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An experiment was conducted to examine the addition of zinc in nutritive rich feed contained lemuru oil to growth and birth weight of Bali cattle. The experiment design used was randomized complete block design. The animal were randomly assigned into four group of treatments, i. e addition of 0, 75, 150 and 225 mg ZnSO4 kg-1 on nutritive rich feed (NRF) containing lemuru oil. The basal diet consist dried grass while NRF consisted of palm sugar, leucaena and glirisidia leaf meal, rice bran fermentation, fish meal, lemuru oil and urea. The crude protein content of NRF was 23%, while total digestible nutrient (TDN) was 70%. Addition ZnSO4 in the diet did not increase significantly zinc status, prostaglandin E2 (PGE2) concentration and ME retention. However, level of ZnSO4 addition increase significantly (P

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SARS-CoV-2 3C-like protease (3CL pro or M pro ) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CL pro expression and viral replication. Unexpectedly, 3CL pro and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis. Bronchoalveolar lavage fluid of wild-type SARS-CoV-2-infected mice contains 3CL pro , which decreases in Gsdmd -/- Gsdme -/- mice. We identify new 3CL pro cut-sites in GSDMD at LQ 29 ↓ 30 SS, which blocks pore formation by 3CL pro cleavage at LH 270 ↓N lying adjacent to the caspase activation site (NFLTD 275 ↓G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CL pro secretion. Extracellular 3CL pro retains activity in serum, dampens platelet activation and aggregation, and inactivates antiviral interferon-λ1. Thus, in countering gasdermin pore formation and pyroptosis in SARS-CoV-2 infection, 3CL pro is secreted with extracellular pathological sequelae., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Cigarette smoke (CS) promotes the development of chronic pulmonary disease and has been associated with increased risk for influenza-related illness. Here, we directly addressed the impact of CS disordered microbiota on the severity of influenza A virus (IAV) infection. Specific and opportunistic pathogen-free (SOPF) C57BL/6J mice were exposed to CS or room air (RA) for 5.5 months. Each exposed mouse was then cohoused with a group of recipient germ-free (GF) mice for 1 month for microbial transfer. Colonized GF mice were then infected intranasally with IAV and disease development was monitored. Upper and lower airway and fecal microbiota were longitudinally investigated by 16S rRNA gene sequencing and bacterial cultures in donor and recipient mice. The bacterial family Streptococcaceae accounted for the largest difference between CS- and RA-exposed microbiota in the oropharynx. Analysis of the oropharynx and fecal microbiota indicated an efficient transfer to coprophagic recipient mice, which replicated the differences in microbiota composition observed in donor mice. Subsequent IAV infection revealed significantly higher weight loss for CS microbiota recipient mice at 8-10 days post infection (dpi) compared to control recipient mice. In addition, H1N1 infection inflicted substantial changes in the microbiota composition, especially at days 4 and 8 after infection. In conclusion, mice with a CS-associated microbiota suffer from higher disease severity upon IAV infection compared to mice colonized with a normal SOPF microbiota. Our data suggest that independently of CS exposure and concomitant structural lung damage, microbial distortion due to CS exposure may impact the severity of IAV disease course.IMPORTANCEIt has been reported that chronic exposure to CS is associated with a disordered microbiota composition. In this study, we colonized germ-free (GF) mice with the microbiota from SOPF mice which were chronically exposed to CS or RA. This allowed disentangling the effect of the disordered microbiota from the immune-modulating effects of actual CS exposure. We observed a successful transfer of the microbiotas after cohousing including specific microbiota differences induced by CS exposure in formerly GF mice, which were never exposed to CS. We then investigated the effects of IAV infection on the disease course and microbiotas of formerly GF mice. We found that mice with CS-associated microbiota reveal worse disease course compared to the control group. We hypothesize that CS-induced disordering of the microbiota may, indeed, impact the severity of influenza A disease., Competing Interests: The authors declare no conflict of interest.

Background: One of the most prevalent bacteria that cause nosocomial infections is Pseudomonas aeruginosa. Fluoroquinolones (FQ) and aminoglycosides are vital antipseudomonal drugs, but resistance is increasingly prevalent. The study sought to investigate the diverse mechanisms underlying FQ and aminoglycoside resistance in various P. aeruginosa strains particularly during the COVID-19 crisis., Methods: From various clinical and environmental samples, 110 P. aeruginosa isolates were identified and their susceptibility to several antibiotic classes was evaluated. Molecular techniques were used to track target gene mutations, the presence of genes encoding for quinolone resistance, modifying enzymes for aminoglycosides and resistance methyltransferase (RMT). Efflux pump role was assessed phenotypically and genotypically. Random amplified polymorphic DNA (RAPD) analysis was used to measure clonal diversity., Results: QnrS was the most frequently encountered quinolone resistance gene (37.5%) followed by qnrA (31.2%) and qnrD (25%). Among aminoglycoside resistant isolates, 94.1% harbored modifying enzymes genes, while RMT genes were found in 55.9% of isolates. The aac(6')-Ib and rmtB were the most prevalent genes (79.4% and 32.3%, respectively). Most FQ resistant isolates overexpressed mexA (87.5%). RAPD fingerprinting showed 63.2% polymorphism., Conclusions: Aminoglycosides and FQ resistance observed in this study was attributed to several mechanisms with the potential for cross-contamination existence so, strict infection control practices are crucial., (© 2024. The Author(s).)

