Spatial single-cell mass spectrometry defines zonation of the hepatocyte proteome.

Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.

A Standardized and Reproducible Workflow for Membrane Glass Slides in Routine Histology and Spatial Proteomics.

Defining the molecular phenotype of single cells in situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection (LMD) of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and LMD for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with LMD. In this study, we describe a novel method for handling glass membrane slides that enables automated eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed "G-HIER." We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry-based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced deep visual proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging, and sophisticated downstream spatial omics technologies.

Translation from unconventional 5' start sites drives tumour initiation.

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.

A Regression-Based Analysis of Ribosome-Profiling Data Reveals a Conserved Complexity to Mammalian Translation.

A fundamental goal of genomics is to identify the complete set of expressed proteins. Automated annotation strategies rely on assumptions about protein-coding sequences (CDSs), e.g., they are conserved, do not overlap, and exceed a minimum length. However, an increasing number of newly discovered proteins violate these rules. Here we present an experimental and analytical framework, based on ribosome profiling and linear regression, for systematic identification and quantification of translation. Application of this approach to lipopolysaccharide-stimulated mouse dendritic cells and HCMV-infected human fibroblasts identifies thousands of novel CDSs, including micropeptides and variants of known proteins, that bear the hallmarks of canonical translation and exhibit translation levels and dynamics comparable to that of annotated CDSs. Remarkably, many translation events are identified in both mouse and human cells even when the peptide sequence is not conserved. Our work thus reveals an unexpected complexity to mammalian translation suited to provide both conserved regulatory or protein-based functions.

Phylogenetic Analysis of Francisella tularensis Group A.II Isolates from 5 Patients with Tularemia, Arizona, USA, 2015-2017.

We examined 5 tularemia cases in Arizona, USA, during 2015-2017. All were caused by Francisella tularensis group A.II. Genetically similar isolates were found across large spatial and temporal distances, suggesting that group A.II strains are dispersed across long distances by wind and exhibit low replication rates in the environment.

Using Molecular Characterization to Support Investigations of Aquatic Facility-Associated Outbreaks of Cryptosporidiosis - Alabama, Arizona, and Ohio, 2016.

Cryptosporidiosis is a nationally notifiable gastrointestinal illness caused by parasitic protozoa of the genus Cryptosporidium, which can cause profuse, watery diarrhea that can last up to 2-3 weeks in immunocompetent patients and can lead to life-threatening wasting and malabsorption in immunocompromised patients. Fecal-oral transmission of Cryptosporidium oocysts, the parasite's infectious life stage, occurs via ingestion of contaminated recreational water, drinking water, or food, or following contact with infected persons or animals, particularly preweaned bovine calves (1). The typical incubation period is 2-10 days. Since 2004, the annual incidence of nationally notified cryptosporidiosis has risen approximately threefold in the United States (1). Cryptosporidium also has emerged as the leading etiology of nationally notified recreational water-associated outbreaks, particularly those associated with aquatic facilities (i.e., physical places that contain one or more aquatic venues [e.g., pools] and support infrastructure) (2). As of February 24, 2017, a total of 13 (54%) of 24 states reporting provisional data detected at least 32 aquatic facility-associated cryptosporidiosis outbreaks in 2016. In comparison, 20 such outbreaks were voluntarily reported to CDC via the National Outbreak Reporting System for 2011, 16 for 2012, 13 for 2013, and 16 for 2014. This report highlights cryptosporidiosis outbreaks associated with aquatic facilities in three states (Alabama, Arizona, and Ohio) in 2016. This report also illustrates the use of CryptoNet, the first U.S. molecularly based surveillance system for a parasitic disease, to further elucidate Cryptosporidium chains of transmission and cryptosporidiosis epidemiology. CryptoNet data can be used to optimize evidence-based prevention strategies. Not swimming when ill with diarrhea is key to preventing and controlling aquatic facility-associated cryptosporidiosis outbreaks (https://www.cdc.gov/healthywater/swimming/swimmers/steps-healthy-swimming.html).

Misfolded proteins induce aggregation of the lectin Yos9.

