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With the emergence of the COVID-19 pandemic and shortage of adequate personal protective equipment (PPE), hospitals implemented inpatient telemedicine measures to ensure operational readiness and a safe working environment for clinicians. The utility and sustainability of inpatient telemedicine initiatives need to be evaluated as the number of COVID-19 inpatients is expected to continue declining. In this viewpoint, we describe the use of a rapidly deployed inpatient telemedicine workflow at a large academic medical center and discuss the potential impact on PPE savings. In early 2020, videoconferencing software was installed on patient bedside iPads at two academic medical center teaching hospitals. An internal website allowed providers to initiate video calls with patients in any patient room with an activated iPad, including both COVID-19 and non–COVID-19 patients. Patients were encouraged to use telemedicine technology to connect with loved ones via native apps or videoconferencing software. We evaluated the use of telemedicine technology on patients’ bedside iPads by monitoring traffic to the internal website. Between May 2020 and March 2021, there were a total of 1240 active users of the Video Visits website (mean 112.7, SD 49.0 connection events per month). Of these, 133 (10.7%) connections were made. Patients initiated 63 (47.4%) video calls with family or friends and sent 37 (27.8%) emails with videoconference connection instructions. Providers initiated a total of 33 (24.8%) video calls with the majority of calls initiated in August (n=22, 67%). There was a low level of adoption of inpatient telemedicine capability by providers and patients. With sufficient availability of PPE, inpatient providers did not find a frequent need to use the bedside telemedicine technology, despite a high census of patients with COVID-19. Compared to providers, patients used videoconferencing capabilities more frequently in September and October 2020. We did not find savings of PPE associated with the use of inpatient telemedicine.

Context.--Urinalysis instrument-specific dip strips offer physicians qualitative results for actionable analytes (protein, glucose, leukocyte esterase, nitrates, hemoglobin, and ketones). Objective.--To explain a strategy implemented to support clinical decision-making by providing urine quantification of protein, glucose, white blood cells (WBCs), and red blood cells because of urine strip shortages. Design.--During shortages, we implemented an automated algorithm that triggered sending urine samples to the automation line for quantification of protein and glucose and ensured that urine microscopy was performed to obtain WBC and red blood cell counts. The algorithm printed 2 labels so nursing staff would collect 2 specimens. We monitored the turnaround time from the specimen being received in the laboratory to result verification, ensured that the culture reflex order was triggered, and tracked complaints by physicians regarding not having usual urinalysis results. Prior to implementation, correlation between sample types for protein and glucose measurement was found acceptable. Results.--The algorithm was put in place twice during 2022. The turnaround time of urine microscopic study was identical to that obtained when the urinalysis was done with the strips; however, the quantification of glucose and protein took approximately 30 minutes more. Urine reflex cultures were triggered correctly with the algorithm, as they were derived entirely from a WBC count higher than 10 per high-power field. During the shortage period we had only 1 complaint, by a physician wanting to have results of nitrates. Conclusions.--During urine strip shortages, we successfully implemented a diversion algorithm that provided actionable urinalysis analytes in a timely manner with minimal provider complaints., doi: 10.5858/arpa.2023-0217-OA The COVID-19 pandemic has caused shortages of many resources required for laboratory testing, such as swabs, collection tubes, reagents, and even personnel. Mitigation strategies have been used to [...]

* Context.--Genetic profiling data of prostatic adenocarcinoma are derived from predominantly White patients. In African Americans, prostatic adenocarcinoma has a poorer prognosis, raising the possibility of distinct genetic alterations. Objective.--To investigate the genomic alterations of prostatic adenocarcinoma metastatic to regional lymph nodes in African American patients, with an emphasis on SPOP mutation. Design.--We retrospectively reviewed African American patients with pN1 prostatic adenocarcinoma managed with radical prostatectomy and lymph node dissection. Comprehensive molecular profiling was performed, and androgen receptor signaling scores were calculated. Results.--Nineteen patients were included. The most frequent genetic alteration was SPOP mutations (5 of 17; 29.4% [95% CI: 10.3-56.0]). While most alterations were associated with a high androgen receptor signaling score, mutant SPOP was exclusively associated with a low median and interquartile range (IQR) androgen receptor signaling score (0.788 [IQR 0.765-0.791] versus 0.835 [IQR 0.828-0.842], P = .003). In mutant SPOP, mRNA expression of SPOP inhibitor G3BP1 and SPOP substrates showed a significantly decreased expression of AR (33.40 [IQR 28.45-36.30] versus 59.53 [IQR 53.10-72.83], P = .01), TRIM24 (3.95 [IQR 3.28-5.03] versus 9.80 [IQR 7.39-11.70], P = .008), and NCOA3 (15.19 [IQR 10.59-15.93] versus 21.88 [IQR 18.41-28.33], P = .046). Conclusions.--African American patients with metastatic prostate adenocarcinoma might have a higher prevalence of mutant SPOP (30%), compared to ~10% in unselected cohorts with lower expressions of SPOP substrates. In our study, in patients with mutant SPOP, the mutation was associated with decreased SPOP substrate expression and androgen receptor signaling, raising concern for suboptimal efficacy of androgen deprivation therapy in this subset of patients., Prostatic adenocarcinoma (PCa) is the third most common cancer in the United States, with approximately 1 in 9 men developing PCa during their lifetime. (1) At presentation, most PCas are [...]