The majority of people with dementia live in low or middle-income countries (LMICs) where resources that play a crucial role in brain health, such as quality education, are still not widely available. In Brazil, illiteracy remains a prevalent issue, especially in communities with lower socioeconomic status (SES). The PROAME study set out to explore basic education in illiterate adults as a means to improve cognitive reserve., Objective: This manuscript aims to explore the relationship between SES and learning, as well as cognitive outcomes, in an older illiterate population., Methods: This six-month clinical trial (NCT04473235) involved 108 participants, of which 77 concluded all assessments, enrolled in late-life basic education. SES assessments included Quality of Urban Living Index, Municipal Human Development Index and Household SES calculated for each participant. Cognitive assessments encompassed the Free and Cued Selective Reminding Test (FCSRT), a word list to assess reading, and the Beta III matrix., Results: The sample consisted primarily of women, with a mean age of 58.5. Participants improved their reading (p=0.01) and their FCSRT (p=0.003). Regarding episodic memory, women outperformed men (p=0.007) and younger participants improved more than their older counterparts (p=0.001). There was no association observed between SES and cognitive outcomes., Conclusion: Irrespective of SES, participants demonstrated positive outcomes after attending basic education. These findings highlight that late life education could be an important non-pharmacologic preventative measure, especially in LMICs., Competing Interests: Disclosure: The authors report no conflicts of interest.

We describe an unusual case of bilateral pulmonary venous thrombosis in a pregnant woman in her mid 30s, who presented at 34 weeks of gestation with symptoms of sudden onset chest pain, shortness of breath and near syncope attacks. The patient was treated with enoxaparin and made an excellent clinical and hemodynamic recovery., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Road deposited sediments (RDS) are important sinks of potentially toxic elements (PTEs), which may have a significant impact on human health. A systematic review of published papers on the PTEs occurrence in RDS was carried out. The main goal was to assess the global RDS contamination by PTEs and human health risks linked with anthropogenic activities. A systematic search was made to collect information about the most cited PTEs in the published literature and perform a statistical analysis. Subsequently, health risks were assessed for 35 different areas worldwide. PTE concentrations showed high variability, and means were multiple times higher than the corresponding consensus-based threshold effect concentrations (5.2-, 10.3-, 5.3-, 3-, 7.3-, and 3.6-fold higher for Zn, Pb, Ni, Cr, Cu, and Cd, respectively). PTEs concentrations were ranked as Zn > Pb > Cu > Mn > Cr > Ni > Cd. Non carcinogenic risks followed the trend Pb > Cu > Zn > Cd. Lead is responsible for the highest significant non carcinogenic risk to human health. Unacceptable exposition to carcinogenic risks is present in most areas. The top carcinogenic risk areas were Singapore > Beijing > Yixing > Shanghai > Zhuzhou for adult male, Dresden > Singapore > Ulsan > Huludao for adult females, and Dresden > Singapore > Ulsan > Huludao for children. Highest chromium and nickel carcinogenic risks occurred in Singapore, Cd in Dresden, and Cu in Huludao. Highest RDS contamination was seen in industrial areas due to pollutants deposition. Highest Zn, Cu, Cd, and Pb concentrations occur in densely urbanized areas due to heavy-duty vehicular exhausts., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Little is known about the life cycle and mode of transmission of Dientamoeba fragilis . Recently it was suggested that fecal–oral transmission of cysts may play a role in the transmission of D. fragilis . In order to establish an infection, D. fragilis is required to remain viable when exposed to the pH of the stomach. In this study, we investigated the ability of cultured trophozoites to withstand the extremes of pH. We provide evidence that trophozoites of D. fragilis are vulnerable to highly acidic conditions. We also investigated further the ultrastructure of D. fragilis cysts obtained from mice and rats by transmission electron microscopy. These studies of cysts showed a clear cyst wall surrounding an encysted parasite. The cyst wall was double layered with an outer fibrillar layer and an inner layer enclosing the parasite. Hydrogenosomes, endoplasmic reticulum and nuclei were present in the cysts. Pelta-axostyle structures, costa and axonemes were identifiable and internal flagellar axonemes were present. This study therefore provides additional novel details and knowledge of the ultrastructure of the cyst stage of D. fragilis .

