Long-acting lenacapavir protects macaques against intravenous challenge with simian-tropic HIV.

BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.

Increasing Medication Access by Promoting Appropriate Use of Multi-dose Vials.

Medication shortage is a problem that affects patients, providers, and institutions of all sizes and scope across the United States. The objective of this quality improvement project was to promote the appropriate use of multi-dose vials (MDVs) by anesthesia providers at an independent plastic surgery office. Multi-dose vials can be used to decrease waste and potentially cost, thus increasing access to necessary medications for the patients at this practice. A focus group was used to obtain an understanding of barriers to the use of MDVs at this practice. A focused E-learning module on safe use based on established guidelines was then created, and a simplified flow sheet was implemented and placed in medication preparation areas as a cognitive aid. The education and flow sheet focused on identification and preparation of the medication area, proper identification of MDVs versus single-use vials, hand hygiene, proper beyond-use labeling, septum cleaning, use of a new sterile syringe and needle, and administration time frames. Provider feedback included high levels of satisfaction with the E-learning module. Our comparison of the use of ketamine from MDVs during the pre- and postimplementation phases showed a 14% increase in the number of doses used per vial. This finding suggests that were similar practices implemented at a larger site with MDVs of medications other than ketamine, resources could be impacted to manage shortages and increase access to medications.

Gene-Specific Linear Trends Constrain Transcriptional Variability of the Toll-like Receptor Signaling.

Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1ß involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.

A biobank of small cell lung cancer CDX models elucidates inter- and intratumoral phenotypic heterogeneity.

Although small cell lung cancer (SCLC) is treated as a homogeneous disease, biopsies and preclinical models reveal heterogeneity in transcriptomes and morphology. SCLC subtypes were recently defined by neuroendocrine transcription factor (NETF) expression. Circulating-tumor-cell-derived explant models (CDX) recapitulate donor patients' tumor morphology, diagnostic NE marker expression and chemotherapy responses. We describe a biobank of 38 CDX models, including six CDX pairs generated pretreatment and at disease progression revealing complex intra- and intertumoral heterogeneity. Transcriptomic analysis confirmed three of four previously described subtypes based on ASCL1, NEUROD1 and POU2F3 expression and identified a previously unreported subtype based on another NETF, ATOH1. We document evolution during disease progression exemplified by altered MYC and NOTCH gene expression, increased 'variant' cell morphology, and metastasis without strong evidence of epithelial to mesenchymal transition. This CDX biobank provides a research resource to facilitate SCLC personalized medicine.

Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation.

Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.

Examination of marine and vegetable oil oxidation data from a multi-year, third-party database.

Fish oil (FO) products constitute good sources of omega-3 fats. Oxidation data from a large third-party database of 1900 + globally-sourced FO samples were assessed. In FO products, for peroxide value (PV), 13.9% exceeded 5 mEq O2/kg (2.2% >10); for acid value (AcV) 2.1% exceeded 3 mg KOH/g, while for p-anisidine value (pAV) in unflavoured oils, 6.1% exceeded 20, (3.8% >30), and 8.8% exceeded TOTOX limits (26). Additionally, we compared FO with other dietary oils. The FO median PV was similar to those of algal and sunflower oils, 4.8-fold greater than krill oil, and 5.2-fold less than extra-virgin olive oil. The median pAV differed non-significantly among oils. The FO median AcV was similar to those of algal and extra-virgin olive oils, 3.4-fold greater than sunflower oil, and 11.9-fold less than krill oil. This study has provided new insight that retail FO products predominantly meet regulatory guidelines and are comparable in oxidative status to other dietary oils.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1ß instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

Puf3p induces translational repression of genes linked to oxidative stress.

In response to stress, the translation of many mRNAs in yeast can change in a fashion discordant with the general repression of translation. Here, we use machine learning to mine the properties of these mRNAs to determine specific translation control signals. We find a strong association between transcripts acutely translationally repressed under oxidative stress and those associated with the RNA-binding protein Puf3p, a known regulator of cellular mRNAs encoding proteins targeted to mitochondria. Under oxidative stress, a PUF3 deleted strain exhibits more robust growth than wild-type cells and the shift in translation from polysomes to monosomes is attenuated, suggesting puf3Δ cells perceive less stress. In agreement, the ratio of reduced:oxidized glutathione, a major antioxidant and indicator of cellular redox state, is increased in unstressed puf3Δ cells but remains lower under stress. In untreated conditions, Puf3p migrates with polysomes rather than ribosome-free fractions, but this is lost under stress. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) of Puf3p targets following affinity purification shows Puf3p-mRNA associations are maintained or increased under oxidative stress. Collectively, these results point to Puf3p acting as a translational repressor in a manner exceeding the global translational response, possibly by temporarily limiting synthesis of new mitochondrial proteins as cells adapt to the stress.