Genome-wide CRISPR-based screening is a powerful tool in forward genetics, enabling biologic discovery by linking a desired phenotype to a specific genetic perturbation. However, hits from a genome-wide screen require individual validation to reproduce and accurately quantify their effects outside of a pooled experiment. Here, we describe a step-by-step protocol to rapidly assess the effects of individual sgRNAs from CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. All steps, including cloning, lentivirus generation, cell transduction, and phenotypic readout, can be performed entirely in 96-well plates. The system is highly flexible in both cell type and selection system, requiring only that the phenotype(s) of interest be read out via flow cytometry. We expect that this protocol will provide researchers with a rapid way to sift through potential screening hits, and prioritize them for deeper analysis in more complex in vitro or even in vivo systems. Graphical abstract.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Subcutaneous panniculitis-like T-cell lymphoma is a cutaneous lymphoma characterized by CD8+ T-cell infiltrate in the subcutis that is rare in children. Acute lymphoblastic lymphoma is the most common pediatric malignancy and often presents with fevers and pancytopenia. Herein, we report 2 pediatric patients presenting with subcutaneous panniculitis-like T-cell lymphoma and B-cell acute lymphoblastic lymphoma, distinct hematologic malignancies arising from different lymphoid lineages, with no identifiable germline cancer predisposition.

Although human interactions with cats are often even typically analyzed in the context of domesticity, with a focus on what sorts of interactions might make both people and cats "happy at home," a large number of cats in the world live, for one reason or another, beyond the bounds of domesticity. Human interactions with these more or less free-living cats raise deeply controversial questions about how both the cats and the people they interact with should be sensibly managed, and about the moral imperatives that ought to guide the management of their interactions through the laws and public policies regulating both human interactions with pets and with wildlife. We review the geography of human interactions with cats living beyond the bounds of domesticity. We acknowledge the contributions made to ideas about how to manage cats by the animal protection movement. We review the tensions that have emerged over time between advocates for the eradication of free-living cats, because of the impacts they have on native wildlife species, and those who have imagined alternatives to eradication, most notably one or another variant of trap-neuter-return (TNR). The conflict over how best to deal with cats living beyond the bounds of domesticity and their wildlife impacts raises the prospect of stalemate, and we canvass and critique possibilities for moving beyond that stalemate.

Black carbon (BC) absorbs solar radiation, leading to a strong but uncertain warming effect on climate. A key challenge in modeling and quantifying BC's radiative effect on climate is predicting enhancements in light absorption that result from internal mixing between BC and other aerosol components. Modeling and laboratory studies show that BC, when mixed with other aerosol components, absorbs more strongly than pure, uncoated BC; however, some ambient observations suggest more variable and weaker absorption enhancement. We show that the lower-than-expected enhancements in ambient measurements result from a combination of two factors. First, the often used spherical, concentric core-shell approximation generally overestimates the absorption by BC. Second, and more importantly, inadequate consideration of heterogeneity in particle-to-particle composition engenders substantial overestimation in absorption by the total particle population, with greater heterogeneity associated with larger model-measurement differences. We show that accounting for these two effects-variability in per-particle composition and deviations from the core-shell approximation-reconciles absorption enhancement predictions with laboratory and field observations and resolves the apparent discrepancy. Furthermore, our consistent model framework provides a path forward for improving predictions of BC's radiative effect on climate.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current clinical therapies and known causes of familial hypercholesterolemia. We show that Cold Shock Domain-Containing Protein E1 (CSDE1) enhances hepatic LDLR mRNA decay via its 3’ untranslated region to regulate atherogenic lipoproteins in vivo . Using parallel phenotypic genome-wide CRISPR interference screens, we found 40 specific regulators of the LDLR left unidentified by observational human genetics. Among these, we show that CSDE1 regulates the LDLR at least as strongly as the mechanistically distinct pathways exploited by the best available clinical therapies: statins and PCSK9 inhibitors. Additionally, we show that hepatic gene silencing of Csde1 treats diet-induced dyslipidemia in mice better than that of Pcsk9 . Our results reveal the therapeutic potential of manipulating a newly identified key factor in the post-transcriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. We anticipate that our approach of modelling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, will serve as a generalizable template for the identification of therapeutic targets in other human disease states. One Sentence Summary A genome-wide CRISPR screen identifies CSDE1 as a key regulator of hepatic LDLR mRNA decay in vivo , making it a promising target for heart disease. Graphical Abstract