Cytosolic proliferating cell nuclear antigen (PCNA) is involved in neutrophil survival and function, in which it acts as a scaffold and associates with proteins involved in apoptosis, NADPH oxidase activation, cytoskeletal dynamics, and metabolism. While the PCNA interactome has been characterized in neutrophils under homeostatic conditions, less is known about neutrophil PCNA in pathophysiological contexts. Granulocyte colony-stimulating factor (G-CSF) is a cytokine produced in response to inflammatory stimuli that regulates many aspects of neutrophil biology. Here, we used isolated normal-density neutrophils from G-CSF-treated haemopoietic stem cell donors (GDs) as a model to understand the role of PCNA during inflammation. Proteomic analysis of the neutrophil cytosol revealed significant differences between GDs and healthy donors (HDs). PCNA was one of the most upregulated proteins in GDs, and the PCNA interactome was significantly different in GDs compared with HDs. Importantly, while PCNA associated with almost all enzymes involved in glycolysis in HDs, these associations were decreased in GDs. Functionally, neutrophils from GDs had a significant increase in glycolysis compared with HDs. Using p21 competitor peptides, we showed that PCNA negatively regulates neutrophil glycolysis in HDs but had no effect on GD neutrophils. These data demonstrate that G-CSF alters the PCNA scaffold, affecting interactions with key glycolytic enzymes, and thus regulates glycolysis, the main energy pathway utilized by neutrophils. By this selective control of glycolysis, PCNA can organize neutrophils functionality in parallel with other PCNA mechanisms of prolonged survival. PCNA may therefore be instrumental in the reprogramming that neutrophils undergo in inflammatory or tumoral settings., Competing Interests: Conflict of interest statement. I.W. and K.R.M. have received funding from CSL Innovation Ltd for research on antagonists of haemopoietic growth factors. The authors declare that the research was conducted in the absence of any other commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Weight gain can trigger mechanisms that increase blood pressure. Nevertheless, obesity causes structural changes in the myocardium, including increased ventricular mass, atrial dilatation, and diastolic and systolic dysfunction. Additionally, blood pressure variations, like morning surge (MS) in obese hypertensive patients may have clinical relevance in cardiovascular events. Although morning blood pressure surge is a physiological phenomenon, excess MS can be considered an independent risk factor for cardiovascular events., Objective: To evaluate MS values and their association with left ventricular hypertrophy (LVH) and nocturnal dipping (ND) in obese and non-obese hypertensive patients., Methods: A cross-sectional study that evaluated BP measurements by ambulatory blood pressure monitoring (ABPM) and the presence of LVH by echocardiography in 203 hypertensive outpatients, divided into two groups: 109 non-obese and 94 obese hypertensives patients. The significance level was set at 0.05 in two-tailed tests., Results: A MS above 20 mmHg by ABPM was detected in 59.2% of patients in the non-obese group and 40.6% in the obese group. LVH was found in 18.1% and 39.3% of patients in the non-obese and obese groups, respectively, p<0.001. In the "obese group", it was observed that a MS>16 mmHg was associated with LVH, [prevalence ratio: 2.80; 95%CI (1.12-6.98), p=0.03]. For the non-obese group, the cut-off point of MS for this association was >22 mmHg., Conclusion: High MS was positively associated with LVH, with a particular behavior in the hypertensive obese group.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance., (© 2022. The Author(s).)

The severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is β-coronavirus that is responsible for the pandemic coronavirus disease 2019 (COVID-19) all over the world. The rapid spread of the novel SARS CoV-2 worldwide is raising a significant global public health issue with nearly 61.86 million people infected and 1.4 million deaths. To date, no specific drugs are available for the treatment of COVID-19. The inhibition of proteases essential for the proteolytic treatment of viral polyproteins is a conventional therapeutic strategy for conquering viral infections. In the study, molecular docking approach was used to screen potential drug compounds among the phytochemicals of Vitex negundo L. against COVID-19 infection. Molecular docking analysis showed that oleanolic acid forms a stable complex and other phyto-compounds ursolic acid, 3β-acetoxyolean-12-en-27-oic acid and isovitexin of V. negundo natural compounds form a less-stable complex. When compared with the control the synergistic interaction of these compounds shows inhibitory activity against papain-like protease (PL pro ) of SARS CoV-2 (COVID-19). The molecular dynamics (MD) simulation (50 ns) were performed on the complexes of PL pro and the phyto-compounds viz . oleanolic acid, ursolic acid, 3β-acetoxyolean-12-en-27-oic acid and isovitexin followed by the binding free energy calculations using MM-GBSA and these molecules have stable interactions with PL pro protein binding site. The MD simulation study provides more insight into the functional properties of the protein-ligand complex and suggests that these molecules can be considered as a potential drug molecule against COVID-19. In this pandemic situation, these herbal compounds provide a rich resource to produce new antivirals against COVID-19.Communicated by Ramaswamy H. Sarma.

Corroborating data sustain the pleiotropic effect of nerve growth factor (NGF) in the protection of the visual system from dangerous stimuli, including ultraviolet (UV). Since UV exposure might promote ocular surface changes (conjunctival inflammation and matrix rearrangement), as previously reported from in vivo studies sustaining some protective NGF effects, in vitro cultures of human conjunctival fibroblasts (FBs) were developed and exposed to a single UV exposure over 15 min (0.277 W/m 2 ), either alone or supplemented with NGF (1-10-100 ng/mL). Conditioned media and cell monolayers were collected and analyzed for protein release (ELISA, ELLA microfluidic) and transcript expression (real-time PCR). A specific "inflammatory to remodeling" pattern (IL8, VEGF, IL33, OPN, and CYR61) as well as a few epigenetic transcripts (known as modulator of cell differentiation and matrix-remodeling ( DNMT3a , HDAC1, NRF2 and KEAP1 )) were investigated in parallel. UV-exposed FBs (i), showed no proliferation or significant cytoskeleton rearrangement; (ii), displayed a trkA NGFR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect NTR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect DNMT3a or HDAC1 at lowest NGF doses and did not change NRF2 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkA KEAP1 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkA NGFR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.NTR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.

