPs are novel innate immune sensors that form a non-canonical inflammasome and facilitate LPS presentation to caspase-4 and -11. Caspases recognise their substrates through substrate binding-pockets and use exosites to increase substrate selectivity.Future directions: Research to identify and characterise novel caspase substrates will expand our understanding of inflammatory caspases in health and disease. Future research will address how inflammatory caspases activity is controlled by endogenous mechanisms and inhibitors. Targeting caspase exosites may allow for the development of more specific pharmacological inhibitors.


Aberrant activity of oncogenic rat sarcoma virus (RAS) protein promotes tumor growth and progression. RAS-driven cancers comprise more than 30% of all human cancers and are refractory to frontline treatment strategies. Since direct targeting of RAS has proven challenging, efforts have been centered on the exploration of inhibitors for RAS downstream effector kinases. Two major RAS downstream signaling pathways, including the Raf/MEK/Erk cascade and the phosphatidylinositol-3-kinase (PI3K) pathway, have become compelling targets for RAS-driven cancer therapy. However, the main drawback in the blockade of a single RAS effector is the multiple levels of crosstalk and compensatory mechanisms between these two pathways that contribute to drug resistance against monotherapies. A growing body of evidence reveals that the sequential or synergistic inhibition of multiple RAS effectors is a more convenient route for the efficacy of cancer therapy. Herein, we revisit the recent developments and discuss the most promising modalities targeting canonical RAS downstream effectors for the treatment of RAS-driven cancers. Rat sarcoma virus (RAS) is involved in distinct cellular processes, including cell division, proliferation, migration and cellular differentiation. RAS is tethered to the plasma membrane and acts as a nexus to relay mitogenic stimuli from receptor tyrosine kinases (RTKs), thereby activating a plethora of downstream signaling pathways. RAS proto-oncogenes encode four members of evolutionary conserved, membrane-associated small GTP-binding proteins . The activity of RAS is tightly regulated by a molecular switch between GTP-bound active and GDP-bound inactive conformation. Two classes of regulatory proteins maintain the GTPase activity of RAS proteins: guanine exchange factors (GEFs) catalyze the exchange of GDP for GTP, and GTPase activating proteins (GAPs) dictate the spatial regulation of RAS by accelerating the hydrolysis of bound GTP to GDP and inorganic phosphate .RAS hyperactivation is frequently observed in a large spectrum of cancer types, including pancreatic ductal adenocarcinomas (PDACs), colorectal myelomas, lung adenocarcinomas and endometrial carcinomas. RAS-activating mutations and epidermal growth factor (EGF) hyper-signaling, which potentiates cell proliferation, are common in cancer. RAS proteins are overactive in nearly 30% of all cancers and are associated with resistance to frontline monotherapies . Among RG12C mutant has been tractable in blocking the oncogenic signal [G12C via a disulfide bond and inhibit SOS1-catalyzed nucleotide exchange [G12C mutant cell lines, which can be enhanced by concomitant inhibition of the PI3K cascade [G12C inhibitors will not likely confer a benefit to a significant number of patients.In the last three decades, tremendous efforts have been expended on targeting RAS-driven tumors. In an effort to inhibit mutant RAS function, RAS can be directly targeted by molecules disrupting its interaction with SOS1 (SOS Ras/Rac Guanine Nucleotide Exchange Factor 1) or with its effectors such as Raf and PI3K (phosphatidylinositol-3-kinase). However, RAS proteins are often termed “undruggable” due to the consecutive failures in the development of direct RAS inhibitors. Various alternative ways to battle against this perception have been implemented ,7,8. Thuc signal ,10,11,12exchange . In the  cascade ,15. Furt cascade . HoweverOncogenic RAS signaling can be blocked indirectly by targeting the attachment of RAS to the inner plasma membrane or inhibiting RAS-dependent metabolic processes such as autophagy and macropinocytosis . A varieTargeting RAS effector signaling cascades or synthetic lethal interactors of mutant RAS are other indirect strategies being pursued to conquer RAS-mutant cancers . The forRAS engages with two major downstream effector signaling pathways, including Raf/MEK/Erk (MAPK) and phosphatidylinositol-3-kinase (PI3K) cascades. These pathways are associated with specific cancer-related phenotypes, including transcriptional reprogramming, promoted cell survival and proliferation, suppressed apoptosis and enhanced invasiveness of the cells . MitogenThe RAS-Erk signaling network, which is the central regulator for cell cycle progression and proliferation, is a prominent mediator of RAS-dependent cancer growth. RAS stimulates cell proliferation by increasing the concentration