Genetic variations at loci involved in the immune response are risk factors for hepatocellular carcinoma.

UNLABELLED: Primary liver cancer is the third most common cause of cancer-related death worldwide, with a rising incidence in Western countries. Little is known about the genetic etiology of this disease. To identify genetic factors associated with hepatocellular carcinoma (HCC) and liver cirrhosis (LC), we conducted a comprehensive, genome-wide variation analysis in a population of unrelated Asian individuals. Copy number variation (CNV) and single nucleotide polymorphisms (SNPs) were assayed in peripheral blood with the high-density Affymetrix SNP6.0 microarray platform. We used a two-stage discovery and replication design to control for overfitting and to validate observed results. We identified a strong association with CNV at the T-cell receptor gamma and alpha loci (P < 1 × 10(-15)) in HCC cases when contrasted with controls. This variation appears to be somatic in origin, reflecting differences between T-cell receptor processing in lymphocytes from individuals with liver disease and healthy individuals that is not attributable to chronic hepatitis virus infection. Analysis of constitutional variation identified three susceptibility loci including the class II MHC complex, whose protein products present antigen to T-cell receptors and mediate immune surveillance. Statistical analysis of biologic networks identified variation in the "antigen presentation and processing" pathway as being highly significantly associated with HCC (P = 1 × 10(-11)). SNP analysis identified two variants whose allele frequencies differ significantly between HCC and LC. One of these (P = 1.74 × 10(-12)) lies in the PTEN homolog TPTE2. CONCLUSION: Combined analysis of CNV, individual SNPs, and pathways suggest that HCC susceptibility is mediated by germline factors affecting the immune response and differences in T-cell receptor processing.

Multiobjective evolutionary optimisation for surface-enhanced Raman scattering.

In most optimisation experiments, a single parameter is first optimised before a second and then third one are subsequently modified to give the best result. By contrast, we believe that simultaneous multiobjective optimisation is more powerful; therefore, an optimisation of the experimental conditions for the colloidal SERS detection of L-cysteine was carried out. Six aggregating agents and three different colloids (citrate, borohydride and hydroxylamine reduced silver) were tested over a wide range of concentrations for the enhancement and the reproducibility of the spectra produced. The optimisation was carried out using two methods, a full factorial design (FF, a standard method from the experimental design literature) and, for the first time, a multiobjective evolutionary algorithm (MOEA), a method more usually applied to optimisation problems in computer science. Simulation results suggest that the evolutionary approach significantly out-performs random sampling. Real experiments applying the evolutionary method to the SERS optimisation problem led to a 32% improvement in enhancement and reproducibility compared with the FF method, using far fewer evaluations.

Long space missions, gene therapy, and the vital role of magnesium: a three-pronged plan for the next 50 years.

Since pharmaceuticals cannot be used in space until liver and kidney dysfunctions are corrected, and with invariable malabsorption, it appears there is no alternative other than to use subcutaneous magnesium (Mg) replacements in the presence of deficiencies and use of gene therapy. I suggest beginning with the correction of as many as four gene deficiencies: atrial natriuretic peptide (ANP), nitric oxide (NO), vascular endothelial growth factor (VEGF), and erythropoietin (EPO), all as well as Mg related to perfusion and angiogenesis. There is no evidence of significant lunar radiation levels in the absence of a solar storm. It could then be determined whether this has resulted in correction of liver and kidney dysfunction. If this persists, serial additions of gene therapy will be required determining the effect of each individual gene trial on organ function. Microgravity and endothelial gaps with leaks trigger reduced plasma volume. Partial correction by use of a plasma volume substitute and development of a delivery device may reduce complexity of gene therapy. Research would be conducted both on Earth and in microgravity, with the development of subcutaneous pharmaceuticals and Mg, and a space walk-reliable subcutaneous silicon device, given that no replenishable subcutaneous device is presently available. A three-pronged approach provides a plan for the next 50 years: A. complete correction of a Mg deficit; B. partial replacement with plasma volume substitutes, and C. multiple gene factor strategy.

Rrp1b, a new candidate susceptibility gene for breast cancer progression and metastasis.

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

The BRCA1 Ashkenazi founder mutations occur on common haplotypes and are not highly correlated with anonymous single nucleotide polymorphisms likely to be used in genome-wide case-control association studies.

