Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries.

Reducing disparities is vital for equitable access to precision treatments in cancer. Socioenvironmental factors are a major driver of disparities, but differences in genetic variation likely also contribute. The impact of genetic ancestry on prioritization of cancer targets in drug discovery pipelines has not been systematically explored due to the absence of pre-clinical data at the appropriate scale. Here, we analyze data from 611 genome-scale CRISPR/Cas9 viability experiments in human cell line models to identify ancestry-associated genetic dependencies essential for cell survival. Surprisingly, we find that most putative associations between ancestry and dependency arise from artifacts related to germline variants. Our analysis suggests that for 1.2-2.5% of guides, germline variants in sgRNA targeting sequences reduce cutting by the CRISPR/Cas9 nuclease, disproportionately affecting cell models derived from individuals of recent African descent. We propose three approaches to mitigate this experimental bias, enabling the scientific community to address these disparities.

Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy.

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F1-ATPase inhibitor ATP5IF1. In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Predicting response to immune checkpoint blockade in NSCLC with tumour-only RNA-seq.

BACKGROUND: Targeted RNA sequencing (RNA-seq) from FFPE specimens is used clinically in cancer for its ability to estimate gene expression and to detect fusions. Using a cohort of NSCLC patients, we sought to determine whether targeted RNA-seq could be used to measure tumour mutational burden (TMB) and the expression of immune-cell-restricted genes from FFPE specimens and whether these could predict response to immune checkpoint blockade. METHODS: Using The Cancer Genome Atlas LUAD dataset, we developed a method for determining TMB from tumour-only RNA-seq and showed a correlation with DNA sequencing derived TMB calculated from tumour/normal sample pairs (Spearman correlation = 0.79, 95% CI [0.73, 0.83]. We applied this method to targeted sequencing data from our patient cohort and validated these results against TMB estimates obtained using an orthogonal assay (Spearman correlation = 0.49, 95% CI [0.24, 0.68]). RESULTS: We observed that the RNA measure of TMB was significantly higher in responders to immune blockade treatment (P = 0.028) and that it was predictive of response (AUC = 0.640 with 95% CI [0.493, 0.786]). By contrast, the expression of immune-cell-restricted genes was uncorrelated with patient outcome. CONCLUSION: TMB calculated from targeted RNA sequencing has a similar diagnostic ability to TMB generated from targeted DNA sequencing.

Movement if Life-Optimizing Patient Access to Total Joint Arthroplasty: Alcohol and Substance Abuse Disparities.

Alcohol use disorders (AUDs) and substance use disorders (SUDs) place patients undergoing total joint arthroplasty at notable risk for complications. AUD and SUD disproportionately affect vulnerable communities and often coexist. Following is a discussion of the presence of these disorders in vulnerable populations and approaches to screening for them to optimize care and reduce the risks of joint arthroplasty surgery. 25.1% of American adults report binge drinking in the past year, and 5.8% of American adults carry a diagnosis of AUD. Alcohol consumption and AUD disproportionately affect American Indians/Alaskan Natives, and heavy episodic drinking is highest in Latinx and American Indians. AUD is higher in those who are unemployed, have lower education level, and those who are single/divorced. Alcohol use in the preoperative period is associated with difficulty maintaining blood pressure during surgery, infections, wound disruptions, and increased length of stay. In addition, patients with AUD or unhealthy alcohol use have a greater comorbidity burden, including liver disease and dementia, that predisposes them to poor surgical outcomes. Optimization in these vulnerable populations include proper screening, cessation programs, psychosocial interventions, assessment of support systems, and pharmacologic interventions. 38% of adults battle a drug use disorder. Twenty-one million Americans have at least one addiction, but only 10% receive treatment. Rates of opioid use and opioid-related deaths have continued to rise. Recreational drug use is highest in American Indians. Marijuana use is highest in Black and Latinx lesbian, gay, and bisexual women. Overall, substance use is associated with depression and anxiety; discrimination based on race, ethnicity, sex, or sexual preference is also deeply interwoven with depression, anxiety, and substance use. Preoperative use of opioids is the number one predictor of prolonged chronic postoperative opioid use. Optimization in these vulnerable groups begins with appropriate screening, followed by psychosocial interventions, social work and substance abuse counseling, and pharmacologic therapies.