A substantial fraction of nascent proteins delivered into the endoplasmic reticulum (ER) never reach their native conformations. Eukaryotes use a series of complementary pathways to efficiently recognize and dispose of these terminally misfolded proteins. In this process, collectively termed ER-associated degradation (ERAD), misfolded proteins are retrotranslocated to the cytosol, polyubiquitinated, and degraded by the proteasome. Although there has been great progress in identifying ERAD components, how these factors accurately identify substrates remains poorly understood. The targeting of misfolded glycoproteins in the ER lumen for ERAD requires the lectin Yos9, which recognizes the glycan species found on terminally misfolded proteins. In a role that remains poorly characterized, Yos9 also binds the protein component of ERAD substrates. Here, we identified a 45-kDa domain of Yos9, consisting of residues 22-421, that is proteolytically stable, highly structured, and able to fully support ERAD in vivo. In vitro binding studies show that Yos9(22-421) exhibits sequence-specific recognition of linear peptides from the ERAD substrate, carboxypeptidase Y G255R (CPY*), and binds a model unfolded peptide ΔEspP and protein Δ131Δ in solution. Binding of Yos9 to these substrates results in their cooperative aggregation. Although the physiological consequences of this substrate-induced aggregation remain to be seen, it has the potential to play a role in the regulation of ERAD.

Organic Compounds Responsible for the Fouling of Ultrafiltration Membrane Treating Algae-Laden Water.

Fouling comparisons of the organic fractions in surface and algae-laden waters make it possible to determine the main compounds responsible for the fouling of ultrafiltration (UF) membranes. This study examined the fouling of UF membranes and its relationship to the characteristics of the organic fractions found in drinking-water supply. Four types of water were prepared by combining natural organic matter (NOM) from lake water with algal organic matter (AOM) from four algae species commonly found in freshwater. Liquid chromatography-organic carbon detection (LC-OCD) and a fluorescence excitation-emission matrix (FEEM) were used to analyze the feed water and permeate to assess the interactions between and fouling behavior of the organic fractions. The results showed that the interaction of large-molecular-weight AOMs on the membrane surfaces and their transport through the membrane pores were the main fouling mechanisms. Polysaccharides followed by protein-like substances were the organic compounds responsible for the fouling of the UF membranes. The fouling affinity of these substances was attributed to two processes, the adsorption of their carboxyl, hydroxyl and cationic groups on the membrane surfaces, and the molecular complexation of their organic groups. The humic substances' retention was marginal and attributed to the synergetic effects of the polysaccharides and proteins.

Association Between Mortality Due to Nasopharyngeal Carcinoma and Race in the United States From 2007 to 2016.

BACKGROUND: Asians and Pacific Islanders (API) exhibit increased incidence of nasopharyngeal carcinoma (NPC). However, they are often excluded when the disease is studied. Risk-factors and incidence are well-researched while cancer-specific mortality trends remain unclear. We aimed to determine whether insurance status modifies the association between race and cancer-specific mortality in NPC patients. METHODS: This retrospective cohort study used secondary data analysis from the Surveillance, Epidemiology, and End Results Program database. Patients ≥18 years with histologically confirmed primary NPC from 2007 - 2016 were included. The main outcome assessed was 5-year survival and the main exposure variable was race (API, white, black). Insurance status was classified into uninsured, any Medicaid, and insured (with any insurance). Potential confounders included age, sex, marital status, stage at diagnosis, and surgical treatment. Adjusted Cox regression analysis was used to calculate hazard ratios (HR) and corresponding 95% confidence intervals (CI). RESULTS: 1610 patients were included (72.98% male, 27.02% female). 49.8% were API, 40.5% were Whites, and 9.8% Blacks. Maximum follow-up was 5-years. The adjusted hazards of 5-year cancer-specific death for API and Blacks compared with Whites were 0.77 (95% CI 0.62 - 0.96) and 0.92 (95% CI 0.65 - 1.31), respectively. Cases decreased with age in API and Blacks. 8.2% of cases had localized disease, 45.3% had local spread, and 44.6% had distant metastasis. Insurance status did not modify the association between race and mortality. CONCLUSION: Race is an important prognostic factor to account for in NPC patients. Investigating risk-factors and subtypes stratified by race may explain our findings.

Progression From Patterns A to B in Wellens Syndrome: Electrocardiographic Progression in Wellens Syndrome.

