Chronic rhinosinusitis (CRS) is a common clinical syndrome that produces significant morbidity and costs to our health system. The study of CRS has progressed from an era focused on phenotype to include endotype based information. Phenotypic classification has identified clinical heterogeneity in CRS based on endoscopically observed features such as presence of nasal polyps, presence of comorbid or systemic diseases and timing of disease onset. More recently, laboratory-based findings have established CRS endotype based upon specific mechanisms or molecular biomarkers. Understanding the basis of widespread heterogeneity in the manifestations of CRS is advanced by findings that the three main endotypes, Type 1, 2 and 3, orchestrate the expression of three distinct large sets of genes. The development and use of improved methods of endotyping disease in the clinic is ushering in an expansion of the use of biological therapies targeting Type 2 inflammation now and perhaps other inflammatory endotypes in the near future. The purpose of this review is to discuss the phenotypic and endotypic heterogeneity of CRS from the perspective of advancing the understanding of the pathogenesis and improvement of treatment approaches and outcomes.

The utilization and popularization of biodiesel are always limited by its poor cold flow properties. Both bio-based alcohol and diesel from direct coal liquefaction (DDCL) has potential to enhance the cold flow properties of biodiesel. Ternary blends of waste cooking oil biodiesel (BWCO) with DDCL and bio-based ethanol (ET) or 1-butanol (BT) for improving the cold flow properties of biodiesel. The pour point (PP), cold filter plugging point (CFPP), and cloud point (CP) of BWCO-ET, BWCO-BT, and BWCO-DDCL binary blends, and BWCO-ET-DDCL and BWCO-BT-DDCL ternary blends were comparatively assessed. Ternary phase diagrams were also applied into analyze the blending effect of the three components on the cold flow properties of biodiesel. Results showed that both DDCL, ET and BT can remarkably enhance the cold flow properties of BWCO. BT and DDCL presented a better synergistic depression effect. For ternary blends in 20:10:70 blending ratio, BWCO-BT-DDCL exhibited the lowest PP, CFPP, and CP of −23 °C, −19 °C, and −17 °C, respectively. The crystallization behavior and crystal morphology of blended fuels are also observed via a polarizing optical microscope, and find that DDCL together with BT in biodiesel can effectively retard the aggregation of large crystals and inhibit crystals growth.

One of the hallmarks of COVID-19 is the cytokine storm that provokes primarily pneumonia followed by systemic inflammation. Emerging evidence has identified a potential link between elevated levels of interleukin-17A (IL-17A) and disease severity and progression. Considering that per se IL-17A can activate several inflammatory pathways, it is plausible to hypothesize an involvement of this cytokine in COVID-19 clinical outcomes. Thus, this cytokine can represent a marker of disease progression and/or a target to develop therapeutic strategies. This hypothesis paper aims to propose this “unique” cytokine as a silent amplifier of the COVID-19 immune response and (potentially) related therapy.

A case series of 22 patients with confirmed SARS-CoV-2 infection and Corona Virus Disease 2019 (COVID-19) symptoms was followed in a primary care clinic. We provided quantitative and longitudinal profiling for SARS-CoV-2 IgM and IgG with a point of care device. Half the patients had a history of anosmia or ageusia. IgM and IgG responses were highly heterogeneous quantitatively and temporally. We determined that clinical symptomatology of chemosensory loss correlated with lesser but sustained titers of IgM and IgG whereas normal chemosensation correlated with transiently higher but rapidly declining antibody levels.

