Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

6-Azaspiro[2.5]octanes as small molecule agonists of the human glucagon-like peptide-1 receptor.

Activation of the glucagon-like peptide-1 (GLP-1) receptor stimulates insulin release, lowers plasma glucose levels, delays gastric emptying, increases satiety, suppresses food intake, and affords weight loss in humans. These beneficial attributes have made peptide-based agonists valuable tools for the treatment of type 2 diabetes mellitus and obesity. However, efficient, and consistent delivery of peptide agents generally requires subcutaneous injection, which can reduce patient utilization. Traditional orally absorbed small molecules for this target may offer improved patient compliance as well as the opportunity for co-formulation with other oral therapeutics. Herein, we describe an SAR investigation leading to small-molecule GLP-1 receptor agonists that represent a series that parallels the recently reported clinical candidate danuglipron. In the event, identification of a benzyloxypyrimidine lead, using a sensitized high-throughput GLP-1 agonist assay, was followed by optimization of the SAR using substituent modifications analogous to those discovered in the danuglipron series. A new series of 6-azaspiro[2.5]octane molecules was optimized into potent GLP-1 agonists. Information gleaned from cryogenic electron microscope structures was used to rationalize the SAR of the optimized compounds.

Small molecule inhibitors of PCSK9. SAR investigations of head and amine groups.

Our previous work on the optimization of a new class of small molecule PCSK9 mRNA translation inhibitors focused on empirical optimization of the amide tail region of the lead PF-06446846 (1). This work resulted in compound 3 that showed an improved safety profile. We hypothesized that this improvement was related to diminished binding of 3 to non-translating ribosomes and an apparent improvement in transcript selectivity. Herein, we describe our efforts to further optimize this series of inhibitors through modulation of the heterocyclic head group and the amine fragment. Some of the effort was guided by an emerging cryo electron microscopy structure of the binding mode of 1 in the ribosome. These efforts led to the identification of 15 that was deemed suitable for evaluation in a humanized PCSK9 mouse model and a rat toxicology study. Compound 15 demonstrated a dose dependent reduction of plasma PCSK9 levels. The rat toxicological profile was not improved over that of 1, which precluded 15 from further consideration as a clinical candidate.

Disentangling working memory from multiple-object tracking: Evidence from dual-task interferences.

Multiple object tracking (MOT) is generally regarded as a pure attention-consuming task that draws heavily on attention resources. In the present study, we adopted a cross-channel visual-audio dual-task paradigm, i.e., the MOT task combined with the concurrent auditory N-back working memory task, to test whether working memory indeed plays a necessary role in the process of multiple tracking, as well as to further identify the specific types of working memory components involved in this process. Experiments 1a and 1b examined the relationship between the MOT task and nonspatial object working memory (OWM) processing by manipulating the tracking load and working memory load, respectively. Results in both experiments indicated that the concurrent nonspatial OWM task did not have a significant effect on the tracking capacity of the MOT task. In contrast, Experiments 2a and 2b examined the relationship between the MOT task and spatial working memory (SWM) processing by a similar approach. Results in both experiments indicated that the concurrent SWM task significantly impaired the tracking capacity of the MOT task, showing a gradual decrease with increasing SWM load. Overall, our study provides empirical evidence that multiple object tracking does involve working memory, primarily related to spatial working memory rather than nonspatial object working memory, which sheds more light on the mechanisms of multiple object tracking.

Correction: Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

[This corrects the article DOI: 10.1371/journal.pbio.2001882.].

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

Discovery of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides as small molecule inhibitors of PCSK9.

A series of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides were identified as small molecule PCSK9 mRNA translation inhibitors. Analogues from this new chemical series, such as 4d and 4g, exhibited improved PCSK9 potency, ADME properties, and in vitro safety profiles when compared to earlier lead structures.

Liver-Targeted Small-Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis.

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.

Regio- and Enantioselective Synthesis of Azole Hemiaminal Esters by Lewis Base Catalyzed Dynamic Kinetic Resolution.

We report a modular three-component dynamic kinetic resolution (DKR) that affords enantiomerically enriched hemiaminal esters derived from azoles and aldehydes. The novel and scalable reaction can be used to synthesize valuable substituted azoles in a regioselective manner by capping (e.g., acylation) of the equilibrating azole-aldehyde adduct. With the use of a prolinol-derived DMAP catalyst as the chiral Lewis base, the products can be obtained in high chemical yield and with high enantiomeric excess. The DKR was performed on a multikilogram scale to produce a tetrazole prodrug fragment for a leading clinical candidate that posed formidable synthesis challenges.