of Raf kinases to the plasma membrane, where effector kinases are activated . Here, aThe other well-described downstream pathway of RAS is the PI3K-mTOR signaling cascade, which controls a number of cellular events including cell cycle progression, protein synthesis, metabolism and survival . PI3K siV600 mutant-driven melanoma. These inhibitors hinder catalytically active B-RafV600 mutant monomers with specificity for the ATP-binding site, but not non-V600 mutants [So far, three B-Raf kinase inhibitors, including vemurafenib ,42, dabr mutants ,47,48. H mutants . When RaIn the presence of oncogenic RAS, B-Raf inhibition has been shown to lead to the formation of RAS-dependent B-Raf/C-Raf heterodimers and drive tumorigenesis through C-Raf activation . A signiV600 inhibitors through distinct mechanisms [G12V mutant spindle cell sarcoma which was refractory to multi-kinase inhibitor treatment in a phase I clinical trial (NCT02437227) [In an effort to suppress B-Raf activity without stimulating cancer growth, second-generation Raf inhibitors including type II pan-Raf inhibitors and paradox-breakers were developed to hinder dimerization-driven transactivation. These inhibitors possess minimal paradoxical activation compared to approved B-Rafchanisms . Of notechanisms ,56. Examchanisms ,58. Sevechanisms , acute mchanisms , colorecchanisms ,62, pancchanisms  and thyrchanisms , and arechanisms . Howeverchanisms ,66, but chanisms . Unlike 2437227) . Similar2437227) . These r2437227) . Moreove2437227) . LXH254 V600E background, taking into account the thermodynamics and kinetics of inhibitor–protein interactions, post-translational modifications and structural determinants [V600E cells [V600E and RAS mutations [The efficacy of pan-Raf inhibitors is promising, but nevertheless not striking for anti-RAS cancer therapy, which requires excellent drug combination for a robust response from dozens of alternatives. In this regard, Rukhlenko et al. built a system biology-based dynamic model to analyze the synergy of Raf inhibitor combinations in an oncogenic RAS and/or B-Rafrminants . They fo0E cells . The datutations , thus prV600E and evaluating pathway reactivation in RAS-mutant cell lines. These inhibitors dock on Raf proteins to impair Raf dimerization [Q61L mutation, compared to vemurafenib [Another class of B-Raf inhibitors, paradox breakers, have been developed utilizing the structure of vemurafenib as a skeleton, with various chemical modifications . Candidarization ,75,76 anrization . The parurafenib . Given tV600 tumor cells [D594G:C-Raf dimers [Transmission of signals from B-Raf to MEK requires the dimerization of B-Raf protomers in both normal cells and non-B-Rafor cells . To circor cells . To targor cells . Similaror cells . In a suf dimers , thus prG12V-addicted non-small-cell lung cancer (NSCLC) in mice. While the ablation of C-Raf from these cells inhibited tumor development, B-Raf did not show the same effect, indicating that the loss of C-Raf cannot be compensated by other Raf isoforms in K-RASG12V-addicted NSCLCs [G13D-mutant cells [Among Raf isoforms, B-Raf has taken the center stage in targeted cancer therapy due to its high incidence of mutations in various types of cancer. Remarkably, another member of the Raf family, C-Raf , has been shown to have an essential role to relay upstream signals to MEK and Erk in K-RASd NSCLCs . In lined NSCLCs . The losd NSCLCs . Disappont cells . Therefont cells . On the nt cells .600E/K melanoma. However, tumor responses to MEK inhibitors have been generally transient due to the rapid emergence of resistance in RAS-addicted cancers. Furthermore, in a subset of cancers in which MEK inhibitors have shown a notable clinical activity, patients have suffered from off-target effects, including dermatological and gastrointestinal toxicities [G12C tumors have been shown as more sensitive to selumetinib compared to KRASG12D in lung cancer mouse models. This finding underscores the heterogeneity of tumors harboring different KRAS mutations [Since attempts to target Raf kinases have suffered from acquired drug resistance that limits the effectiveness of inhibitors, the attention turned to the inhibition of MEK kinase. Many kinase inhibitors have been developed to compete for ATP by directly binding to this conserved site, which limits the selectivity of the molecule. Therefore, a number of allosteric MEK inhibitors have been developed for enhanced selectivity. These MEK inhibitors bind to a unique site close to the ATP-binding pocket of MEK, not competitively to the ATP-binding site. Thus, allosteric MEK inhibitors only bind to MEK selectively and inhibit the function of the kinase by inducing a conformational change that locks the enzyme in a catalytically inactive state . High sexicities . Inhibitxicities  and reacxicities ,93. The xicities . For exaxicities . Similarxicities ,97. In Nxicities . Howeverutations .Due to the limited efficacy of MEK inhibitors in mono-agent settings, efforts have focused on