Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Efficient Spectrum Occupancy Prediction Exploiting Multidimensional Correlations through Composite 2D-LSTM Models.

In cognitive radio systems, identifying spectrum opportunities is fundamental to efficiently use the spectrum. Spectrum occupancy prediction is a convenient way of revealing opportunities based on previous occupancies. Studies have demonstrated that usage of the spectrum has a high correlation over multidimensions, which includes time, frequency, and space. Accordingly, recent literature uses tensor-based methods to exploit the multidimensional spectrum correlation. However, these methods share two main drawbacks. First, they are computationally complex. Second, they need to re-train the overall model when no information is received from any base station for any reason. Different than the existing works, this paper proposes a method for dividing the multidimensional correlation exploitation problem into a set of smaller sub-problems. This division is achieved through composite two-dimensional (2D)-long short-term memory (LSTM) models. Extensive experimental results reveal a high detection performance with more robustness and less complexity attained by the proposed method. The real-world measurements provided by one of the leading mobile network operators in Turkey validate these results.

[Helicobacter pylori infection detected during upper gastrointestinal endoscopy]. / Disminución en la frecuencia de infección por Helicobacter pylori en endoscopías digestivas altas.

BACKGROUND: Helicobacter pylori (HP) is the most widespread chronic human infection worldwide and the most important pathogenic factor of gastric cancer. The calculated prevalence at the Clinical Hospital of the University of Chile from 2002 to 2005 was 44.9%. AIM: To determine the current prevalence of HP in patients undergoing an upper gastrointestinal endoscopy (UGI) and analyze its distribution according to age and endoscopic findings. MATERIAL AND METHODS: We reviewed 3.433 UGI performed during the year 2015, selecting those in which rapid urease test (RUT) was done. A positive RUT or a positive gastric biopsy (GB) were considered as HP infection. RESULTS: RUT was done in 1862 UGI (55%) performed in patients aged 51 ± 17 years, (66% women). In 23% of these endoscopies, the RUT was positive. A GB was obtained 43% of endoscopies and 30% were positive for HP. In 105 patients the RUT was negative and the GB positive (rendering a 19.5% false negative rate). HP was detected by RUT and GB in 29% of endoscopies. The highest prevalence of infection (38.1%) was found between 40 and 49 years. HP infection had odds ratio of 4.24 for nodular gastropathy, 2.63 for gastric ulcer and 2.14 for duodenal ulcer (p < 0.05). CONCLUSIONS: HP prevalence in our center decreased significantly from 44.9% to 28.9% in 11 years. False negative RUT results may bias this finding. The use of proton pump inhibitors and antimicrobials that can interfere with the detection of HP should be registered to properly analyze the results of the RUT.

Discovery of benzophosphadiazine drug candidate IDX375: A novel hepatitis C allosteric NS5B RdRp inhibitor.

Hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells, and as a consequence is an attractive target for selective inhibition. This paper describes the discovery of a novel family of HCV NS5B non-nucleoside inhibitors inspired by the bioisosterism between sulfonamide and phosphonamide. Systematic structural optimization in this new series led to the identification of IDX375, a potent non-nucleoside inhibitor that is selective for genotypes 1a and 1b. The structure and binding domain of IDX375 were confirmed by X-ray co-crystalisation study.

The discovery of IDX21437: Design, synthesis and antiviral evaluation of 2'-α-chloro-2'-ß-C-methyl branched uridine pronucleotides as potent liver-targeted HCV polymerase inhibitors.

Herein we describe the discovery of IDX21437 35b, a novel RPd-aminoacid-based phosphoramidate prodrug of 2'-α-chloro-2'-ß-C-methyluridine monophosphate. Its corresponding triphosphate 6 is a potent inhibitor of the HCV NS5B RNA-dependent RNA polymerase (RdRp). Despite showing very weak activity in the in vitro Huh-7 cell based HCV replicon assay, 35b demonstrated high levels of active triphosphate 6 in mouse liver and human hepatocytes. A biochemical study revealed that the metabolism of 35b was mainly attributed to carboxyesterase 1 (CES1), an enzyme which is underexpressed in HCV Huh-7-derived replicon cells. Furthermore, due to its metabolic activation, 35b was efficiently processed in liver cells compared to other cell types, including human cardiomyocytes. The selected RP diastereoisomeric configuration of 35b was assigned by X-ray structural determination. 35b is currently in Phase II clinical trials for the treatment of HCV infection.

Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study.

Time-efficient germ cell transplantation from goldfish (Carassius auratus) into adult common carp (Cyprinus carpio).