We describe a clinical case series of 3 patients whose electrocardiogram evolved from type A Wellens syndrome to a type B. We emphasize that the diagnosis and treatment for both patterns is the same and that these findings suggest the evolution of the same disease.

Effects of Oral Cannabidiol on Health and Fitness in Healthy Adults: An 8-Week Randomized Trial.

BACKGROUND: There is a lack of research on the effects of cannabidiol (CBD) on health-related fitness, physical activity, cognitive health, psychological wellbeing, and concentrations of C-reactive protein (CRP) in healthy individuals. CBD has potential anti-inflammatory and neuroprotective effects. METHODS: This study aimed to investigate the effects of 8 weeks of CBD on the above-mentioned measures in healthy individuals. Forty-eight participants were randomized into two groups receiving either oral capsules of 50 mg of CBD or a calorie-matched placebo daily. Participants completed pre- and post-intervention assessments, including blood draws, body composition, fitness, physical activity, and self-reported surveys. RESULTS: There were no significant differences between groups regarding body composition, aerobic fitness, muscular strength, physical activity, cognitive health, psychological wellbeing, and resting CRP concentrations. However, the placebo group experienced a decline in mean peak power and relative peak power compared to the CBD group. CONCLUSIONS: The results suggest that 8 weeks of CBD supplementation may prevent declines in anaerobic fitness over time. However, long-term CBD supplementation may not be beneficial for altering measures of health-related fitness, mental health, and inflammation in healthy individuals.

A comparison of resource use of insured and uninsured venezuelan migrants: evidence from the hospital setting.

BACKGROUND: There is no characterization of resource use in the hospital setting for immigrants in Colombia, we aimed to describe the resource use by Venezuelan immigrants, comparing those enrolled in the national health insurance system with those with and without the ability to pay. METHODS: Retrospective review in the billing data system of our Hospital from 2011 to 2020. We collected information for 6,837 hospital episodes associated with 1,022 Venezuelan patients, hospital's billing information for all services rendered was extracted. RESULTS: The mean cost per patient event were 4,595 USD for those without the ability to pay, costing 2.37 times more than a legal resident insured. Care in the ICU, inpatient days, surgery, and OB-GYN department consume most resources provided to vulnerable migrants. DISCUSSION: Enrolment in the national health insurance may allow better access to health services by vulnerable Venezuelan migrants and thus reduce resource use for the health system.

The potential of PGPR and Trichoderma-based bioproducts and resistant cultivars as tools to manage clubroot disease in cruciferous crops.

The objective of this research was to determine the potential use of eco-friendly technologies to reduce the clubroot disease caused by Plasmodiophora brassicae, the main constraint of cruciferous crops worldwide. Two commercial bioproducts were evaluated in susceptible broccoli, one based on the PGPR consortium (Bacillus amyloliquefaciens, Bacillus pumilus, and Agrobacterium radiobacter K84) and the other one based on Trichoderma koningiopsis Th003 (Tricotec® WG). Additionally, the resistant broccoli cv. Monclano® was tested under two concentrations of resting spores (RS) of P. brassicae, 1 × 103 and 1 × 105 RS g-1 of soil. The first phase of evaluations with broccoli was carried out under a greenhouse, while susceptible broccoli, cauliflower, and red cabbage were included in a subsequent field phase. Tebuconazole + Trifloxystrobin mixture and Fluazinam were included as positive controls. The effectiveness of the bioproducts depended on the nature of the biocontrol agent, the concentration of P. brassicae, and the dose of treatment. Tricotec® showed consistent plant growth promotion but no biocontrol effect against clubroot, and the rhizobacteria-based bioproduct significantly reduced the disease in both greenhouse and field experiments. Higher disease severity was observed with the higher dose of Tricotec®. Under field conditions, the rhizobacteria reduced the incidence progress by 26%, 39%, and 57% under high, medium, and low pressure of the pathogen, respectively. However, no reduction of clubroot severity under high pressure of the pathogen was observed. Complete inhibition of club formation in roots was achieved via the fungicide, but a phytotoxic effect was observed under greenhouse conditions. Fungicides reduced the incidence progress of clubroot, but not the severity under high inoculum pressure in the field. The fungicides, the bacterial treatment, and the combination of bioproducts tended to delay the progress of the disease compared with the negative control and Tricotec alone. The resistant broccoli showed a low level of disease under high concentrations of P. brassicae (less than 10% incidence and up to 2% severity). These results suggested the overall potential of commercial tools based on the PGPR consortium and plant resistance to control P. brassicae. The integration of control measures, the role of Trichoderma spp. in P. brassicae-cruciferous pathosystems, and the need to recover highly infested soils will be discussed.