BACKGROUND: We studied linkage disequilibrium (LD) patterns at the BRCA1 locus, a susceptibility gene for breast and ovarian cancer, using a dense set of 114 single nucleotide polymorphisms in 5 population groups. We focused on Ashkenazi Jews in whom there are known founder mutations, to address the question of whether we would have been able to identify the 185delAG mutation in a case-control association study (should one have been done) using anonymous genetic markers. This mutation is present in approximately 1% of the general Ashkenazi population and 4% of Ashkenazi breast cancer cases. We evaluated LD using pairwise and haplotype-based methods, and assessed correlation of SNPs with the founder mutations using Pearson's correlation coefficient. RESULTS: BRCA1 is characterized by very high linkage disequilibrium in all populations spanning several hundred kilobases. Overall, haplotype blocks and pair-wise LD bins were highly correlated, with lower LD in African versus non-African populations. The 185delAG and 5382insC founder mutations occur on the two most common haplotypes among Ashkenazim. Because these mutations are rare, even though they are in strong LD with many other SNPs in the region as measured by D-prime, there were no strong associations when assessed by Pearson's correlation coefficient, r (maximum of 0.04 for the 185delAG). CONCLUSION: Since the required sample size is related to the inverse of r, this suggests that it would have been difficult to map BRCA1 in an Ashkenazi case-unrelated control association study using anonymous markers that were linked to the founder mutations.

Systematic analysis of genetic alterations in tumors using Cancer Genome WorkBench (CGWB).

Systematic investigations of genetic changes in tumors are expected to lead to greatly improved understanding of cancer etiology. To meet the analytical challenges presented by such studies, we developed the Cancer Genome WorkBench (http://cgwb.nci.nih.gov), the first computational platform to integrate clinical tumor mutation profiles with the reference human genome. A novel heuristic algorithm, IndelDetector, was developed to automatically identify insertion/deletion (indel) polymorphisms as well as indel somatic mutations with high sensitivity and accuracy. It was incorporated into an automated pipeline that detects genetic alterations and annotates their effects on protein coding and 3D structure. The ability of the system to facilitate identifying genetic alterations is illustrated in three projects with publicly accessible data. Mutagenesis in tumor DNA replication leading to complex genetic changes in the EGFR kinase domain is suggested by a novel deletion-insertion combination observed in paired tumor-normal lung cancer resequencing data. Automated analysis of 152 genes resequenced by the SeattleSNPs group was able to identify 91% of the 1251 indel polymorphisms discovered by SeattleSNPs. In addition, our system discovered 518 novel indels in this data set, 451 of which were found to be valid by manual inspection of sequence traces. Our experience demonstrates that CGWB not only greatly improves the productivity and the accuracy of mutation identification, but also, through its data integration and visualization capabilities, facilitates identification of underlying genetic etiology.

SNPdetector: a software tool for sensitive and accurate SNP detection.

Identification of single nucleotide polymorphisms (SNPs) and mutations is important for the discovery of genetic predisposition to complex diseases. PCR resequencing is the method of choice for de novo SNP discovery. However, manual curation of putative SNPs has been a major bottleneck in the application of this method to high-throughput screening. Therefore it is critical to develop a more sensitive and accurate computational method for automated SNP detection. We developed a software tool, SNPdetector, for automated identification of SNPs and mutations in fluorescence-based resequencing reads. SNPdetector was designed to model the process of human visual inspection and has a very low false positive and false negative rate. We demonstrate the superior performance of SNPdetector in SNP and mutation analysis by comparing its results with those derived by human inspection, PolyPhred (a popular SNP detection tool), and independent genotype assays in three large-scale investigations. The first study identified and validated inter- and intra-subspecies variations in 4,650 traces of 25 inbred mouse strains that belong to either the Mus musculus species or the M. spretus species. Unexpected heterozygosity in CAST/Ei strain was observed in two out of 1,167 mouse SNPs. The second study identified 11,241 candidate SNPs in five ENCODE regions of the human genome covering 2.5 Mb of genomic sequence. Approximately 50% of the candidate SNPs were selected for experimental genotyping; the validation rate exceeded 95%. The third study detected ENU-induced mutations (at 0.04% allele frequency) in 64,896 traces of 1,236 zebra fish. Our analysis of three large and diverse test datasets demonstrated that SNPdetector is an effective tool for genome-scale research and for large-sample clinical studies. SNPdetector runs on Unix/Linux platform and is available publicly (http://lpg.nci.nih.gov).

Intravenous cyclosporine for the treatment of severe steroid refractory ulcerative colitis: what is the cost?