Background The delivery of Radiation therapy (RT) needs a level of coordination and planning which can be challenging in resource-limited settings. In this retrospective study we describe the profile of children undergoing radiation and analyze deviation from accepted norms. Procedure Data on all children (< 18 years age) with cancer who completed RT from Jan 2009 to Dec 2019 were retrieved. Diagnostic groups with more than 5 patients, were included in the analysis for deviations in RT (Time to start (TTS); total dose delivered (TDD) in Grays; and time to complete (TTC)). We investigated reasons for deviation. Results 207 received RT as front-line treatment (68% Indian, 59% male and 32% received treatment preceding RT outside). Most common diagnosis were brain tumors (44%), lymphoma (13%). leukemia and soft tissue sarcoma (10% each). 23.6% had deviation in TTS, 6.0% in TDD and 24.7% in TTC while 43.4% had at least one deviation in any of these three parameters. Deviation in TTS varied significantly by location of preceding treatment and by cancer (greatest deviation in sarcomas) with issues around access to healthcare the most common reason. Deviation in TTC varied significantly by cancer (greatest deviation in sarcomas and medulloblastoma) with myelotoxicity the most common reason. Conclusions Our study adds to the limited literature on RT quality for children with cancer in LMIC. Certain cancers (sarcomas and medulloblastoma) and patient groups (preceding treatment outside our institute) had the maximum deviation. Barriers to accessing care and myelotoxicity were the two main reasons for this deviation.

Legends of Allergy/Immunology: Robert P. Schleimer

Plant phenology is manifested in the seasonal timing of flowering and vegetative processes, but also has ontogenetic aspects. The adaptive basis of seasonal phenology has been considered mainly in terms of climatic drivers. However, some biotic factors as likely evolutionary influences on plants’ phenology appear to have been under-researched. Several specific cases of putative biotic factors driving plant phenology are outlined, involving both herbivores and pathogens. These illustrate the diversity of likely interactions rather than any systematic coverage or review. Emphasis is on woody perennials, in which phenology is often most multi-faceted and complicated by the ontogenetic aspect. The timing and duration of shoot flushing may in at least some cases contribute to defencses against herbivores, largely through brief periods of ‘predator satiation’ when plant tissues have highest food value. However, the complete seasonal leaf fall that characterizes deciduous plants may be a very important defencse against some pathogens. Whether biotic influences drive acquisition or ‘biotic lock-in’ of deciduousness is considered. In one case; of leaf rusts in poplars, countervailing influences of the rusts and climate suggest lock-in. Often, however, biotic and environmental influences likely reinforce each other. Wide re-examination of plant phenology, accommodating the roles of biotic factors and their interplays with environments as additional adaptive drivers, is advocated, towards developing and applying hypotheses that are observationally or experimentally testable.

The evolution of floral traits is largely attributed to pollinator-mediated selection; however, the importance of pollinators as selective agents in pollen-limited environments is poorly resolved. In pollen-limited arctic and subarctic regions, selection is expected to either favor floral traits that increase pollinator attraction or promote reproductive assurance through selfing. We quantified phenotypic selection on floral traits in two arctic and two subarctic populations of Parrya nudicaulis. Additionally, we measured selection in plants in both open-pollination and pollen-augmentation treatments to estimate selection imposed by pollinators in one population. Seed production was found to be limited by pollen availability and strong directional selection on flower number was observed. We did not detect consistently greater magnitudes of selection on floral traits in the arctic relative to the subarctic populations. Directional selection for more pigmented flowers in one arctic population was observed however. In some populations, selection on flower color was found to interact with other traits. We did not detect consistently stronger selection gradients across all traits for plants exposed to pollinator selection relative to those in the pollen-augmentation treatment; however directional selection tended to be higher for some floral traits in open-pollinated plants.

Background: Although congenital erythrocytosis (CE), an inherited disorder, impairs pediatric quality of life, physicians often overlook high hemoglobin (Hgb) levels and its symptoms due to lack of knowledge of age-adjusted pediatric Hgb levels and CE’s rarity. Methods: In a retrospective, single-center study, data from hospital records of patients (<18 years) diagnosed with CE were evaluated. Results: Forty patients from 39 families (male: female ratio = 7:1) had been diagnosed with CE in a 20-year period, at a mean age of 15.31 ± 2.49 years (8.34–17.92) and with mean Hgb levels of 17.4 ± 1.34 g/dL (14.63–23.0). No serum erythropoietin levels exceeded the reference levels. Although the most common symptom was headache (80.0%), 40.0% of patients presented with at least one gastrointestinal symptom (e.g., nausea, vomiting, abdominal pain, and rectal bleeding), and 75.0% exhibited plethora. None had leukocytosis, thrombocytosis, abnormal capillary oxygen saturation, JAK2 mutation, and venous blood gas analysis and Hgb electrophoresis revealed no abnormalities. While 43.6% of patients had family histories of CE, 28.2% had 15–48-year-old relatives who had experienced myocardial infarction, stroke, and/or sudden death. Six asymptomatic patients were detected incidentally. When symptoms of hyperviscosity were present, aspirin was prescribed, and phlebotomy performed. No thrombotic episodes occurred as a result. Conclusion: To detect CE, physicians should assess Hgb levels in consideration of normal age-adjusted levels in children. Pediatric patients with CE may also present with gastrointestinal symptoms. Although no thrombotic episode occurred among the patients, their family histories included life-threatening thrombotic episodes, even in adolescents.