Forests play a critical role in the hydrologic cycle, impacting the surface and groundwater dynamics of watersheds through transpiration, interception, shading, and modification of the atmospheric boundary layer. It is therefore critical that forest dynamics are adequately represented in watershed models, such as the widely applied Soil and Water Assessment Tool (SWAT). SWAT's default parameterization generally produces unrealistic forest growth predictions, which we address here through an improved representation of forest dynamics using species-specific re-parameterizations. We applied this methodology to the two dominant pine species in the southeastern U.S., loblolly pine (Pinus taeda L.) and slash pine (Pinus elliotti). Specifically, we replaced unrealistic parameter values related to tree growth with physically meaningful parameters derived from publicly available remote-sensing products, field measurements, published literature, and expert knowledge. Outputs of the default and re-parameterized models were compared at four pine plantation sites across a range of management, soil, and climate conditions. Results were validated against MODIS-derived leaf area index (LAI) and evapotranspiration (ET), as well as field observations of total biomass. The re-parameterized model outperformed the default model in simulating LAI, biomass accumulation, and ET at all sites. The two parametrizations also resulted in substantially different mean annual water budgets for all sites, with reductions in water yield ranging from 13 to 45% under the new parameterization, highlighting the importance of properly parameterizing forest dynamics in watershed models. Importantly, our re-parameterization methodology does not require alteration to the SWAT code, allowing it to be readily adapted and applied in ongoing and future watershed modeling studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mast cells are multifunctional immune cells scattered in tissues near blood vessels and mucosal surfaces where they mediate important reactions against parasites and contribute to the pathogenesis of allergic reactions. Serine proteases released from secretory granules upon mast cell activation contribute to these functions by modulating cytokine activity, platelet activation and proteolytic neutralization of toxins. The forced release of granule proteases into the cytosol of mast cells to induce cell suicide has recently been proposed as a therapeutic approach to reduce mast cell numbers in allergic diseases, but the molecular pathways involved in granule-mediated mast cell suicide are incompletely defined. To identify intrinsic granule proteases that can cause mast cell death, we used mice deficient in cytosolic serine protease inhibitors and their respective target proteases. We found that deficiency in Serpinb1a, Serpinb6a, and Serpinb9a or in their target proteases did not alter the kinetics of apoptosis induced by growth factor deprivation in vitro or the number of peritoneal mast cells in vivo . The serine protease cathepsin G induced marginal cell death upon mast cell granule permeabilization only when its inhibitors Serpinb1a or Serpinb6a were deleted. In contrast, the serine protease granzyme B was essential for driving apoptosis in mast cells. On granule permeabilization, granzyme B was required for caspase-3 processing and cell death. Moreover, cytosolic granzyme B inhibitor Serpinb9a prevented caspase-3 processing and mast cell death in a granzyme B-dependent manner. Together, our findings demonstrate that cytosolic serpins provide an inhibitory shield preventing granule protease-induced mast cell apoptosis, and that the granzyme B-Serpinb9a-caspase-3 axis is critical in mast cell survival and could be targeted in the context of allergic diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burgener, Brügger, Leborgne, Sollberger, Basilico, Kaufmann, Bird and Benarafa.)

Background: Predicting the mutation statuses of 2 essential pathogenic genes [epidermal growth factor receptor ( EGFR ) and Kirsten rat sarcoma ( KRAS )] in non-small cell lung cancer (NSCLC) based on CT is valuable for targeted therapy because it is a non-invasive and less costly method. Although deep learning technology has realized substantial computer vision achievements, CT imaging being used to predict gene mutations remains challenging due to small dataset limitations., Methods: We propose a multi-channel and multi-task deep learning (MMDL) model for the simultaneous prediction of EGFR and KRAS mutation statuses based on CT images. First, we decomposed each 3D lung nodule into 9 views. Then, we used the pre-trained inception-attention-resnet model for each view to learn the features of the nodules. By combining 9 inception-attention-resnet models to predict the types of gene mutations in lung nodules, the models were adaptively weighted, and the proposed MMDL model could be trained end-to-end. The MMDL model utilized multiple channels to characterize the nodule more comprehensively and integrate patient personal information into our learning process., Results: We trained the proposed MMDL model using a dataset of 363 patients collected by our partner hospital and conducted a multi-center validation on 162 patients in The Cancer Imaging Archive (TCIA) public dataset. The accuracies for the prediction of EGFR and KRAS mutations were, respectively, 79.43% and 72.25% in the training dataset and 75.06% and 69.64% in the validation dataset., Conclusions: The experimental results demonstrated that the proposed MMDL model outperformed the latest methods in predicting EGFR and KRAS mutations in NSCLC., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/qims-20-600). The authors have no conflicts of interest to declare., (2021 Quantitative Imaging in Medicine and Surgery. All rights reserved.)