The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Activation of JAK/STAT signaling is thought to be a central component of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor Food and Drug Administration (FDA) approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Herein, we validated in vitro, in stromal-responsive human myeloma cell lines, and in vivo, in orthotopic disseminated xenograft models of myeloma, that tofacitinib showed efficacy in myeloma models. Furthermore, tofacitinib strongly synergized with venetoclax in coculture with bone marrow stromal cells but not in monoculture. Surprisingly, we found that ruxolitinib, an FDA approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. Transcriptome analysis and unbiased phosphoproteomics revealed that bone marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib reverses the growth-promoting effects of the tumor microenvironment. As tofacitinib is already FDA approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.

Interleukin-2 (IL-2) stimulates both activated CD4+ and CD8+ T cells to proliferate. IL-2 signals through an identical receptor complex and promotes the same dose-dependent phosphorylation of the canonical transcription factor STAT5 in both cell types. Despite this, CD8+ T cells enter the S phase earlier and proliferate to a greater extent than do CD4+ T cells in response to IL-2. We identified distinct IL-2 signaling dynamics in CD4+ and CD8+ T cells. In IL-2-stimulated CD8+ T cells, STAT5 phosphorylation increased rapidly and was sustained for 6 hours. In contrast, CD4+ T cells had a biphasic response, with maxima at 15 min and 2 to 4 hours after stimulation. Both cell types required vesicular trafficking, but only CD4+ T cells required new protein synthesis to maintain high phosphorylation of STAT5. Two subunits of the IL-2 receptor, IL-2Rβ and IL-2Rγ, were twice as abundant in CD8+ T cells than in CD4+ T cells. Reduction of IL-2Rβ abundance by 50% was sufficient to convert CD8+ T cells to a CD4+ T cell-like signaling pattern and delay S phase entry. These results suggest that the larger pool of IL-2Rβ chains in CD8+ T cells is required to sustain IL-2 signaling and contributes to the quantitatively greater proliferative response to IL-2 relative to that of CD4+ T cells. This cell type-specific difference in IL-2Rβ abundance appears to tune responses, potentially preventing extensive, autoimmune proliferation of CD4+ T cells, while still enabling sufficient proliferation of CD8+ T cells to control viral infections.

Lymphocytes integrate Ag and cytokine receptor signals to make cell fate decisions. Using a specific reporter of TCR signaling that is insensitive to cytokine signaling, Nur77-eGFP, we identify a sharp, minimal threshold of cumulative TCR signaling required for proliferation in CD4 and CD8 T cells that is independent of both Ag concentration and affinity. Unexpectedly, IL-2 reduces this threshold in CD8 but not CD4 T cells, suggesting that integration of multiple mitogenic inputs may alter the minimal requirement for TCR signaling in CD8 T cells. Neither naive CD4 nor naive CD8 T cells are responsive to low doses of IL-2. We show that activated CD8 T cells become responsive to low doses of IL-2 more quickly than CD4 T cells, and propose that this relative delay in turn accounts for the differential effects of IL-2 on the minimal TCR signaling threshold for proliferation in these populations. In contrast to Nur77-eGFP, c-Myc protein expression integrates mitogenic signals downstream of both IL-2 and the TCR, yet marks an invariant minimal threshold of cumulative mitogenic stimulation required for cell division. Our work provides a conceptual framework for understanding the regulation of clonal expansion of CD8 T cells by subthreshold TCR signaling in the context of mitogenic IL-2 signals, thereby rendering CD8 T cells exquisitely dependent upon environmental cues. Conversely, CD4 T cell proliferation requires an invariant minimal intensity of TCR signaling that is not modulated by IL-2, thereby restricting responses to low-affinity or low-abundance self-antigens even in the context of an inflammatory milieu.