A subtle aortic dissection can be challenging to detect despite the availability of multiple diagnostic modalities. Whilst rare, the inability to detect this variant of aortic dissection can lead to a dismal prognosis. We present an extremely rare case of a subtle aortic dissection with supraannular aortic root intimal tear and acute severe aortic regurgitation in a patient with a bicuspid aortic valve. Initial concerns were either aortic dissection or infective endocarditis. Despite advanced multimodality preoperative imaging, diagnosis was made intraoperatively and a Bentall procedure with a mechanical aortic valve was performed. As current data is limited, a literature review concerning subtle aortic dissection is provided.

Hexagonal chains are a special class of catacondensed benzenoid system and phenylene chains are a class of polycyclic aromatic compounds. Recently, A family of Sombor indices was introduced by Gutman in the chemical graph theory. It had been examined that these indices may be successfully applied on modeling thermodynamic properties of compounds. In this paper, we study the expected values of the Sombor indices in random hexagonal chains, phenylene chains, and consider the Sombor indices of some chemical graphs such as graphene, coronoid systems and carbon nanocones.

Abnormal ostial morphology and acute take-off are considered potential mechanisms of flow obstruction in the anomalous coronary artery. We present a proof of concept describing how three-dimensional transoesophageal echocardiography can be used to visualise the coronary ostium and proximal course in patients with anomalous coronary artery.

The outcome of coronary artery bypass surgery depends on complete revascularization. In our paper, we attempt to demonstrate that Off-pump coronary artery bypass (OPCAB) is applicable to coronary heart disease patients with low LVEF. Low LVEF does not affect cardiac revascularization. Low LVEF is an independent risk factor for the outcome of CABG patients, but it does not mean that the OPCAB procedure leads to poor outcomes. In our hospital, we used on-pump CABG or conventional bypass surgery for coronary heart disease patients with low LVEF before 2010.With the accumulation of cases, OPCAB is now used in more than 95% of coronary artery bypass grafts in our center. Our data suggest that OPCAB is safe and reliable for patients with low LVEF.

Theoretical calculations involving singlet molecular oxygen (O2(1g)) are challeng- ing due to their inherent multi-reference character. We have tested the quality of re- stricted and unrestricted DFT geometries obtained for the reaction between singlet oxy- gen and a series of alkenes (propene, 2-methylpropene, trans-butene, 2-methylbutene and 2,3-dimethylbutene) which are able to follow the ene-reaction. The electronic en- ergy of the obtained geometries are rened using 3 dierent methods which account for the multi-reference character of singlet oxygen. The results show that the mechanism for the ene-reaction is qualitatively dierent when either one or two allylic-hydrogen groups are available for the reaction. When one allylic-hydrogen group is available the UDFT calculations predict a stepwise addition forming a biradical intermediate, while, the RDFT calculations predict a concerted reaction where both hydrogen abstrac- tion and oxygen addition occur simultaneously. When two allylic-hydrogen groups are available for the reaction then UDFT and RDFT predict the same reaction mechanism, namely that the reaction occurs as a stepwise addition without a stable intermediate between the two transition states. The calculated rate constants are in reasonable agreement with experimental data, except for trans-butene where the calculated rate constant is three orders of magnitude lower than the experimental one. In conclusion we nd that the simple bypassing scheme tested in this paper is a robust approach for calculations of reaction involving singlet oxygen in the limit that the transition state processes low multi-reference character. 2

We present the derivation of a new response method termed rst order po- larization propagator approximation. The electronic structure is given by a density functional representation. We provide a detailed derivation of the method along with explicit expressions for the relevant integrals and matrix elements.