Aptamers are oligonucleotides that bind with high affinity to target molecules of interest. One such target is glycated hemoglobin (gHb), a biomarker for assessing glycemic control and diabetes diagnosis. By the coupling of aptamers with surface plasmon resonance (SPR) sensing surfaces, a fast, reliable and inexpensive assay for gHb can be developed. In this study, we tested the affinity of SPR-sensing surfaces, composed of aptamers and antifouling self-assembled monolayers (SAMs), to hemoglobin (Hb) and gHb. First, we developed a gHb-targeted aptamer (GHA) through a modified Systematic Evolution of Ligands by EXponential (SELEX) enrichment process and tested its affinity to gHb using the Nano-Affi protocol. GHA was used to produce three distinct SAM-SPR-sensing surfaces: (Type-1) a SAM of GHA directly attached to a sensor surface; (Type-2) GHA attached to a SAM of 11-mercaptoundecanoic acid (11MUA) on a sensor surface; (Type-3) GHA attached to a binary SAM of 11MUA and 3,6-dioxa-8-mercaptooctan-1-ol (DMOL) on a sensor surface. Type-2 and Type-3 surfaces were characterized by cyclic voltammetry and electrochemical impedance spectroscopy to confirm that GHA bound to the underlying SAMs. The adsorption kinetics for Hb and gHb interacting with each SPR sensing surface were used to quantify their respective affinities. The Type-1 surface without antifouling modification had a dissociation constant ratio (K D,Hb /K D,gHb ) of 9.7, as compared to 809.3 for the Type-3 surface, demonstrating a higher association of GHA to gHb for sensor surfaces with antifouling modifications than those without. The enhanced selectivity of GHA to gHb can likely be attributed to the inclusion of DMOL in the SAM-modified surface, which reduced interference from nonspecific adsorption of proteins. Results suggest that pairing aptamers with antifouling SAMs can significantly improve their target affinity, potentially allowing for the development of novel, low cost, and fast assays.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - coronavirus disease 2019 (COVID-19) has raised a severe global public health issue and creates a pandemic situation. The present work aims to study the molecular -docking and dynamic of three pertinent medicinal plants i.e. Eurycoma harmandiana , Sophora flavescens and Andrographis paniculata phyto-compounds against SARS-COV-2 papain-like protease (PL pro ) and main protease (M pro )/3-chymotrypsin-like protease (3CL pro ). The interaction of protein targets and ligands was performed through AutoDock-Vina visualized using PyMOL and BIOVIA-Discovery Studio 2020. Molecular docking with canthin-6-one 9-O-beta-glucopyranoside showed highest binding affinity and less binding energy with both PL pro and M pro /3CL pro proteases and was subjected to molecular dynamic (MD) simulations for a period of 100ns. Stability of the protein-ligand complexes was evaluated by different analyses. The binding free energy calculated using MM-PBSA and the results showed that the molecule must have stable interactions with the protein binding site. ADMET analysis of the compounds suggested that it is having drug-like properties like high gastrointestinal (GI) absorption, no blood-brain barrier permeability and high lipophilicity. The outcome revealed that canthin-6-one 9-O-beta-glucopyranoside can be used as a potential natural drug against COVID-19 protease., Competing Interests: The authors declare that there is no conflict of interest., (© 2021 Published by Elsevier B.V.)

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes. They quickly respond to antigenic stimulation by producing copious amounts of cytokines and chemokines. iNKT precursors differentiate into three subsets iNKT1, iNKT2, and iNKT17 with specific cytokine production signatures. While key transcription factors drive subset differentiation, factors that regulate iNKT subset homeostasis remain incompletely defined. Transcriptomic analyses of thymic iNKT subsets indicate that Serpinb1a is one of the most specific transcripts for iNKT17 cells suggesting that iNKT cell maintenance and function may be regulated by Serpinb1a. Serpinb1a is a major survival factor in neutrophils and prevents cell death in a cell-autonomous manner. It also controls inflammation in models of bacterial and viral infection as well as in LPS-driven inflammation. Here, we examined the iNKT subsets in neutropenic Serpinb1a -/- mice as well as in Serpinb1a -/- mice with normal neutrophil counts due to transgenic re-expression of SERPINB1 in neutrophils. In steady state, we found no significant effect of Serpinb1a -deficiency on the proliferation and numbers of iNKT subsets in thymus, lymph nodes, lung, liver and spleen. Following systemic activation with α-galactosylceramide, the prototypic glycolipid agonist of iNKT cells, we observed similar serum levels of IFN-γ and IL-4 between genotypes. Moreover, splenic dendritic cells showed normal upregulation of maturation markers following iNKT cell activation with α-galactosylceramide. Finally, lung instillation of α-galactosylceramide induced a similar recruitment of neutrophils and production of iNKT-derived cytokines IL-17, IFN-γ, and IL-4 in wild-type and Serpinb1a -/- mice. Taken together, our results indicate that Serpinb1a, while dominantly expressed in iNKT17 cells, is not essential for iNKT cell homeostasis, subset differentiation and cytokine release., (Copyright © 2020 Leborgne, Taddeo, Freigang and Benarafa.)