Germ cell transplantation in fish is a promising technique for surrogate broodstock parents with broader application in aquaculture and conserving endangered and valuable genetic resources. Herein, we describe the establishment of an intrapapillary xenogeneic transplant of germ cells from sexually mature goldfish (C. auratus) males into common carp (C. carpio) males cytoablated with a thermochemical treatment (two doses of busulfan at 40 mg/kg at 35°C). To analyze the presence and development of donor germ cells in recipient testes, donor germ cells were labeled with PKH26, a fluorescent cell membrane dye, before transplantation. Our results demonstrated that thermochemical treatment caused effective spermatogenesis suppression and pronounced germ cell loss. Moreover, transplanted spermatogonial cells were able to colonize the recipients' testes, resume spermatogenesis, and generate spermatozoa within eight weeks after germ cell transplantation. These findings suggested that recipient testes provided suitable conditions for the survival, colonization, proliferation, and differentiation of donor spermatogonia from a related species. This study indicated that recipients' testes exhibited a high degree of plasticity to accept and support xenogeneic donor germ cells, which were able to form sperm in a short time frame. This approach has significant implications for assisted animal reproduction, biotechnology, conservation, and the production of valuable genetic resources and endangered fish species.

A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-ß and BMP signaling in the Fsh-mediated spermatogonial fate.

Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-ß superfamily. On the other hand, the specific roles of TGF-ß and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-ß (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-ß or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-ß inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-ß and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-ß and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-ß subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-ß (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

The type II RAF inhibitor tovorafenib in relapsed/refractory pediatric low-grade glioma: the phase 2 FIREFLY-1 trial.

BRAF genomic alterations are the most common oncogenic drivers in pediatric low-grade glioma (pLGG). Arm 1 (n = 77) of the ongoing phase 2 FIREFLY-1 (PNOC026) trial investigated the efficacy of the oral, selective, central nervous system-penetrant, type II RAF inhibitor tovorafenib (420 mg m-2 once weekly; 600 mg maximum) in patients with BRAF-altered, relapsed/refractory pLGG. Arm 2 (n = 60) is an extension cohort, which provided treatment access for patients with RAF-altered pLGG after arm 1 closure. Based on independent review, according to Response Assessment in Neuro-Oncology High-Grade Glioma (RANO-HGG) criteria, the overall response rate (ORR) of 67% met the arm 1 prespecified primary endpoint; median duration of response (DOR) was 16.6 months; and median time to response (TTR) was 3.0 months (secondary endpoints). Other select arm 1 secondary endpoints included ORR, DOR and TTR as assessed by Response Assessment in Pediatric Neuro-Oncology Low-Grade Glioma (RAPNO) criteria and safety (assessed in all treated patients and the primary endpoint for arm 2, n = 137). The ORR according to RAPNO criteria (including minor responses) was 51%; median DOR was 13.8 months; and median TTR was 5.3 months. The most common treatment-related adverse events (TRAEs) were hair color changes (76%), elevated creatine phosphokinase (56%) and anemia (49%). Grade ≥3 TRAEs occurred in 42% of patients. Nine (7%) patients had TRAEs leading to discontinuation of tovorafenib. These data indicate that tovorafenib could be an effective therapy for BRAF-altered, relapsed/refractory pLGG. ClinicalTrials.gov registration: NCT04775485 .

Structural analysis of hepatitis C virus core-E1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase.

The maturation of the hepatitis C virus (HCV) core protein requires proteolytic processing by two host proteases: signal peptidase (SP) and the intramembrane-cleaving protease signal peptide peptidase (SPP). Previous work on HCV genotype 1a (GT1a) and GT2a has identified crucial residues required for efficient signal peptide processing by SPP, which in turn has an effect on the production of infectious virus particles. Here we demonstrate that the JFH1 GT2a core-E1 signal peptide can be adapted to the GT3a sequence without affecting the production of infectious HCV. Through mutagenesis studies, we identified crucial residues required for core-E1 signal peptide processing, including a GT3a sequence-specific histidine (His) at position 187. In addition, the stable knockdown of intracellular SPP levels in HuH-7 cells significantly affects HCV virus titers, further demonstrating the requirement for SPP for the maturation of core and the production of infectious HCV particles. Finally, our nuclear magnetic resonance (NMR) structural analysis of a synthetic HCV JFH1 GT2a core-E1 signal peptide provides an essential structural template for a further understanding of core processing as well as the first model for an SPP substrate within its membrane environment. Our findings give deeper insights into the mechanisms of intramembrane-cleaving proteases and the impact on viral infections.

Reconstitution of the entire hepatitis C virus life cycle in nonhepatic cells.