Self-Control, Openness, Personal Need for Structure and Compensatory Control Change: A Serial Mediation Investigation.

In the aftermath of the COVID-19 pandemic, numerous studies have indicated that individuals are confronting a diminished sense of control. Compensatory control theory suggests that individuals strive to mitigate this loss by modifying their behavior. The present study aims to investigate the relationship between self-control and compensatory control change during the COVID-19 pandemic, as well as the mediating effects of openness and the personal need for structure. Participants completed an online questionnaire consisting of Personal Need for Structure Scale, Self-Control Scale, Openness Scale and Compensatory Control Change Scale. The results showed that the compensatory control change increased after the outbreak. Moreover, a serial mediation was found: openness and the personal need for structure partially mediated the relationship between self-control and compensatory control change. The results indicate that the COVID-19 pandemic has led to an increase in compensatory control behaviors, especially among those with pronounced self-control. High self-control individuals are found to exhibit greater openness, reducing their personal need for structure, in effect enhancing their compensatory control change. These findings highlight the critical role of self-control in sustaining a sense of control, which is vital for understanding psychological health management in the context of public health events.

A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

A new nasal cavity nursing methods application in patients with mechanical ventilation.

OBJECTIVE: To compare different nasal cavity nursing methods on mechanically ventilated patients. METHODS: According to acute physiology and chronic health evaluation (APACHEII), 615 cases of mechanically ventilated patients were divided into group A, group B and group C by stratified random method. Traditional oral nursing plus aspirating secretions from oral cavity and nasal cavity q6h were done in group A. Based on methods in group A, normal saline was used for cleaning nasal cavity in group B. Besides the methods in group A, atomizing nasal cleansing a6h was also used in group C. Incidence rate of Ventilator-Associated Pneumonia (VAP) and APACHE II scores after administrating were compared. The correlation between APACHE II score and outcomes was analyzed by Spearman-rank correlation. RESULTS: In group A, incidence of VAP was 36.76%, group B was 30.24%, group C was 20.38%, and the difference was statistically significant. APACHE II scores in group C were significantly lower compared with group A and B. APACHE II score was negatively correlated with clinical outcomes. CONCLUSIONS: For mechanically ventilated patients, nasal nursing can't be ignored and the new atomizing nasal cleaning is an effective method for VAP prevention.

Pathogenicity Studies of NADC34-like Porcine Reproductive and Respiratory Syndrome Virus LNSY-GY and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus GXGG-8011 in Piglets.

The porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses to the swine industry. The U.S., China, and Peru have reported NADC30-like or NADC34-like PRRSV-infected piglets, which have been identified as the cause of a significant number of abortions in clinics. Although the pathogenicity of NADC30-like PRRSV and NADC34-like PRRSV in piglets exhibits significant variability globally, studies on their pathogenicity in China are limited. In this study, the animal experiments showed that within 8-14 days post-infection, both piglets infected with NADC30-like PRRSV GXGG-8011 and those infected with NADC34-like PRRSV LNSY-GY exhibited significant weight loss compared to the control piglets. Additionally, the viremia of the LNSY-GY persisted for 28 days, while the viremia of piglets infected with the GXGG-8011 lasted for 17 days. Similarly, the duration of viral shedding through the fecal-oral route after the LNSY-GY infection was longer than that observed after the GXGG-8011 infection. Furthermore, post-infection, both the LNSY-GY and GXGG-8011 led to pronounced histopathological lesions in the lungs of piglets, including interstitial pneumonia and notable viral colonization. However, the antibody production in the LNSY-GY-infected group occurred earlier than that in the GXGG-8011-infected group. Our research findings indicate that LNSY-GY is a mildly pathogenic strain in piglets, whereas we speculate that the GXGG-8011 might be a highly pathogenic strain.

People in Tight Cultures and Tight Situations Wear Masks More: Evidence From Three Large-Scale Studies in China.