The rapid global spread of SARS-CoV-2 and the resulting Coronavirus disease (COVID-19) pandemic has led to urgent efforts to contain and mitigate transmission. The use of face masks became a primary measure to limit the spread, which increased the production and consequent waste generation. It is estimated that globally 129 billion single-use face masks are discharged into the environment every month. Besides to add to already great concerns over plastic pollution, contaminated face masks not properly handled also poses a health risk. The health and environmental consequences caused by the inadequate disposal of single-use face masks are treated in this study as an eminent risk to be properly communicated. This study framed the waste generation as a side impact of the COVID-19 pandemic and analysed how the communication has been made regarding the correct disposal of single-use masks, with focus in users of public transport in Stockholm region. National and regional authorities are recognized as the main responsibles for the communication about correct disposal, together with manufacturers and suppliers of single-use face masks. Disclosure and informative material of four government authorities and instruction manuals of eight brands of single-use face masks are analysed according to: i) presence / absence of information (about the disposal); ii) type of communication (visual or written); iii) message transmitted and; ix) availability in other languages. It was found guidance about correct disposal of face mask in two thirds of the existing communication, that is mainly written and with few visual aids. Half of communication is made only in Swedish and with no sufficient focus on the importance of correct disposal neither with reference to trash bins with lid. It suggests that the current communication about how to dispose single-use masks safely may not be enough, which is affecting the perception of risk by the population and worsening the health and environmental consequences caused by the inadequate disposal.

Background: This study investigated the impact of transplanting center donor acceptance patterns on usage of extended-criteria donors (ECDs) and posttransplant outcomes following orthotopic heart transplantation (OHT). Methods: The Scientific Registry of Transplant Recipients was queried to identify heart donor offers and adult, isolated OHT recipients in the United States from 1/1/2013-10/17/2018. Centers were stratified into 3 equal-size terciles based on donor heart acceptance rates (<13.7%, 13.7%-20.2%, >20.2%). Overall survival was compared between recipients of ECDs (≥40 years, left ventricular ejection fraction <60%, distance ≥500 miles, hepatitis B, hepatitis C or human immunodeficiency virus, or ≥50 offers) and recipients of traditional-criteria donors, and among transplanting terciles. Results: A total of 85,505 donor heart offers were made to 133 centers with 15,264 (17.9%) accepted for OHT. High-acceptance programs (>20.2%) more frequently accepted donors with LVEF <60%, HIV, HCV, and/or HBV, ≥50 offers, or distance >500 miles from the transplanting center (each p<0.001). Posttransplant survival was comparable across all three terciles (p=0.11). One- and five-year survival were also similar across terciles when examining recipients of all five ECD factors. Acceptance tier and increasing acceptance rate were not found to have any impact on mortality in multivariable modeling. Of ECD factors, only age ≥40 years was found to have increased hazards for mortality (HR 1.33, 95% CI 1.22-1.46, p<0.001). Conclusions: Of recipients of ECD hearts, outcomes are similar across center-acceptance terciles. Educating less aggressive programs to increase donor acceptance and ECD utilization may yield higher national rates of OHT without major impact on outcomes.

The timing of germination is a key life-history trait in plants, which is strongly affected by the strength of seed dormancy. Continental-wide variation in seed dormancy has been related to differences in climate and the timing of conditions suitable for seedling establishment. However, for predictions of adaptive potential and consequences of climatic change, information is needed regarding the extent to which seed dormancy varies within climatic regions and the factors driving such variation We planted 17 Italian and 28 Fennoscandian populations of Arabidopsis thaliana in the greenhouse and at two field sites in Italy and Sweden. To identify possible drivers of among-population variation in seed dormancy, we examined the relationship between seed dormancy and climate at the sites where populations were originally sampled. Seed dormancy was on average stronger in the Italian compared to the Fennoscandian populations, but also varied widely within both regions. Estimates of seed dormancy in the three maternal environments were positively correlated, but seeds had on average stronger dormancy when produced in the greenhouse than at the two field sites. Among Fennoscandian populations, seed dormancy tended to increase with increasing summer temperature and decreasing precipitation at the site of origin. In the smaller sample of Italian populations, no significant association was detected between mean seed dormancy and climate at the site of origin. The correlation between seed dormancy and climatic factors in Fennoscandia suggests that at least some of the among-population variation is adaptive and that climate change will affect selection on this trait.