Interventional- and amputation-stage muscle proteomes in the chronically threatened ischemic limb.

BACKGROUND: Despite improved surgical approaches for chronic limb-threatening ischemia (CLTI), amputation rates remain high and contributing tissue-level factors remain unknown. The purpose of this study was twofold: (1) to identify differences between the healthy adult and CLTI limb muscle proteome, and (2) to identify differences in the limb muscle proteome of CLTI patients prior to surgical intervention or at the time of amputation. METHODS AND RESULTS: Gastrocnemius muscle was collected from non-ischemic controls (n = 19) and either pre-interventional surgery (n = 10) or at amputation outcome (n = 29) CLTI patients. All samples were subjected to isobaric tandem-mass-tag-assisted proteomics. The mitochondrion was the primary classification of downregulated proteins (> 70%) in CLTI limb muscles and paralleled robust functional mitochondrial impairment. Upregulated proteins (> 38%) were largely from the extracellular matrix. Across the two independent sites, 39 proteins were downregulated and 12 upregulated uniformly. Pre-interventional CLTI muscles revealed a robust upregulation of mitochondrial proteins but modest functional impairments in fatty acid oxidation as compared with controls. Comparison of pre-intervention and amputation CLTI limb muscles revealed mitochondrial proteome and functional deficits similar to that between amputation and non-ischemic controls. Interestingly, these observed changes occurred despite 62% of the amputation CLTI patients having undergone a prior surgical intervention. CONCLUSIONS: The CLTI proteome supports failing mitochondria as a phenotype that is unique to amputation outcomes. The signature of pre-intervention CLTI muscle reveals stable mitochondrial protein abundance that is insufficient to uniformly prevent functional impairments. Taken together, these findings support the need for future longitudinal investigations aimed to determine whether mitochondrial failure is causally involved in amputation outcomes from CLTI.

Preventing Home Medication Administration Errors.

Medication administration errors that take place in the home are common, especially when liquid preparations are used and complex medication schedules with multiple medications are involved; children with chronic conditions are disproportionately affected. Parents and other caregivers with low health literacy and/or limited English proficiency are at higher risk for making errors in administering medications to children in their care. Recommended strategies to reduce home medication errors relate to provider prescribing practices; health literacy-informed verbal counseling strategies (eg, teachback and showback) and written patient education materials (eg, pictographic information) for patients and/or caregivers across settings (inpatient, outpatient, emergency care, pharmacy); dosing-tool provision for liquid medication measurement; review of medication lists with patients and/or caregivers (medication reconciliation) that includes prescription and over-the-counter medications, as well as vitamins and supplements; leveraging the medical home; engaging adolescents and their adult caregivers; training of providers; safe disposal of medications; regulations related to medication dosing tools, labeling, packaging, and informational materials; use of electronic health records and other technologies; and research to identify novel ways to support safe home medication administration.

Contrast-enhanced ultrasound of the spleen, pancreas and gallbladder in children.

Gray-scale and color/power Doppler ultrasound (US) are the first-line imaging modalities to evaluate the spleen, gallbladder and pancreas in children. The increasing use of contrast-enhanced ultrasound (CEUS) as a reliable and safe method to evaluate liver lesions in the pediatric population promises potential for imaging other internal organs. Although CEUS applications of the spleen, gallbladder and pancreas have been well described in adults, they have not been fully explored in children. In this manuscript, we present an overview of the applications of CEUS for normal variants and diseases affecting the spleen, gallbladder and pancreas. We highlight a variety of cases as examples of how CEUS can serve in the diagnosis and follow-up for such diseases in children. Our discussion includes specific examination techniques; presentation of the main imaging findings in various benign and malignant lesions of the spleen, gallbladder and pancreas in children; and acknowledgment of the limitations of CEUS for these organs.