Studies have found large differences in masks use during the pandemic. We found evidence that cultural tightness explains mask use differences and this association was more robust in tight situations like subways. In Study 1, we observed 23,551 people's actual mask use in public places around China. People wore masks more in tight situations; however, differences did not extend to outdoor streets and public parks, where norms are looser. We replicated this finding using a data from 15,985 people across China. Finally, in a preregistered study we observed mask use with the removal of COVID-19 restrictions, people still wore masks more in tight situations like subways than in loose situations of parks. These findings suggest that norm tightness has a lasting association with people's health-protective behaviors, especially in tight situations. It provides insight into how different cultures might respond with future pandemics and in what situations people adopt health-protective behaviors.

An fMRI study of visual geometric shapes processing.

Cross-modal correspondence has been consistently evidenced between shapes and other sensory attributes. Especially, the curvature of shapes may arouse the affective account, which may contribute to understanding the mechanism of cross-modal integration. Hence, the current study used the functional magnetic resonance imaging (fMRI) technique to examine brain activity's specificity when people view circular and angular shapes. The circular shapes consisted of a circle and an ellipse, while the angular shapes consisted of a triangle and a star. Results show that the brain areas activated by circular shapes mainly involved the sub-occipital lobe, fusiform gyrus, sub and middle occipital gyrus, and cerebellar VI. The brain areas activated by angular shapes mainly involve the cuneus, middle occipital gyrus, lingual gyrus, and calcarine gyrus. The brain activation patterns of circular shapes did not differ significantly from those of angular shapes. Such a null finding was unexpected when previous cross-modal correspondence of shape curvature was considered. The different brain regions detected by circular and angular shapes and the potential explanations were discussed in the paper.

Treatment of corneal dermoid with lenticules from small incision lenticule extraction surgery: a surgery assisted by fibrin glue.

AIM: To observe the clinical efficacy of the combined use of small incision lenticule extraction (SMILE)-derived lenticule patches in corneal dermoid excision, with fixation of the lenticule patches assisted by fibrin glue. METHODS: Seventeen eyes of 17 patients with corneal dermoid were treated with dermoid removal combined with SMILE-derived lenticule transplantation. All lenticule patches were fixed by fibrin glue. Ocular changes were assessed using slit lamp microscopy and anterior-segmental optical coherence tomography. The best-corrected visual acuity (BCVA) and ocular dioptric variations were examined preoperatively and postoperatively. Intraocular pressure (IOP) was also monitored in all visited time. RESULTS: Totally, 18 lenticule patches were used on 17 eyes of 17 cornea dermoid patients. The mean follow-up time was 11.47±5.28mo. All lenticule patches were successfully glued, kept on its location and maintained transparent during the follow-up time, with a consecutive epithelial cover for 1wk. Nine of the patients could coordinate visual and optometry exam well. Their preoperative BCVA is 0.60±0.35 in decimal, significantly improved to 0.80±0.26 in decimal at 6mo postoperatively (Z=-2.392, P=0.017), but the changes of their corneal astigmatism diopters showed no significance, with 2.22±1.91 D preoperatively, and 2.28±1.31 D at 6mo postoperatively (Z=-0.135, P=0.893). Limbal pannus formation occurred in 4 (23.52%) cases and decreased with the application of tacrolimus eyedrops. IOP increased in 2 (11.76%) cases, but well decreased by timolol maleate eyedrops. All the adult patients or guardians of minor patients were satisfied with the cosmetic improvement. CONCLUSION: Dermoid excision combined with transplantation of SMILE-derived lenticule patches using fibrin glue is a safe and effective novel tectonic keratoplasty procedure for corneal dermoid.

A plasma proteomic approach in patients with heart failure after acute myocardial infarction: insights into the pathogenesis and progression of the disease.

Aims: The pathogenesis of disease progression targets for patients with heart failure after acute myocardial infarction was investigated by using plasma proteomics. Methods: The plasma proteomes of acute myocardial infarction patients with (MI-HF) and without (MI-WHF) heart failure were compared. Each group consisted of 10 patients who were matched for age and sex. The peptides were analyzed by 2-dimensional liquid chromatography coupled to tandem mass spectrometry in a high definition mode. Parallel reaction monitoring (PRM) verified the selected target proteins. Results: We identified and quantified 2,589 and 2,222 proteins, respectively, and found 117 differentially expressed proteins (DEPs) (≥1.5-fold), when the MI-HF and MI-WHF groups were compared. Of these 51 and 66 were significantly up-regulated and down-regulated, respectively. The significant DEPs was subjected to protein-protein interaction network analysis which revealed a central role of the NF-κB signaling pathway in the MI-HF patients. PRM verified that MB, DIAPH1, VNN1, GOT2, SLC4A1, CRP, CKM, SOD3, F7, DLD, PGAM2, GOT1, UBA7 and HYOU1 were 14 proteins which were highly expressed in MI-HF patients. Conclusions: These findings showed a group of proteins related to the NF-κB signaling pathway in the pathogenesis of patients with poor outcomes after experiencing MI-HF. These proteins may be useful candidate markers for the diagnosis of MI-HF as well as help to elucidate the pathophysiology of this major cause of mortality in older patients.