Richter's hernia has a misleading presentation with absence of typical intestinal obstructive symptoms leading to delay in making a diagnosis, tendency to strangulation and eventual spontaneous fistula formation. This article explores a case of enterocutaneous fistula that was managed non-operatively until spontaneous closure.

Nuclear Magnetic Resonance (NMR) shielding constants of transition metals in solvated complexes are computed at the relativistic density functional theory (DFT) level. The solvent effects evaluated with subsystem-DFT approaches are compared with the reference solvent shifts predicted from supermolecular calculations. Two subsystem-DFT approaches are analyzed – in the standard frozen density embedding (FDE) scheme the transition metal complexes are embedded in an environment of solvent molecules whose density is kept frozen, in the second approach the densities of the complex and of its environment are relaxed in the “freeze-and-thaw” procedure. The latter approach improves the description of the solvent effects in most cases, nevertheless the FDE deficiencies are rather large in some cases. KEYWORDS — Frozen Density Embedding, NMR shielding constant, solvent shifts, transition-metal complexes

Conservation of large carnivores like leopards requires large and interconnected habitats. Despite the wide geographic range of the leopard globally, only 17% of their habitat is within protected areas. In Nepal, leopards are distributed widely across the country but their status is not adequately studied which compromised the necessary conservation attention for the species. This study carried out sign-based occupancy survey across the Chure (the Himalayan foothills) range (~19,000 km2) to understand the habitat occupancy of leopards along with the covariates affecting their presence. The model-averaged leopard occupancy in the Chure range was 0.5732 (0.0082 SD) with a detection probability of 0.2554 (0.1142 SE). The top model included wild boar, ruggedness, presence of livestock and human population density as covariates. The β coefficient estimate from the model indicated the wild boar was the primary covariate contributing positively to the leopard occupancy followed by the presence of livestock, ruggedness and human population density. The detection probability of leopard was higher outside the protected areas, less in the densely vegetated areas, and higher in the area where there is a presence of livestock. Enhanced law enforcement and mass awareness activities are necessary to reduce poaching/killing of wild ungulates and leopard in the Chure range and to increase leopard occupancy. In addition, maintaining a sufficient natural prey base can contribute to minimize the livestock depredation and hence, decrease the human-leopard conflict in the Chure range.