Contrast-enhanced ultrasound of benign and malignant liver lesions in children.

Contrast-enhanced ultrasound (CEUS) is increasingly being used in children. One of the most common referrals for CEUS performance is characterization of indeterminate focal liver lesions and follow-up of known liver lesions. In this setting, CEUS is performed with intravenous administration of ultrasound contrast agents (UCAs). When injected into a vein, UCA microbubbles remain confined within the vascular network until they dissipate. Therefore, visualization of UCA within the tissues and lesions corresponds to true blood flow. CEUS enables continuous, real-time observation of the enhancement pattern of a focal liver lesion, allowing in most cases for a definite diagnosis and obviating the need for further cross-sectional imaging or other interventional procedures. The recent approval of Lumason (Bracco Diagnostics, Monroe Township, NJ) for pediatric liver CEUS applications has spurred the widespread use of CEUS. In this review article we describe the role of CEUS in pediatric liver applications, focusing on the examination technique and interpretation of main imaging findings of the most commonly encountered benign and malignant focal liver lesions. We also compare the diagnostic performance of CEUS with other imaging modalities for accurate characterization of focal liver lesions.

Noncanonical open reading frames encode functional proteins essential for cancer cell survival.

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

Validity of Hemodynamic Monitoring Using Inert Gas Rebreathing Method in Patients With Chronic Heart Failure and Those Implanted With a Left Ventricular Assist Device.

OBJECTIVE: The present study assessed agreement between resting cardiac output estimated by inert gas rebreathing (IGR) and thermodilution methods in patients with heart failure and those implanted with a left ventricular assist device (LVAD). METHODS AND RESULTS: Hemodynamic measurements were obtained in 42 patients, 22 with chronic heart failure and 20 with implanted continuous flow LVAD (34 males, aged 50 ± 11 years). Measurements were performed at rest using thermodilution and IGR methods. Cardiac output derived by thermodilution and IGR were not significantly different in LVAD (4.4 ± 0.9 L/min vs 4.7 ± 0.8 L/min, P = .27) or patients with heart failure (4.4 ± 1.4 L/min vs 4.5 ± 1.3 L/min, P = .75). There was a strong relationship between thermodilution and IGR cardiac index (r = 0.81, P = .001) and stroke volume index (r = 0.75, P = .001). Bland-Altman analysis showed acceptable limits of agreement for cardiac index derived by thermodilution and IGR, namely, the mean difference (lower and upper limits of agreement) for patients with heart failure -0.002 L/min/m2 (-0.65 to 0.66 L/min/m2), and -0.14 L/min/m2 (-0.78 to 0.49 L/min/m2) for patients with LVAD. CONCLUSIONS: IGR is a valid method for estimating cardiac output and should be used in clinical practice to complement the evaluation and management of chronic heart failure and patients with an LVAD.

RAF1 rearrangements are common in pancreatic acinar cell carcinomas.

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.

PURPOSE: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor. EXPERIMENTAL DESIGN: BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system. RESULTS: Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy. CONCLUSIONS: This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

Chronic mild stress alters synaptic plasticity in the nucleus accumbens through GSK3ß-dependent modulation of Kv4.2 channels.