Structure-Activity Relationship of N-Ethyl-Hexedrone Analogues: Role of the α-Carbon Side-Chain Length in the Mechanism of Action, Cytotoxicity, and Behavioral Effects in Mice.

Synthetic cathinones are ß-keto amphetamine derivatives whose appearance has increased dramatically in the past decades. N-Ethyl substituted cathinones have been proven to potently inhibit dopamine (DA) uptake and induce psychostimulant and rewarding effects in mice. However, little is known about the influence of the alpha-carbon side-chain length of N-ethyl cathinones on their pharmacological and toxicological effects. Thus, the aim of this study was to synthesize and investigate the in vitro and in vivo effects of five N-ethyl substituted cathinones: N-ethyl-cathinone (NEC), N-ethyl-buphedrone (NEB), N-ethyl-pentedrone, N-ethyl-hexedrone (NEH), and N-ethyl-heptedrone. HEK293 cells expressing the human DA or serotonin transporter (hDAT and hSERT) were used for uptake inhibition and binding assays. PC12 cells were used for the cytotoxicity assays. Swiss CD-1 mice were used to study the in vivo psychostimulant, anxiogenic, and rewarding properties. Our results show that all tested cathinones are able to inhibit DA uptake and are DAT-selective. The potency of DA uptake inhibitors increases with the elongation of the aliphatic side chain from methyl to propyl and decreases when increasing from butyl to pentyl, which correlates with an inverted U-shape psychostimulant response in mice at the medium dose tested. On the other hand, an increase in the α-carbon side-chain length correlates with an increase in the cytotoxic properties in PC12 cells, probably due to better membrane penetration. Moreover, all the cathinones tested have shown higher cytotoxicity than methamphetamine. Finally, our study not only demonstrated the rewarding properties of NEC and NEB but also the anxiety-like behavior induced at high doses by all the cathinones tested.

Spatial proteo-transcriptomic profiling reveals the molecular landscape of borderline ovarian tumors and their invasive progression.

Serous borderline tumors (SBT) are epithelial neoplastic lesions of the ovaries that commonly have a good prognosis. In 10-15% of cases, however, SBT will recur as low-grade serous cancer (LGSC), which is deeply invasive and responds poorly to current standard chemotherapy1,2,3. While genetic alterations suggest a common origin, the transition from SBT to LGSC remains poorly understood4. Here, we integrate spatial proteomics5 with spatial transcriptomics to elucidate the evolution from SBT to LGSC and its corresponding metastasis at the molecular level in both the stroma and the tumor. We show that the transition of SBT to LGSC occurs in the epithelial compartment through an intermediary stage with micropapillary features (SBT-MP), which involves a gradual increase in MAPK signaling. A distinct subset of proteins and transcripts was associated with the transition to invasive tumor growth, including the neuronal splicing factor NOVA2, which was limited to expression in LGSC and its corresponding metastasis. An integrative pathway analysis exposed aberrant molecular signaling of tumor cells supported by alterations in angiogenesis and inflammation in the tumor microenvironment. Integration of spatial transcriptomics and proteomics followed by knockdown of the most altered genes or pharmaceutical inhibition of the most relevant targets confirmed their functional significance in regulating key features of invasiveness. Combining cell-type resolved spatial proteomics and transcriptomics allowed us to elucidate the sequence of tumorigenesis from SBT to LGSC. The approach presented here is a blueprint to systematically elucidate mechanisms of tumorigenesis and find novel treatment strategies.

Spontaneous Pulmonary Embolism Leading to Sudden Cardiac Arrest and Perimortem C-Section in a 39-Week Parturient During Induction of Labor: A Case Report.

We report the successful salvage of mother and baby after a perimortem cesarean delivery (PMCD) complicated by a 21-minute asystolic maternal cardiac arrest (MCA) that was precipitated by a pulmonary embolism during the early stages of induction of labor. With rapid PMCD, recovery of maternal quality of life is possible even after prolonged resuscitation.