Poly(beta-amino ester)s as high-yield transfection reagents for recombinant protein productionKathryn M. Luly1,2, Stephen J. Lee2,3, Huilin Yang2,3, Wentao Wang1,2, Seth D. Ludwig2,3, Haley E. Tarbox4, David R. Wilson1,2,5, Jordan J. Green1,2,3,5,6,7,8,9,10*, Jamie B. Spangler1,2,3,6,7,8*1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine,3Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 4Department of Chemistry, Johns Hopkins University, 5Institute for Nanobiotechnology, Johns Hopkins University,6Department of Oncology, Johns Hopkins University School of Medicine, 7Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, 8Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine,9Departments of Neurosurgery and Ophthalmology, Johns Hopkins University School of Medicine, 10Department of Materials Science & Engineering, Johns Hopkins University*Correspondence should be addressed to:Jamie B. Spangler400 N Broadway, Smith 5011, Baltimore, MD 21231(443) 287-1708jamie.spangler@jhu.eduJordan J. Green400 N Broadway, Smith 5017, Baltimore, MD 21231(410) 614-9113green@jhu.eduGrant numbers: R01EY031097, R01EB029455, R01CA228133, P41EB028239, R01CA240339Transient transfection is an essential tool for recombinant protein production, as rapid screening for expression is conducted without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of an alternative class of cationic polymers, poly(beta-amino ester)s (PBAEs), for transient transfection of human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions. In both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and production of a cytosolic reporter compared to PEI. This result extended to secreted proteins, as a model PBAE increased the production of three secreted antibodies compared to PEI at scales ranging from 20-2,000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticles led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types. These results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.Keywords: protein production, transient transfection, poly(beta-amino ester)s, transfection reagentsCurrent research into chemical-based transfection methods focuses largely on optimizing agents for use in the development and production of recombinant proteins. Transient transfection, in which introduced genetic material is not incorporated into the host genome, is especially useful during the high-throughput design and screening of proteins (e.g., candidate biologics) wherein stable expression is not needed. While culture conditions and plasmid design have been popular targets for optimization in transient transfection workflows (Backliwal et al., 2008; Galbraith, Tait, Racher, Birch, & James, 2006), further research into improved transfection reagents has even greater potential for boosting protein yields. Chemical-based transient transfection relies on condensation and encapsulation of plasmid DNA by a biocompatible material into particles which are taken up by target cells; differences in particle size can affect the method of cellular uptake, leading to differences in transfection efficiency (Kim, Sunshine, & Green, 2014). Particles must then escape the endosome and the encapsulating material must degrade to allow for DNA release, nuclear translocation, transcription and subsequent export, and finally translation and processing into fully formed protein (Karlsson, Rhodes, Green, & Tzeng, 2020).Transfection reagent structure and buffering capacity have been demonstrated to influence DNA uptake and escape, making these properties particularly consequential in reagents for transient transfection workflows (Sunshine, Peng, & Green, 2012). Maximizing protonability, for example, facilitates endosomal swelling and consequent rupture via the “proton sponge” effect (Boussif et al., 1995; Bus, Traeger, & Schubert, 2018). Cationic polymers have typically been among the most promising transfection reagents; their charge-based association with DNA into particles offers protection from degradation and offers sufficient buffering capacity to facilitate endosomal escape following cellular uptake (Sunshine et al., 2012).Poly(ethyleneimine) (PEI) is a commercially available cationic polymer used extensively as a transfection reagent that has a high density of protonatable amines, giving rise to high buffering capacity and efficient endosomal escape (Boussif et al., 1995). PEI of average molecular weight 25 kDa is most frequently used in transfection workflows, but its toxicity limits transfection efficiency and, consequently, protein yield (Breunig, Lungwitz, Liebl, & Goepferich, 2007; Yang, Li, Goh, & Li, 2007). Previously, PEI has been conjugated to polyethylene glycol (Petersen et al., 2002) and arginine modified oligo(-alkylaminosiloxane) [P(SiDAAr)n] (Morris & Sharma, 2010) to mitigate cytotoxicity.A promising alternative to PEI, poly(beta-amino ester)s (PBAEs) are a class of cationic polymers used to facilitate efficient gene transferin vitro (Bishop, Kozielski, & Green, 2015). PBAEs are composed of an acrylate base monomer, an amine sidechain, and a terminal end-capping group, each of which can be varied to create a vast library of materials (Akinc, Lynn, Anderson, & Langer, 2003). Hydrolyzable ester linkages allow for degradation of the PBAEs in transfection conditions which allows for use of the polymers at high weight ratios relative to other non-biodegradable materials, maximizing density of buffering amines to facilitate endosomal escape (Sunshine et al., 2012). Their biodegradability also obviates the need for medium replacements or additions, themselves contributors to cell death, which are common where PEI is utilized (Galbraith et al., 2006). These linear polymers are synthesized from inexpensive, commercially available reagents using a two-step polymerization method (Fig. S1A) and are stable long term when stored dry at -20°C (Wilson et al., 2019).Given the high transfection efficacy observed with PBAEs in variousin vitro contexts, we sought to investigate the use of PBAE nanoparticles for transient transfection of suspension cultures in intracellular and secreted protein production workflows (Fig. 1A). We selected four PBAEs with varying base (B), sidechain (S), and end-cap (E) structures to evaluate in comparison with linear 25 kDa PEI: B4-S4-E6 (4-4-6); B4-S5-E7 (4-5-7); B4-S5-E39 (4-5-39); and B5-S3-E6 (5-3-6) (Fig. 1B-C, S1B). Physiochemical characterization of PBAE and PEI nanoparticles in serum-free transfection media indicated that PBAE nanoparticles maintained a smaller size in transfection conditions (approximately 200-350 nm) whereas PEI nanoparticles were prone to aggregation, resulting in sizes over 1 µm (Fig. 1D, S1C). Previous studies indicated that PEI nanoparticles were prone to aggregation in serum-free media due to interactions with salts and a lack of adsorbed proteins that can help stabilize discrete particles and prevent clustering (Ogris et al., 1998; Pezzoli, Giupponi, Mantovani, & Candiani, 2017). Analysis of surface charge revealed that PBAE nanoparticles maintained a positive zeta potential in transfection conditions, whereas PEI nanoparticles exhibited a near neutral surface charge (Fig. 1E, S1C). Shielded surface charge of PEI particles may limit interactions with a charged cell membrane, thus hindering cellular uptake.To determine the optimal DNA dose for production of cytosolic mCherry using various polymer-based transfection agents, we selected a representative PBAE, 2-(3-aminopropylamino)ethanol end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (referred to here as 4-4-6), and compared this PBAE to 25 kDa PEI at doses ranging from 0.5 to 4 µg/mL DNA. The polymers were compared in two mammalian cell lines frequently employed for protein expression: human embryonic kidney 293F (HEK) cells and Chinese hamster ovary-S (CHO) cells. Evaluation of mCherry fluorescence over a span of 5 days indicated that peak mCherry expression occurred using the 4-4-6 polymer at 2 µg/mL and 4 µg/mL DNA doses in HEK and CHO cells, respectively (Fig. S1A). Notably, peak mCherry expression in PEI-based transfections was not comparable to that attained by 4-4-6 at any dose. Subsequent time course studies using the optimized DNA dose that compared additional PBAE structures demonstrated significantly increased mCherry expression using 4-4-6, 4-5-7, and 4-5-39 in HEK cells, and using 4-4-6 and 4-5-7 in CHO cells, compared to PEI-mediated transfection (Fig. 2A, 2C). Transfection efficiency, as measured by mCherry-positive cells on day 5, was significantly increased with all PBAEs tested in HEK cells and with 4-4-6, 4-5-7, and 4-5-39 in CHO cells (Fig. 2B, 2D, S2C). Fluorescence microscopy confirmed the increase in mCherry expression (Fig. 2E, S2D). Cell viability, assessed via MTS assay 24 h following transfection, indicated that PBAEs (especially 4-5-39 and 5-3-6) showed greater toxicity than PEI, though notably at a 20- to 30-fold higher weight ratio (Fig. S2B). Importantly, this reduced viability did not result in inferior mCherry expression relative to PEI, with 4-4-6 and 4-5-7 demonstrating superior expression in both HEK and CHO cells (Fig. 2A, C).To demonstrate that the results of these fluorescent protein expression experiments were replicable at scales relevant to the development and production of secreted proteins, we transfected HEK cell cultures of varying volumes with DNA encoding the recombinant antibody 10H2 (Chuntharapai, Lee, Hébert, & Kim, 1994; Patent No. WO/2020/243489, 2020) using either 4-4-6- or PEI-based particles. Based on SDS-PAGE image analysis of small-scale dose titrations (Fig. S3A-D), DNA was dosed at 1 µg/mL for secreted proteins in HEK cells, with polymer weight adjusted accordingly. At volumes ranging from 20-200 mL, transfection with 4-4-6 yielded between 4.5-fold and 8.2-fold more protein than did transfection with PEI (Fig. 3A). To further demonstrate the scalability of enhanced protein expression using PBAEs, 2 L cultures of HEK cells were transfected with DNA encoding either 10H2 or the bispecific antibody BS2 (Patent No. WO/2020/243489, 2020) using either 4-4-6 or PEI. Both the 10H2 and BS2 antibodies were recovered in significantly higher quantities (4.9-fold and 5.6-fold higher, respectively) when 4-4-6 was utilized compared to PEI (Fig. 3B-C). Superiority of PBAE nanoparticles was reproducible across cell lines; transfection of CHO cells with DNA encoding the recombinant antibody 602 (Krieg, Letourneau, Pantaleo, & Boyman, 2010; Létourneau et al., 2010; Patent No. WO/2020/264321, 2020) at an optimized dose of 4 µg/mL resulted in 3.4-fold more protein recovered when 4-4-6 was used compared to PEI (Fig. S3E-G).Taken together, experiments with both cytosolic and secreted proteins demonstrated that PBAEs lead to significantly enhanced protein yields compared to leading commercial reagent PEI in two cell lines that are widely used for protein production. Storage stability and straightforward synthesis from inexpensive chemical monomers further strengthen their attractiveness for use in recombinant protein production across batch scales. Overall, the favorable properties of PBAEs combined with the results herein suggest that these polymers hold promise as superior reagents for transient transfection that can significantly improve protein production workflows.