Although major depressive disorder (MDD) is highly prevalent, its pathophysiology is poorly understood. Recent evidence suggests that glycogen-synthase kinase 3ß (GSK3ß) plays a key role in memory formation, yet its role in mood regulation remains controversial. Here, we investigated whether GSK3ß activity in the nucleus accumbens (NAc) is associated with depression-like behaviors and synaptic plasticity. We performed whole-cell patch-clamp recordings of medium spiny neurons (MSNs) in the NAc and determined the role of GSK3ß in spike timing-dependent long-term potentiation (tLTP) in the chronic unpredictable mild stress (CUMS) mouse model of depression. To assess the specific role of GSK3ß in tLTP, we used in vivo genetic silencing by an adeno-associated viral vector (AAV2) short hairpin RNA against GSK3ß. In addition, we examined the role of the voltage-gated potassium Kv4.2 subunit, a molecular determinant of A-type K+ currents, as a potential downstream target of GSK3ß. We found increased levels of active GSK3ß and augmented tLTP in CUMS mice, a phenotype that was prevented by selective GSK3ß knockdown. Furthermore, knockdown of GSK3ß in the NAc ameliorated depressive-like behavior in CUMS mice. Electrophysiological, immunohistochemical, biochemical, and pharmacological experiments revealed that inhibition of the Kv4.2 channel through direct phosphorylation at Ser-616 mediated the GSK3ß-dependent tLTP changes in CUMS mice. Our results identify GSK3ß regulation of Kv4.2 channels as a molecular mechanism of MSN maladaptive plasticity underlying depression-like behaviors and suggest that the GSK3ß-Kv4.2 axis may be an attractive therapeutic target for MDD.

Invasive breast cancer over four decades reveals persisting poor metastatic outcomes in treatment resistant subgroup - the "ATRESS" phenomenon.

BACKGROUND: Major advances in breast cancer treatment have led to a reducuction in mortality. However, there are still women who are not cured. We hypothesize there is a sub-group of women with treatment-resistant cancers causing early death. METHODS: Between 1975 and 2006, 5392 women with invasive breast cancer underwent surgery at Guy's Hospital, London. Data on patient demographics, tumour characteristics, treatment regimens, local recurrence, secondary metastasis, and death were prospectively recorded. We considered four time periods (1975-1982, 1983-1990, 1991-1998, 1999-2006). Risks and time to event analysis were performed with Cox proportional hazards model and Kaplan-Meier estimation. RESULTS: Unadjusted hazard ratios for developing metastasis and overall mortality relative to the 1975-1982 cohort decreased steadily to 0.23 and 0.63, respectively in 1999-2006. However, metastasis-free interval shortened, with the proportion of women developing metastasis ≤5 years increasing from 73.9% to 83.0%. Furthermore, median post-metastatic survival decreased from 1.49 years to 0.94 years. Applying our risk criteria identified the presence of ±200 patients in each cohort who developed metastasis early and died within a much shorter time frame. CONCLUSIONS: Advances in treatment have decreased the risk of metastasis and improved survival in women with invasive breast cancer over the last 40 years. Despite this, a subpopulation with shorter metastasis-free and post-metastatic survival who are unresponsive to available treatment remains. This may be due to the ATRESS phenomenon (adjuvant therapy-related shortening of survival) secondary to preselection inherent in adjuvant therapy, successful treatment of less malignant tumour cells and treatment-induced resistance in the remaining tumour clones.

Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.

Assessing the role of toll-like receptor in isolated, standard and enriched housing conditions.