Objective: The inhalation of air-borne toxicants is associated with adverse health outcomes which can be somewhat mitigated by enhancing endogenous anti-oxidant capacity. Carnosine is a naturally occurring dipeptide (β-alanine-L-histidine), present in high abundance in skeletal and cardiac muscle. This multi-functional dipeptide has anti-oxidant properties, can buffer intracellular pH, chelate metals, and sequester aldehydes such as acrolein. Due to these chemical properties, carnosine may be protective against inhaled pollutants which can contain metals and aldehydes and can stimulate the generation of electrophiles in exposed tissues. Thus, assessment of carnosine levels, or levels of its acrolein conjugates (carnosine-propanal and carnosine-propanol) may inform on level of exposure and risk assessment., Methods: We used established mass spectroscopy methods to measure levels of urinary carnosine ( n  = 605) and its conjugates with acrolein ( n  = 561) in a subset of participants in the Louisville Healthy Heart Study (mean age = 51 ± 10; 52% male). We then determined associations between these measures and air pollution exposure and smoking behavior using statistical modeling approaches., Results: We found that higher levels of non-conjugated carnosine, carnosine-propanal, and carnosine-propanol were significantly associated with males ( p  < 0.02) and those of Caucasian ethnicity ( p  < 0.02). Levels of carnosine-propanol were significantly higher in never-smokers ( p  = 0.001) but lower in current smokers ( p  = 0.037). This conjugate also demonstrated a negative association with mean-daily particulate air pollution (PM 2.5 ) levels ( p  = 0.01)., Conclusions: These findings suggest that urinary levels of carnosine-propanol may inform as to risk from inhaled pollutants.

Over the past two decades, researchers have discovered a special form of alternative splicing that produces a circular form of RNA. Although these circular RNAs (circRNAs) have garnered considerable attention in the scientific community for their biogenesis and functions, the focus of current studies has been on the tissue-specific circRNAs that exist only in one tissue but not in other tissues or on the disease-specific circRNAs that exist in certain disease conditions, such as cancer, but not under normal conditions. This approach was conducted in the relative absence of methods that analyze a group of common circRNAs that exist in both conditions, but are more abundant in one condition relative to another (differentially expressed). Studies of differentially expressed circRNAs (DECs) between two conditions would serve as a significant first step in filling this void. Here, we introduce a novel computational tool, seekCRIT (seek for differentially expressed CircRNAs In Transcriptome), that identifies the DECs between two conditions from high-throughput sequencing data. Using rat retina RNA-seq data from ischemic and normal conditions, we show that over 74% of identifiable circRNAs are expressed in both conditions and over 40 circRNAs are differentially expressed between two conditions. We also obtain a high qPCR validation rate of 90% for DECs with a FDR of < 5%. Our results demonstrate that seekCRIT is a novel and efficient approach to detect DECs using rRNA depleted RNA-seq data. seekCRIT is freely downloadable at https://github.com/UofLBioinformatics/seekCRIT. The source code is licensed under the MIT License. seekCRIT is developed and tested on Linux CentOS-7., Competing Interests: The authors have declared that no competing interests exist.

Background: High-throughput RNA sequencing (RNA-seq) has evolved as an important analytical tool in molecular biology. Although the utility and importance of this technique have grown, uncertainties regarding the proper analysis of RNA-seq data remain. Of primary concern, there is no consensus regarding which normalization and statistical methods are the most appropriate for analyzing this data. The lack of standardized analytical methods leads to uncertainties in data interpretation and study reproducibility, especially with studies reporting high false discovery rates. In this study, we compared a recently developed normalization method, UQ-pgQ2, with three of the most frequently used alternatives including RLE (relative log estimate), TMM (Trimmed-mean M values) and UQ (upper quartile normalization) in the analysis of RNA-seq data. We evaluated the performance of these methods for gene-level differential expression analysis by considering the factors, including: 1) normalization combined with the choice of a Wald test from DESeq2 and an exact test/QL (Quasi-likelihood) F-Test from edgeR; 2) sample sizes in two balanced two-group comparisons; and 3) sequencing read depths., Results: Using the MAQC RNA-seq datasets with small sample replicates, we found that UQ-pgQ2 normalization combined with an exact test can achieve better performance in term of power and specificity in differential gene expression analysis. However, using an intra-group analysis of false positives from real and simulated data, we found that a Wald test performs better than an exact test when the number of sample replicates is large and that a QL F-test performs the best given sample sizes of 5, 10 and 15 for any normalization. The RLE, TMM and UQ methods performed similarly given a desired sample size., Conclusion: We found the UQ-pgQ2 method combined with an exact test/QL F-test is the best choice in order to control false positives when the sample size is small. When the sample size is large, UQ-pgQ2 with a QL F-test is a better choice for the type I error control in an intra-group analysis. We observed read depths have a minimal impact for differential gene expression analysis based on the simulated data.