PURPOSE: Intravenous cyclosporine often is used to treat patients with severe steroid refractory colitis secondary to ulcerative colitis in an attempt to avoid urgent total abdominal colectomy. The purpose of this study was to evaluate the success and cost of cyclosporine. METHODS: A retrospective, chart review of all patients from 1996 to 2002 who were treated with cyclosporine and/or had a three-stage ileal pouch-anal anastomosis for severe steroid refractory colitis at our institution was performed. Patients were divided into three groups: TAC and CyA: patients who failed cyclosporine and had urgent total abdominal colectomy on the same admission; TAC no CyA: patients who had an urgent total abdominal colectomy without cyclosporine; and CyA only: patients treated successfully with cyclosporine and discharged without surgery. A subgroup of patients who had an ileal pouch-anal anastomosis was identified from each group. Cost data were obtained from the hospital's financial records. RESULTS: Forty-one patients (25 males) were identified. Twenty-nine patients received cyclosporine for severe steroid refractory colitis. Of these, 18 (62 percent) failed and underwent total abdominal colectomy on the same admission. Eleven (38 percent) responded to the cyclosporine and were discharged. Of the 11, 4 never had surgery, 1 had a three-stage ileal pouch-anal anastomosis, 5 had a two-stage ileal pouch-anal anastomosis, and 1 had a total abdominal colectomy only. Only 14 percent of patients avoided colectomy in the long-term. Complications of cyclosporine occurred in 8 patients (28 percent), and surgical complications occurred in 12 patients. CONCLUSIONS: The highest costs, highest length of stay, and highest number of overall complications were found in the group of patients who failed intravenous cyclosporine and required colectomy during the same hospitalization.

The case for a subcutaneous magnesium product and delivery device for space missions.

Cardiovascular (CV) complications, associated with space flight (SF), are caused by microgravity, hypokinesia and radiation, particularly beyond earth orbit, with all three conducive to oxidative stress. Except for emergencies, pharmaceuticals appear to be contraindicated, because of unpredictable side effects from malabsorption (M) and potential hepatic and renal impairment. Magnesium (Mg) depletion and elevations of cytokines (interleukin 6) occur during SF, conducive to self-sustaining vascular inflammation mechanisms. There are potential endothelial injuries (EI) and reduced Cyclic GMP (a second messenger of nitric oxide: NO) and elevated urinary excretion of C-peptide (insulin resistance: IR). Recent findings that show reductions in vascular endothelial growth factor (VEGF) suggest that this may result from SF-related thrombocytopenia since platelets (P) are the major source of VEGF, and that NO might play a role. Both VEGF and Mg are vital for angiogenesis, endothelial function and reendothelialization. Insulin is necessary for VEGF expression. To prevent SF-related CV complications in the presence of IR and M and with the potential for renal insufficiency, closely monitored subcutaneous (SC) Mg should be provided. The dosage can be monitored by sublingual intracellular Mg assays. Needed is development of a SC Mg reservoir device, which can be replenished before extra-vehicular activities (EVA) and which must be reliable despite vigorous movements during EVA, that can last up to 8 hours. This could also be protective against decompression sickness and EVA-related 100% oxygen requirements before and during this activity, both of which predispost to EI.

An open-label trial of L-glucose as a colon-cleansing agent before colonoscopy.

BACKGROUND: The high volume and poor palatability of standard colon cleansers remain obstacles to colonoscopy for many patients. Significant electrolyte disturbances and fluid balance alterations may occur with available agents. L-glucose, the stereoisomer of D-glucose, has laxative effects that make it potentially useful as a colon-cleansing agent. This study evaluated the safety and efficacy of L-glucose as a bowel cleanser before colonoscopy. METHODS: Thirty healthy individuals (age range 34-70 years) scheduled to undergo outpatient colonoscopy were given 24 g of L-glucose in 8 ounces of water to cleanse the colon. Nonblinded endoscopists rated the quality of the preparation based on established criteria. Laboratory studies were monitored before and after administration of the L-glucose and on the day after colonoscopy; adverse clinical events were also monitored. RESULTS: L-glucose administration resulted in excellent or good preparations in 80% (24/30) of the patients and fair or poor preparations in 20%. Average water consumption was 48 ounces. No adverse event occurred, and no significant laboratory test abnormalities were identified. CONCLUSIONS: Eighty percent of patients who prepared for colonoscopy by ingestion of L-glucose had good or excellent preparations. The L-glucose preparation was palatable, and its efficacy and safety appear equivalent to currently available colon-cleansing agents.