Depression is a common psychiatric disorder that has been poorly understood. Consequently, current antidepressant agents have clinical limitations. Until today, most have exhibited the slow onset of therapeutic action and, more importantly, their effect on remission has been minimal. Thus, the need to find new forms of therapeutic intervention is urgent. The inflammation hypothesis of depression is widely acknowledged and is one that theories the relationship between the function of the immune system and its contribution to the neurobiology of depression. In this research, we utilized an environmental isolation (EI) approach as a valid animal model of depression, employing biochemical, molecular, and behavioral studies. The aim was to investigate the anti-inflammatory effect of etanercept, a tumor necrosis factor-α inhibitor on a toll-like receptor 7 (TLR 7) signaling pathway in a depressive rat model, and compare these actions to fluoxetine, a standard antidepressant agent. The behavioral analysis indicates that depression-related symptoms are reduced after acute administration of fluoxetine and, to a lesser extent, etanercept, and are prevented by enriched environment (EE) housing conditions. Experimental studies were conducted by evaluating immobility time in the force swim test and pleasant feeling in the sucrose preference test. The mRNA expression of the TLR 7 pathway in the hippocampus showed that TLR 7, MYD88, and TRAF6 were elevated in isolated rats compared to the standard group, and that acute treatment with an antidepressant and anti-inflammatory drugs reversed these effects. This research indicates that stressful events have an impact on behavioral well-being, TLR7 gene expression, and the TLR7 pathway. We also found that peripheral administration of etanercept reduces depressive-like behaviour in isolated rats: this could be due to the indirect modulation of the TLR7 pathway and other TLRs in the brain. Furthermore, fluoxetine treatment reversed depressive-like behaviour and molecularly modulated the expression of TLR7, suggesting that fluoxetine exerts antidepressant effects partially by modulating the TLR7 signaling pathway.

Hi-Plex2: a simple and robust approach to targeted sequencing-based genetic screening.

We have previously reported Hi-Plex, a multiplex PCR methodology for building targeted DNA sequencing libraries that offers a low-cost protocol compatible with high-throughput processing. Here, we detail an improved protocol, Hi-Plex2, that more effectively enables the robust construction of small-to-medium panel-size libraries while maintaining low cost, simplicity and accuracy benefits of the Hi-Plex platform. Hi-Plex2 was applied to three panels, comprising 291, 740 and 1193 amplicons, targeting genes associated with risk for breast and/or colon cancer. We show substantial reduction of off-target amplification to enable library construction for small-to-medium-sized design panels not possible using the previous Hi-Plex chemistry.

An investigation on the impact of substrate type, temperature, and iodine on moon jellyfish production.

Jellyfish are a popular public aquarium species, however, their collection from natural populations is undesirable due to impact on species abundance and bycatch. Thus, a sustainable supply of jellyfish bred in-house would be highly desirable. Here we describe an investigation into developing a sustainable moon jellyfish, Aurelia aurita, breeding program by determining the impact of substrate type on reattachment of polyps and the influence of iodine and temperature on strobilation and ephyra production. To test whether reattachment and growth of moon jellyfish polyps are influenced by substrate type polyps were provided with anthropogenic and natural substrates after being dislodged in experimental aquaria. Polyps selectively re-attached to plastics rather than natural materials. However, polyp growth was similar on all tested substrates. We tested whether cooling and addition of iodine affected strobilation. A period of cooling of around 10 °C while also introducing soluble iodine to the polyps enhanced strobilation rate. This method produced ephyra at a reliable rate in captivity negating the need for collection of wild individuals providing a supply of individuals for exhibit and for conservation research within a public aquarium. These results demonstrate that plastics should be adopted as an easier to colonize substrate and the use of cooling with iodine addition can enhance sustainable breeding protocols of moon jellyfish and may be relevant to the production of comparable jellyfish species.

Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration.

The availability of multiple datasets comprising genome-scale RNAi viability screens in hundreds of diverse cancer cell lines presents new opportunities for understanding cancer vulnerabilities. Integrated analyses of these data to assess differential dependency across genes and cell lines are challenging due to confounding factors such as batch effects and variable screen quality, as well as difficulty assessing gene dependency on an absolute scale. To address these issues, we incorporated cell line screen-quality parameters and hierarchical Bayesian inference into DEMETER2, an analytical framework for analyzing RNAi screens ( https://depmap.org/R2-D2 ). This model substantially improves estimates of gene dependency across a range of performance measures, including identification of gold-standard essential genes and agreement with CRISPR/Cas9-based viability screens. It also allows us to integrate information across three large RNAi screening datasets, providing a unified resource representing the most extensive compilation of cancer cell line genetic dependencies to date.