Background: Lung cancer is one of the most common types of cancer, among which lung adenocarcinoma accounts for the largest proportion. Currently, accurate staging is a prerequisite for effective diagnosis and treatment of lung adenocarcinoma. Previous research has used mainly single-modal data, such as gene expression data, for classification and prediction. Integrating multi-modal genetic data (gene expression RNA-seq, methylation data and copy number variation) from the same patient provides the possibility of using multi-modal genetic data for cancer prediction. A new machine learning method called gcForest has recently been proposed. This method has been proven to be suitable for classification in some fields. However, the model may face challenges when applied to small samples and high-dimensional genetic data., Results: In this paper, we propose a multi-weighted gcForest algorithm (MLW-gcForest) to construct a lung adenocarcinoma staging model using multi-modal genetic data. The new algorithm is based on the standard gcForest algorithm. First, different weights are assigned to different random forests according to the classification performance of these forests in the standard gcForest model. Second, because the feature vectors generated under different scanning granularities have a diverse influence on the final classification result, the feature vectors are given weights according to the proposed sorting optimization algorithm. Then, we train three MLW-gcForest models based on three single-modal datasets (gene expression RNA-seq, methylation data, and copy number variation) and then perform decision fusion to stage lung adenocarcinoma. Experimental results suggest that the MLW-gcForest model is superior to the standard gcForest model in constructing a staging model of lung adenocarcinoma and is better than the traditional classification methods. The accuracy, precision, recall, and AUC reached 0.908, 0.896, 0.882, and 0.96, respectively., Conclusions: The MLW-gcForest model has great potential in lung adenocarcinoma staging, which is helpful for the diagnosis and personalized treatment of lung adenocarcinoma. The results suggest that the MLW-gcForest algorithm is effective on multi-modal genetic data, which consist of small samples and are high dimensional.

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a -/- mice. Furthermore, Sb1a.Sb6a -/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a -/- macrophages showed increased IL-1β release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

High throughput RNA sequencing (RNA-seq) technology is increasingly used in disease-related biomarker studies. A negative binomial distribution has become the popular choice for modeling read counts of genes in RNA-seq data due to over-dispersed read counts. In this study, we propose two explicit sample size calculation methods for RNA-seq data using a negative binomial regression model. To derive these new sample size formulas, the common dispersion parameter and the size factor as an offset via a natural logarithm link function are incorporated. A two-sided Wald test statistic derived from the coefficient parameter is used for testing a single gene at a nominal significance level 0.05 and multiple genes at a false discovery rate 0.05. The variance for the Wald test is computed from the variance-covariance matrix with the parameters estimated from the maximum likelihood estimates under the unrestricted and constrained scenarios. The performance and a side-by-side comparison of our new formulas with three existing methods with a Wald test, a likelihood ratio test or an exact test are evaluated via simulation studies. Since other methods are much computationally extensive, we recommend our M1 method for quick and direct estimation of sample sizes in an experimental design. Finally, we illustrate sample sizes estimation using an existing breast cancer RNA-seq data.

Breast cancer (BC) is increasing in incidence and resistance to treatment worldwide. The challenges in limited therapeutic options and poor survival outcomes in BC subtypes persist because of its molecular heterogeneity and resistance to standard endocrine therapy. Recently, high throughput RNA sequencing (RNA-seq) has been used to identify biomarkers of disease progression and signaling pathways that could be amenable to specific therapies according to the BC subtype. However, there is no single generally accepted pipeline for the analysis of RNA-seq data in biomarker discovery due, in part, to the needs of simultaneously satisfying constraints of sensitivity and specificity. We proposed a combined approach using gene-wise normalization, UQ-pgQ2, followed by a Wald test from DESeq2. Our approach improved the analysis based on within-group comparisons in terms of the specificity when applied to publicly available RNA-seq BC datasets. In terms of identifying differentially expressed genes (DEGs), we combined an optimized log2 fold change cutoff with a nominal false discovery rate of 0.05 to further minimize false positives. Using this method in the analysis of two GEO BC datasets, we identified 797 DEGs uniquely expressed in triple negative BC (TNBC) and significantly associated with T cell and immune-related signaling, contributing to the immunotherapeutic efficacy in TNBC patients. In contrast, we identified 1403 DEGs uniquely expressed in estrogen positive and HER2 negative BC (ER+HER2-BC) and significantly associated with eicosanoid, notching and FAK signaling while a common set of genes was associated with cellular growth and proliferation. Thus, our approach to control for false positives identified two distinct gene expression profiles associated with these two subtypes of BC which are distinguishable by their molecular and functional attributes., Competing Interests: The authors have declared that no competing interests exist.

Background: Duchenne muscular dystrophy is an X-chromosome-linked genetic disorder (locus Xp21). Involvement of the cardiovascular system is characterized by fibrous degeneration/replacement of myocytes with consequent ventricular hypertrophy and arterial hypertension., Objective: To assess, by using 24-hour ambulatory blood pressure monitoring, the behavior of blood pressure variables in children and adolescents with a confirmed diagnosis of Duchenne muscular dystrophy., Methods: Prospective observational cohort study, which selected 46 patients followed up on an outpatient basis, divided according to age groups. Blood pressure was classified according to the age percentile. The monitoring interpretation includes systolic and diastolic blood pressure means, systolic and diastolic blood pressure loads, and nocturnal dipping. The blood pressure means were calculated for the 24-hour, wakefulness and sleep periods. Nocturnal dipping was defined as a drop in blood pressure means during sleep greater than 10%. The significance level adopted was p < 0.05., Results: Nocturnal dipping for systolic blood pressure was present in 29.9% of the participants. Approximately 53% of them had attenuated nocturnal dipping, and 15%, reverse nocturnal dipping. The age groups of 9-11 years and 6-8 years had the greatest percentage of attenuation, 19.1% and 14.9%, respectively. Regarding diastolic blood pressure, nocturnal dipping was identified in 53.2% of the children, being extreme in 27.7% of those in the age group of 6-11 years., Conclusions: The early diagnosis of blood pressure changes can allow the appropriate and specific therapy, aimed at increasing the life expectancy of patients with Duchenne muscular dystrophy.