Decreases in resource use after implementing an interdisciplinary management program for patients with placenta accreta spectrum.

OBJECTIVE: Placenta accreta spectrum (PAS) often causes severe morbidity and demands the availability of abundant health resources. Research has shown that the participation of experienced interdisciplinary groups in specialized centers improves clinical outcomes. Our objective is to measure resource use variation after implementing an interdisciplinary management program for this condition. METHODS: Using detailed billing information, hospital care resource use was measured at constant prices for women with PAS who were treated between 2011 and 2019. Cases were classified before (Group 1) and after (Group 2) the implementation of the program. A third group included women with intraoperative MAP findings (Group 3). Comparisons were made using descriptive statistics. RESULTS: The mean reduction in resource use after the program was 16.5% per patient. The program also reduced variability in resource use as measured by the standard deviation and the coefficient of variation, which decreased by 55.2% and 46.3%, respectively. CONCLUSION: The interdisciplinary management of patients affected by PAS in experienced hospitals is associated with a reduction in resource use and variability.

Multiple de novo gene variations in a progeroid phenotype case report: haploinsufficiency mechanisms.

We are presenting the case of a 6-year-old male patient with progeroid phenotype and severe developmental delay referred to Genetic clinic. Given the complex phenotype an extensive metabolic and genetic evaluation was performed including a whole exome sequencing analysis that showed genetic variants in TTR, RELN, MYH6, PHIP, and SYNE2 genes. Patients' mother and brother were analyzed for the genetic variants in MYH6, PHIP and RELN. Both had same variants on PHIP and RELN as our patient, with no apparent phenotypical consequences. Physical examination was remarkable for dysmorphism including plagiocephaly, low set and abnormally shaped ears, up slanted palpebral fissures, hypoplastic alae nasi, and a head circumference two standard deviations below the 3rd percentile (microcephaly). Other characteristics include wrinkled skin, a broad forehead, sparse eyelashes in lower eyelid, short palpebral fissures, upturned nares, thick lips, right occipital plagiocephaly, overfolded helix and prominent anti-helix, protuberant chest, scaphoid abdomen, digitalized thumbs, and kyphosis due to low muscle tone. The patient presented abnormal EEG with evidence of epileptic discharges. A temporal bone CT showed plagiocephaly with flattening of the right occipital bone. Brain MRI showed callosal agenesis with bilateral colpocephaly with temporal horn dilatation, parahippocampal atrophy, lissencephaly and midbrain hypoplasia. The combination of de novo gene variants mentioned above has never been reported nor correlated as the result of haploinsufficiency mechanisms. Thus, we propose haploinsufficiency and loss of heterozygosity as etiological reasons for this patient phenotype. Further proteomic studies are needed to allocate the extense of genetic influence within the clinical manifestations.

Assessment of Inoculation Methods of Thielaviopsis paradoxa (De Seynes) Höhn into Oil Palm Seedlings under Greenhouse Conditions.

Oil palm (Elaeis guineensis Jacq. and Elaeis Oleifera Cortes) is one of the most important oil crops in the world. Colombia is the fourth-largest oil palm producer worldwide. However, oil palm diseases are a significant factor affecting yield. Thielaviopsis paradoxa (De Seynes) Höhn is a pathogen that affects young palm trees, causing spear rot. Four disease establishment methods were studied to replicate, in a controlled environment, the symptoms of the disease found in the field. Young palm trees were inoculated with a suspension of endoconidia using either local infiltration, drip, scissor cut, or direct contact with agar blocks bearing mycelia and conidia. The effects of the inoculation methods were studied in dose-method-disease severity experiments conducted in a greenhouse under controlled conditions. All four methods resulted in T. paradoxa infections and the development of symptoms of the disease. The disease severity was correlated with the method and dose of inoculation. In trials to test Koch's postulates, T. paradoxa was isolated from areas of disease progression in the inoculated trees, but the teleomorph Ceratocystis paradoxa (Dade) Moreau was not observed. A photographic record of the infection process at different times post-infection was compiled. Given that establishing the disease through artificial inoculation is essential for assessing plant pathogenesis, this study determined that the local infiltration method (1 × 106 endoconidia mL-1) and a 3-7 day incubation period were critical for the development of symptoms as severe as those observed in natural infections in the field.