Hepatitis C virus (HCV) is a human hepatotropic virus, but the relevant host factors restricting HCV infection to hepatocytes are only partially understood. We demonstrate that exogenous expression of defined host factors reconstituted the entire HCV life cycle in human nonhepatic 293T cells. This study shows robust HCV entry, RNA replication, and production of infectious virus in human nonhepatic cells and highlights key host factors required for liver tropism of HCV.

Electrochemical study of ricin at glassy carbon electrode.

Ricin, Ricinus communis agglutinin 60 - RCA 60, is a deadly phytotoxic protein which inhibits ribosomes (class II), and there is no known effective antidote in living organisms. Ricin is composed of two polypeptide chains, A and B, linked covalently by a single disulfide bond. The analytical methods for the detection of RCA 60 are commonly laborious, expensive, require skilled labor, and involve sophisticated equipment. Aimed at the development of electroanalytical methods for RCA 60 detection, here we studied the electrochemical oxidation of RCA 60 on a glassy carbon (GC) electrode over a wide pH range, using cyclic voltammetry, differential pulse voltammetry (DPV) and square wave voltammetry (SWV). Two quasi-reversible electrochemical RCA 60 oxidation peaks were identified on the GC electrode by SWV. For values of 2.2 ≤ pH ≤ 10.2, DPV studies revealed that the peak potentials, EP1 and EP2, display a linear dependence with pH and the reaction mechanism involves the transfer of 2H⁺/2e⁻ (peak 1) and 1H⁺/1e⁻ (peak 2). The first and second RCA 60 oxidation steps may correspond to the oxidation of cysteine and tyrosine-tryptophan residues, respectively. The oxidation product of the second RCA 60 oxidation step appears at 7.0 ≤ pH ≤ 11.8. For pH ≥ 10.2, both processes are pH independent, resulting in a pKa of ca. 10.2. A third RCA 60 oxidation peak only appears at acidic pH. RCA 60 samples extracted from different castor seed cultivars showed similar electrochemical behavior, enabling the implementation of an analytical voltammetric method.

2,2'-Dihydroxy-N,N'-(ethane-1,2-di-yl)di-benzamide.

The asymmetric unit of the title compound, C(16)H(16)N(2)O(4), contains one half-mol-ecule, the whole mol-ecule being generated by an inversion center located at the mid-point of the C-C bond of the central ethane group. An intra-molecular O-H⋯O hydrogen bond forms an S(6) ring motif. In the crystal, mol-ecules are connected via N-H⋯O hydrogen bonds, generating infinite chains along [1-10].

Digital health education and training for undergraduate and graduate nursing students: a scoping review protocol.

OBJECTIVE: The objective of this review is to collate and analyze literature reporting on digital health education and training courses, or other pedagogical interventions, for nursing students at the undergraduate and graduate level to identify gaps and inform the development of future educational interventions. INTRODUCTION: In this era of technology-driven health care, upskilling and/or reskilling the nursing workforce is urgently needed for nurses to lead the digital health future and improve patient care. While informatics competency frameworks serve to inform nursing education and practice, they do not address the entire digital health spectrum. INCLUSION CRITERIA: This review will include research studies, theoretical/discussion papers, and reports, as well as gray literature from relevant sources published in the last 10 years. Opinion pieces, editorials, conference proceedings, and papers published in languages other than English will be excluded. METHODS: The JBI methodology for scoping reviews will be followed. Searches will be conducted in Embase, CINAHL, ERIC, MEDLINE, Scopus, and Education Research Complete to retrieve potentially relevant studies. Hand searches of reference lists of included studies will be conducted. Two reviewers will independently screen records against predefined eligibility criteria and consult a third reviewer if conflicts arise. Decisions will be documented using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. Quantitative data will be analyzed using descriptive statistics. Content analysis will be applied to qualitative data to identify categories and themes. Findings will be synthesized and reported in tables and narrative format. REVIEW REGISTRATION NUMBER: Open Science Framework osf.io/42eug.

Experiences of Patients With Chronic Obstructive Pulmonary Disease Using the Apple Watch Series 6 Versus the Traditional Finger Pulse Oximeter for Home SpO2 Self-Monitoring: Qualitative Study Part 2.