BACKGROUND Electrophysiologic Study (EPS) is a diagnostic tool that can further evaluate patients prone to arrhythmic death.OBJECTIVE To shed light on the causes of death in Chagas disease, specially on regards of importance of the VT induction and chances of death, as evaluated by Rassi clinical score. METHODS We evaluated data from 153 patients with Chagas disease from the electrophysiology laboratory from a tertiary center between January of 2011 and January 2013. All patients were evaluated with 1, 2, 3 or 4 ventricular extra-stimuli followed by runs of 10 beats fast ventricular stimulation. If VT or VF is inducible the patients are referred to ICD implant or VT ablation. RESULTS Of a total of 153 patients, 48% were male, mean age 58 ±12 years (24 - 84 years). As for risk of death according to the clinical score, 32% were evaluated as low risk, 35,4% intermediate, 32,6% high risk. NSVT on 24 hour Holter (p=0,009), stimulation on EPS (p<0,001), lower VEFE (p<0,01), cardiomegaly (p<0,001) and high risk on Rassi score (p<0,001) where the more associated variables to ventricular tachyarrithmias. CONCLUSIONS Clinical score is an interesting tool to further stratify patients in higher risk for VT induction during EPS, which is also a marker of higher risk of sudden cardiac death. Some characteristics as Non sustained VT, Pulmonary congestion, cardiomegaly and low voltage QRS were the most relevant determinants for inducible VT on EPS. The duration of NSVT is also important for VT induction in EPS.