the development of combinatorial strategies. MEK inhibitors have been used in combination with other treatment modalities in RAS-mutant cancers, such as conventional chemotherapeutic agents , systemiA unique Raf/MEK inhibitor, VS-6766/CH5126766, has shown activity in xenograft models of RAS-mutated cancers ,112. In The final kinase component of the three-layered MAPK cascade, Erk, is another target to evade compensatory resistance mechanisms. Several ATP-competitive Erk1/2 inhibitors, including ASN007 , LY32149G12C-driven autochthonous sarcoma model. However, this combination could not be successful to revert previously developed resistance, which offers the use of dual MEK and Erk treatment as a first-line therapy [Ravoxertinib (GDC-0994) is an orally available small molecule targeting ERK1/2 kinase  with pro therapy . In addi therapy . DespiteAs another example of intra-pathway dual inhibition of the MAPK pathway, concurrent application of pan-Raf inhibitors and Erk inhibitors proved potent at low doses in cell line, organoid and rat models of PDAC with KRAS mutations . AltogetThe functional activity of Erk requires its translocation to the nucleus. Cytosolic active Erk is phosphorylated by MEK on its specific regulatory residues and undergoes a conformational change that releases Erk from its anchoring proteins. Then, exposed Ser residue located within the nuclear translocation sequence (NTS) is phosphorylated, triggering the interaction of Erk and the beta-like importin, Imp7, thereby escorting Erk to the nucleus, where Erk activates a large number of targets ,143. In Erk5 is a more recent member of the MAPK family, which is most similar to the Erk1/2 subfamily but reveals responses through distinct mechanisms . A numbeAberrations in the PI3K-Akt signaling pathway have been implicated with tumorigenesis and resistance to anti-cancer therapies. Dysregulation of the PI3K signaling can occur through different mechanisms with a subset of mutations in the axis. These alterations include amplification of RTKs (EGFR and HER2), mutations in PI3K subunits , loss/mutation of the phosphatase tensin homolog (PTEN), Akt overexpression or RAS hyperactivation. Singly targeting RAS-induced overactivation of PI3K-Akt signaling remains challenging due to the intrinsic resistance caused by beta catenin  and p90 In the PI3K-Akt signaling, PI3K is the first and direct downstream kinase effector of RAS as a potential drug target. Two major classes of PI3K inhibitors, including isoform-selective inhibitors and pan-PI3K inhibitors, have been developed to antagonize PI3K-Akt signaling. In 2019, the PIK3CA inhibitor BYL719  was approved by the FDA for the treatment of estrogen receptor-positive (ER+) breast cancer patients with PIK3CA mutations . StudiesThe second kinase member in the PI3K-mTOR axis, Akt, consists of three functionally distinct isoforms, including Akt-1, Akt-2 and Akt-3. It has been shown that RAS-induced oncogenic signals are mainly relayed through the Akt-1 isoform in KRAS-mutant lung tumors . In contG12C inhibitor ARS-1620 was increased when combined with mTOR and an IGF1R inhibitor [G12C covalent inhibitors and provide a therapeutic opportunity for patients with KRASG12C mutations in combined settings.Inhibitors targeting mTOR, one of the downstream effectors of RAS, have been clinically tested, however the majority of single-agent therapies targeting mTOR did not result in clear clinical benefits. Inhibition of mTOR has been shown to stimulate feedback activation mechanisms including MEK/Erk or Akt signaling, demanding for combinatorial treatment regimens ,163. A pnhibitor  and PI3Knhibitor  in lung Owing to the adaptive resistance to selective targeting of MAPK signaling in RAS-mutant cancers, the PI3K-mTOR pathway has attracted growing interest by virtue of its complementarity to the MAPK pathway. Not only do these pathways share common inputs but also they can both be activated by oncogenic RAS and appear to provide some compensatory signaling when one or the other is inhibited . The theTumor radio-resistance remains a major obstacle for the treatment of many types of cancer. Both MAPK and PI3K-mTOR pathways have been implicated with radio-resistance of RAS-dependent cancer cells ,176. CliRecently, research has focused intensively on Erk inhibitor-based drug combinations, which seem more attractive due to their ability to mitigate feedback relief. Dual inhibition of Erk and PI3K/mTOR with LY3214996 and LY3023414 inhibitors respectively, has been tolerable and resulted in synergistic inhibition on tumor growth in RAS-driven lung cancer . We awaiA great majority of recent combinatorial strategies targeting MAPK and PI3K/Akt signaling together have been found to be poorly tolerated in clinical studies involving patients with RAS mutations, which diverted the attention to other cellular targets. The inhibition of HSP90, highly expressed in most cancers, in combination with a MEK inhibitor has been identified as a promising therapeutic strategy