BACKGROUND: Amid the rise in mobile health, the Apple Watch now has the capability to measure peripheral blood oxygen saturation (SpO2). Although the company indicated that the Watch is not a medical device, evidence suggests that SpO2 measurements among patients with chronic obstructive pulmonary disease (COPD) are accurate in controlled settings. Yet, to our knowledge, the SpO2 function has not been validated for patients with COPD in naturalistic settings. OBJECTIVE: This qualitative study explored the experiences of patients with COPD using the Apple Watch Series 6 versus a traditional finger pulse oximeter for home SpO2 self-monitoring. METHODS: We conducted individual semistructured interviews with 8 female and 2 male participants with moderate to severe COPD, and transcripts were qualitatively analyzed. All received a watch to monitor their SpO2 for 5 months. RESULTS: Due to respiratory distress, the watch was unable to collect reliable SpO2 measurements, as it requires the patient to remain in a stable position. However, despite the physical limitations and lack of reliable SpO2 values, participants expressed a preference toward the watch. Moreover, participants' health needs and their unique accessibility experiences influenced which device was more appropriate for self-monitoring purposes. Overall, all shared the perceived importance of prioritizing their physical COPD symptoms over device selection to manage their disease. CONCLUSIONS: Differing results between participant preferences and smartwatch limitations warrant further investigation into the reliability and accuracy of the SpO2 function of the watch and the balance among self-management, medical judgment, and dependence on self-monitoring technology.

Acceptance of the Apple Watch Series 6 for Telemonitoring of Older Adults With Chronic Obstructive Pulmonary Disease: Qualitative Descriptive Study Part 1.

BACKGROUND: The Apple Watch is not a medical device per se; it is a smart wearable device that is increasingly being used for health monitoring. Evidence exists that the Apple Watch Series 6 can reliably measure blood oxygen saturation (SpO2) in patients with chronic obstructive pulmonary disease under controlled circumstances. OBJECTIVE: This study aimed to better understand older adults' acceptance of the Watch as a part of telemonitoring, even with these advancements. METHODS: This study conducted content analysis on data collected from 10 older adults with chronic obstructive pulmonary disease who consented to wear the Watch. RESULTS: Using the Extended Unified Theory of Acceptance and Use of Technology model, results showed that participants experienced potential health benefits; however, the inability of the Watch to reliably measure SpO2 when in respiratory distress was concerning. Participants' level of tech savviness varied, which caused some fear and frustration at the start, yet all felt supported by family and would have explored more features if they owned the Watch. All agreed that the Watch is mainly a medical tool and not a gadget. CONCLUSIONS: To conclude, although the Watch may enhance their physical health and well-being, results indicated that participants are more likely to accept the Watch if it ultimately proves to be useful when experiencing respiratory distress.

Maternal γδ T cells shape offspring pulmonary type 2 immunity in a microbiota-dependent manner.

Immune development is profoundly influenced by vertically transferred cues. However, little is known about how maternal innate-like lymphocytes regulate offspring immunity. Here, we show that mice born from γδ T cell-deficient (TCRδ-/-) dams display an increase in first-breath-induced inflammation, with a pulmonary milieu selectively enriched in type 2 cytokines and type 2-polarized immune cells, when compared with the progeny of γδ T cell-sufficient dams. Upon helminth infection, mice born from TCRδ-/- dams sustain an increased type 2 inflammatory response. This is independent of the genotype of the pups. Instead, the offspring of TCRδ-/- dams harbors a distinct intestinal microbiota, acquired during birth and fostering, and decreased levels of intestinal short-chain fatty acids (SCFAs), such as pentanoate and hexanoate. Importantly, exogenous SCFA supplementation inhibits type 2 innate lymphoid cell function and suppresses first-breath- and infection-induced inflammation. Taken together, our findings unravel a maternal γδ T cell-microbiota-SCFA axis regulating neonatal lung immunity.

Cannabidiol improves Nile tilapia cichlid fish welfare.

Cannabidiol (CBD) is a substance derived from Cannabis sativa, widely studied in medicine for controlling neural diseases in humans. Besides the positive effects on humans, it also presents anxiolytic proprieties and decreases aggressiveness and stress in mammals. Therefore, CBD has the potential to increase welfare in reared animals, as it seems to reduce negative states commonly experienced in artificial environments. Here, we tested the effect of different CBD doses (0, 1, 10 and 20 mg/kg) on aggressiveness, stress and reproductive development of the Nile tilapia (Oreochromis niloticus) a fish reared worldwide for farming and research purposes. CBD mixed with fish food was offered to isolated fish for 5 weeks. The 10 mg/kg dose decreased fish's aggressiveness over time, whereas 20 mg/kg attenuated non-social stress. Both doses decreased the baseline cortisol level of fish and increased the gonadosomatic index. However, CBD 1 and 10 mg/kg doses decreased the spermatozoa number. No CBD dose affected feeding ingestion and growth variables, showing that it is not harmful to meat production amount. Despite the effect on spermatozoa, CBD supplementation exhibits high potential to benefit animals' lives in artificial environments. Therefore, we showed for the first time that CBD could be used as a tool to increase non-mammal welfare, presenting a great potential to be explored in other husbandry and captivity species.