Adult sex ratio and fecundity are key population parameters in sustainable wildlife management, but inferring these requires estimates of the density of at least three age/sex classes of the population (male and female adults and juveniles). We used an array of 36 wildlife camera traps during 2–3 weeks in autumn prior to harvest during two consecutive years, and recorded white-tailed deer adult males, adult females and fawns from the pictures. Simultaneously, we collected fecal DNA (fDNA) from 92 20mx20m plots placed in 23 clusters of four plots between the camera traps. We identified individuals from fDNA samples with microsatellite markers and estimated the total sex ratio and population density using Spatial Capture Recapture (SCR). The fDNA-SCR analysis concluded equal sex ratio in the first year and female bias in the second year, and no difference in space use between sexes (fawns and adults combined). Camera information was analyzed in a Spatial Capture (SC) framework assuming an informative prior for animals’ space use, either (1) as estimated by fDNA-SCR (same for all age/sex classes), (2) as assumed from the literature (space use of adult males larger than adult females and fawns), (3) by inferring adult male space use from individually-identified males from the camera pictures. These various SC approaches produced plausible inferences on fecundity, but also inferred total density to be lower than the estimate provided by fDNA-SCR in one of the study years. SC approaches where adult male and female were allowed to differ in their space use suggested the population had a female-biased adult sex ratio. In conclusion, SC approaches allowed estimating the pre-harvest population parameters of interest and provided conservative density estimates.