for KRAS-mutant NSCLC. Several clinical trials have been conducted to examine the effect of AUY922, an HSP90 inhibitor, on solid tumors, and revealed the low cellular toxicity of the agent ,185. OwiOne of the key hallmarks of cancer is the metabolic reprogramming of tumor cells, which meet the need of increased biomass by altering their metabolism to grow . RAS sigOne of the most important metabolisms related to cancer is the mevalonate pathway, which is an essential metabolic pathway for cholesterol biosynthesis. Tumorigenesis requires increased mevalonate pathway flux to meet the need for precursors. The mevalonate synthesis inhibitors, Statins, suppress protein prenylation and mediate several cellular events associated with cancer hallmarks, including proliferation, survival and metastasis. Statins have been previously shown to induce apoptosis by suppressing Erk and Akt activation . BlockadTremendous progress has been made in understanding the genetic architecture, the biological heterogeneity and the distinct molecular pathways driven by RAS oncogenes. Although none of these findings could be successfully extrapolated into cancer therapy yet, future progress will be built on the foundation of this deeper understanding of tumor response. The more detailed assessment of these finely balanced signaling networks could help researchers refine their approach and allow greater focus on rational drug combinations. These observations underscore that a focus should be placed on in-depth optimization of timing, dosing schedules and treatment sequences to remedy toxicities and side effects of drugs. Evidently, there is also still a need to improve clinical tools for accurately stratifying patients based on their molecular status. In this regard, the specific markers associated with drug sensitivity and acquired resistance in aiding therapeutic interventions for RAS-driven cancer entities are of utmost importance.Beyond small-molecule inhibitors, some innovative therapeutic approaches, such as oncolytic virus-mediated gene-editing , mRNA vaIn the near future, all these efforts will likely bear fruit for the treatment of RAS-driven cancers. Each strategy developed on the basis of scientific research will form another pillar for cancer treatment and serve as an opportunity to develop efficient and well-tolerated combinations in clinical practice.


As of April 16, 2021, U.S. correctional and detention facilities reported 399,631 cases of COVID-19 in incarcerated persons, resulting in 2,574 deaths  with approximately 120 work sites in multiple industries  and a rural work camp with work sites in the agricultural sector. IDOC provided transportation to and from work sites. A total of 382 COVID-19 cases were identified among incarcerated persons, including 76 (20%) cases in one facility housing women only, and 306 (80%) cases in four facilities housing men only. The median patient age was 37 years (range = 21–69 years). Among COVID-19 patients, 218 (57.1%) were non-Hispanic White persons, 40 (10.5%) were Hispanic or Latino persons, 10 (2.6%) were Black persons, and nine (2.4%) were American Indian or Alaska Native persons; race/ethnicity data were missing for 105 (27.5%) patients. No hospitalizations or deaths occurred.IDOC facilities provided various housing arrangements for 108–276 persons; the number of COVID-19 cases at each facility ranged from nine to 148 . The totInformation on work-release placements was available for CRC A and the work camp. The first COVID-19 case in CRC A was identified on July 14, 2020, in an incarcerated person working at a food processing plant. A COVID-19 outbreak had previously been identified among nonincarcerated employees at this workplace; IDOC was not aware of the ongoing outbreak until notified by public health officials on July 22, 2020, which prompted ongoing communication among IDOC and public health partners. Subsequent IDHW guidance recommended that correctional facilities require work-release sites to notify them of COVID-19 cases among employees and suspend work-release during COVID-19 outbreaks until all close contacts were quarantined and tested.COVID-19 mitigation measures at all 13 IDOC-operated correctional facilities included intensified cleaning and mandatory use of face masks for staff members and incarcerated persons (hand soap and four reusable face masks distributed to each incarcerated person), and periodic SARS-CoV-2 mass testing. Universal temperature checks and symptom screenings were conducted daily and at entry. New admissions were quarantined for 14 days and tested for SARS-CoV-2 before transfer to general housing. The percentage of positive test results from mass testing at IDOC facilities with work-release ranged from 1% to 92% . All casMitigation strategies at IDOC facilities with work-release programs included 1) providing temperature checks and symptom screening before incarcerated persons departed to work sites and upon their return; 2) ensuring that face masks were worn during