Purpose: Based on evidence that nerve growth factor (NGF) exerts healing action on damaged corneal, retinal, and cutaneous tissues, the present study sought to assess whether topical NGF application can prevent and/or protect epithelial cells from deleterious effects of ultraviolet (UV) radiation., Methods: Eyes from 40 young-adult Sprague Dawley rats and cutaneous tissues from 36 adult nude mice were exposed to UVA/B lamp for 60 min, either alone or in the presence of murine NGF. Corneal, retinal, and cutaneous tissues were sampled/processed for morphological, immunohistochemical, and biomolecular analysis, and results were compared statistically., Results: UV exposure affected both biochemical and molecular expression of NGF and trkA NGFR in corneal, retinal, and cutaneous tissues while UV exposure coupled to NGF treatment enhanced NGF and trkA NGFR expression as well as reduced cell death., Conclusions: Overall, the findings of this in vivo/ex vivo study show the NGF ability to reduce the potential UV damage. Although the mechanism underneath this effect needs further investigation, these observations prospect the development of a pharmacological NGF-based therapy devoted to maintain cell function when exposed to phototoxic UV radiation.

The goal of this study is to develop a model that explains the relationship between microRNAs, transcription factors, and their co-target genes. This relationship was previously reported in gene regulatory loops associated with 24 hour (24h) and 7 day (7d) time periods following ischemia-reperfusion injury in a rat's retina. Using a model system of retinal ischemia-reperfusion injury, we propose that microRNAs first influence transcription factors, which in turn act as mediators to influence transcription of genes via triadic regulatory loops. Analysis of the relative contributions of direct and indirect regulatory influences on genes revealed that a substantial fraction of the regulatory loops (69% for 24 hours and 77% for 7 days) could be explained by causal mediation. Over 40% of the mediated loops in both time points were regulated by transcription factors only, while about 20% of the loops were regulated entirely by microRNAs. The remaining fractions of the mediated regulatory loops were cooperatively mediated by both microRNAs and transcription factors. The results from these analyses were supported by the patterns of expression of the genes, transcription factors, and microRNAs involved in the mediated loops in both post-ischemic time points. Additionally, network motif detection for the mediated loops showed a handful of time specific motifs related to ischemia-reperfusion injury in a rat's retina. In summary, the effects of microRNAs on genes are mediated, in large part, via transcription factors.

Perivascular spaces play a pivotal role in the exchange between cerebrospinal and interstitial fluids, and in the clearance of waste in the CNS, yet their precise anatomical components are not well described. The aim of this study was to characterise the ultrastructure of perivascular spaces and their role in the transport of fluid, in the spinal cord of healthy rats, using transmission electron microscopy. The distribution of cerebrospinal fluid tracers injected into the subarachnoid space was studied using light, confocal and electron microscopy. Perivascular spaces were found around arterioles and venules, but not capillaries, throughout the spinal cord white and grey matter. They contained fibroblasts and collagen fibres, and were continuous with the extracellular spaces of the surrounding tissue. At 5 min post injection, tracers were seen in the subarachnoid space, the peripheral white matter, the perivascular spaces, basement membranes, extracellular spaces of the surrounding tissue, and surprisingly, in the lumen of blood vessels, suggesting trans-vascular clearance. These findings point out an unrecognised outflow pathway for CNS fluids, with potential implications for volume regulation in health and disease states, but also clinically for the detection of CNS-derived biomarkers in plasma, the immune response and drug pharmacokinetics.

Purpose: Increasing evidence suggests that nerve growth factor (NGF) exerts protective effects against retinal degeneration in animal models of retinitis pigmentosa (RP). This study aims at investigating the effects of intravitreal injection of recombinant human NGF (rhNGF) on retinal photoreceptors apoptosis in an animal model of RP, the Royal College of Surgeons (RCS) rats., Methods: Thirty-six RCS rats were treated with intravitreal injection of rhNGF or murine NGF (mNGF) or vehicle at 20 postnatal days (pd) and sacrificed at 40 pd. The eyes were enucleated and evaluated by histology, flow cytometric analysis for rhodopsin expression, Western blot for TrkA and activated (phosphorylated) TrkA (pTrkA) levels, and TUNEL assay for apoptosis' detection., Results: RCS rats showed a significant retinal degeneration associated with cell apoptosis at 40 pd when compared to wild-type animals. Histology showed that rhNGF intravitreal treatment significantly increased retinal thickness when compared to untreated eyes. Photoreceptors' number evaluated by flow cytometry was significantly increased in both intravitreal rhNGF- and mNGF-treated groups when compared to untreated eyes. This protective effect was associated with an increase in TrkA and activated pTrkA levels and an inhibition of apoptosis. Intravitreal NGF injection was well tolerated and did not show clinical and histological signs of adverse effects., Conclusions: Intravitreal rhNGF injection proved safe and effective in favoring retinal cell survival in RCS rats. This is the first report showing that the novel rhNGF already proved safe in a phase I study exerts a biologic effect similar to the well-characterized mNGF-induced retinal protection. These results may trigger further studies to investigate rhNGF administration for the treatment of progressive degenerative retinal disorders such as retinitis pigmentosa.