Identification of novel series of pyrazole and indole-urea based DFG-out PYK2 inhibitors.

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide-urea and a pyrazole-urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.

The effects of inorganic anions on degradation kinetics and isotope fractionation during the transformation of tris(2-chloroethyl) phosphate (TCEP) by UV/persulfate.

Tris(2-chloroethyl) phosphate (TCEP), as a typical chlorinated flame retardant, is attracting more attention as a carcinogen. Although persulfate-based oxidation exhibits good performance in removing refractory organic pollutants, the kinetics of persulfate-based remediation are affected by inorganic anions, which causes inaccurate remediation efficiency. This study combines steady-state radical concentration modelling with isotope fractionation to investigate the effects of inorganic anions on TCEP degradation by UV/persulfate (UV/PS). In the absence of anions during UV/PS system, the observed degradation rate was (9.7 ± 0.1) × 10-5 s-1, which was approximately 93 % attributed to sulfate radical (SO4-•) oxidation based on radical modelling. Carbon isotope fractionation, coupled with the identification of transformation products by mass spectrometry, suggests a carbon bond split during TCEP degradation with a carbon isotopic fractionation value (ε) of -1.6 ± 0.2 ‰ (± 95 % confidence intervals). With respect to co-existing anions in UV/PS system, the addition of chloride (Cl-) had a negligible effect on degradation rates, while the addition of hydrogencarbonate (HCO3-) caused them to decrease, and the addition of hydrogenphosphate (HPO42-) caused them to increase. Radical modelling suggested that SO4-• was transformed to chlorine radicals (Cl•/Cl2-•), phosphate radicals (HPO4-•), and carbonate radicals (CO3-•). Furthermore, the overlapping 95 % confidence intervals (C.I.) and the statistical tests (p > 0.05) both agree that Cl- and HPO42- gain identical ε values. Nevertheless, when HCO3- coexisted in the UV/PS system, the ε values were distinct. The addition of HCO3- would result in ε variation of TCEP in the UV activated PS process, which should receive more attention when applying remediation.

How do humans group non-rigid objects in multiple object tracking?: Evidence from grouping by self-rotation.

Previous studies on perceptual grouping found that people can use spatiotemporal and featural information to group spatially separated rigid objects into a unit while tracking moving objects. However, few studies have tested the role of objects' self-motion information in perceptual grouping, although it is of great significance to the motion perception in the three-dimensional space. In natural environments, objects always move in translation and rotation at the same time. The self-rotation of the objects seriously destroys objects' rigidity and topology, creates conflicting movement signals and results in crowding effects. Thus, this study sought to examine the specific role played by self-rotation information on grouping spatially separated non-rigid objects through a modified multiple object tracking (MOT) paradigm with self-rotating objects. Experiment 1 found that people could use self-rotation information to group spatially separated non-rigid objects, even though this information was deleterious for attentive tracking and irrelevant to the task requirements, and people seemed to use it strategically rather than automatically. Experiment 2 provided stronger evidence that this grouping advantage did come from the self-rotation per se rather than surface-level cues arising from self-rotation (e.g. similar 2D motion signals and common shapes). Experiment 3 changed the stimuli to more natural 3D cubes to strengthen the impression of self-rotation and again found that self-rotation improved grouping. Finally, Experiment 4 demonstrated that grouping by self-rotation and grouping by changing shape were statistically comparable but additive, suggesting that they were two different sources of the object information. Thus, grouping by self-rotation mainly benefited from the perceptual differences in motion flow fields rather than in deformation. Overall, this study is the first attempt to identify self-motion as a new feature that people can use to group objects in dynamic scenes and shed light on debates about what entities/units we group and what kinds of information about a target we process while tracking objects.