transport; 3) requiring employers to provide a COVID-19 safety plan; 4) documenting work-site safety measures, including physical distancing, mask use, and hand hygiene; and 5) conducting employer site checks to confirm safety standards were being maintained. Three IDOC facilities with work-release programs erected temporary housing structures to create more space for isolation and quarantine. Work-release was suspended temporarily at three facilities  to help control outbreaks .Collaborative public health response initiatives were also implemented. IDHW hosted weekly calls with representatives from IDOC, the health care contractor, local public health districts, and Boise VA Medical Center laboratory to share information on cases, clinical capacity, mass testing, and public health guidance. IDOC regularly provided lists of CRC work sites to IDHW; public health officials notified IDOC of work sites considered to be high-risk for COVID-19 transmission  or those experiencing active outbreaks. These collaborations increased testing availability and prompted IDOC to reassign some work-release participants to lower-risk work sites.CDC COVID-19 guidance advises correctional facilities to consider suspending work-release programs, especially when the work-release assignment is in a congregate setting, such as a food processing plant (Prompt isolation of persons with COVID-19, quarantine of close contacts, and mass testing helped control SARS-CoV-2 transmission in IDOC correctional facilities operating work-release programs. Mass testing of incarcerated persons detected more cases than did symptom-based or close contact testing. Most COVID-19 cases occurred in asymptomatic persons, providing further evidence that symptom screening alone is insufficient for case detection (Challenges for outbreak control in correctional facilities with work-release programs are similar to those usually faced by correctional and detention facilities, including congregate living and lack of available housing to quarantine close contacts individually (The findings in this report are subject to at least six limitations. First, information on individual work assignments of COVID-19 patients was only available for two correctional facilities. Second, it is unknown whether SARS-CoV-2 transmission from incarcerated workers to nonincarcerated employees occurred. Third, COVID-19 mitigation measures in the surrounding communities were not assessed. Fourth, testing practices might have varied across facilities, and mass testing was not conducted at set intervals. Fifth, self-reporting of symptoms was considered unreliable, and the presence of symptoms before or after testing was not recorded. Finally, findings are not generalizable to all correctional and detention facilities with work-release programs.The benefits of work-release programs include the increased likelihood of postrelease employment and decreased recidivism (Correctional and detention facilities face unique challenges for controlling transmission of SARS-CoV-2. Work-release programs, which place incarcerated persons in community businesses, might pose additional risks.As of November 30, 2020, a total of 382 outbreak-related COVID-19 cases were identified among incarcerated persons at five Idaho correctional facilities with work-release programs; two outbreaks were linked to work at food processing plants.Correctional facilities operating work-release programs should implement measures to reduce SARS-CoV-2 transmission, including mass testing and working with public health officials to identify high-risk work sites. Incarcerated persons participating in work-release should be included in COVID-19 vaccination plans.


ABSTRACT IMPACT: We hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases. OBJECTIVES/GOALS: The role of IFNLR1 receptor dynamics and plasticity in regulating the type-III IFN response is largely unknown. As a specific, powerful component of innate immunity, understanding how the type-III IFN system is regulated could lead to the development of novel therapeutic targets and strategies to face a multitude of viral illnesses. METHODS/STUDY POPULATION: To facilitate our investigation, we will generate doxycycline-inducible FLAG-tagged IFNLR1-expression plasmids representing all known transcriptional variants. These plasmids will allow us to: 1) Evaluate the effect of IFNLR1 surface abundance on the type-III IFN transcriptional profile and 2) Assess the extent of IFNLR1-FLAG co-localization with several notable intracellular structures using immunofluorescence, before and after stimulation with IFNL3. RESULTS/ANTICIPATED RESULTS: We have successfully generated three IFNLR1-FLAG transcriptional variants and confirmed inducible-expression and function in vitro. We are currently assessing the role of surface abundance, internalization, differential isoform expression, and trafficking. DISCUSSION/SIGNIFICANCE OF FINDINGS: By completing this study, we hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases.