Dithiothreitol-functionalized perovskite-based visual sensing array capable of distinguishing food oils.

At present, the combination of fingerprint recognition methods and environmentally friendly and economical analytical instruments is becoming increasingly important in the food industry. Herein, a dithiothreitol (DTT)-functionalized CsPbBr3-based colorimetric sensor array is developed for qualitatively differentiating multiple food oils. In this sensor array composition, two types of iodides (octadecylammonium iodide (ODAI) and ZnI2) are used as recognition elements, and CsPbBr3 is used as a signal probe for the sensor array. Different food oils oxidize iodides differently, resulting in different amounts of remaining iodides. Halogen ion exchange occurs between the remaining iodides and CsPbBr3, leading to different colors observed under ultraviolet light, enabling a unique fingerprint for each food oil. A total of five food oils exhibit their unique colorimetric array's response patterns and were successfully differentiated by linear discriminant analysis (LDA), realizing 100% classification accuracy.

Chimeric Viruses Enable Study of Antibody Responses to Human Rotaviruses in Mice.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human-rotavirus-specific immunity to the outer capsid, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

The neonatal Fc receptor and DPP4 are human astrovirus receptors.

Human astroviruses (HAstV) are major global causes of gastroenteritis, but little is known about host factors required for their cellular entry. Here, we utilized complementary CRISPR-Cas9-based knockout and activation screening approaches and identified neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV infection of human intestinal epithelial cells. Disruption of FcRn or DPP4 reduced HAstV infection in permissive cells and, reciprocally, overexpression of these factors in non-permissive cells was sufficient to promote infection. We observed direct binding between FcRn and HAstV virions as well as purified spike protein. Finally, inhibitors for DPP4 and FcRn currently in clinical use prevent HAstV infection in cell lines and primary human enteroids. Thus, our results reveal mechanisms of HAstV entry as well as druggable targets. One-Sentence Summary: Targeting FcRn or DPP4 using available therapies effectively prevents human astrovirus infection in human enteroid cultures.

Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice.

Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

MYADM binds human parechovirus 1 and is essential for viral entry.

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

SARS-CoV-2 Omicron BA.1 Variant Infection of Human Colon Epithelial Cells.

The Omicron variant of SARS-CoV-2, characterized by multiple subvariants including BA.1, XBB.1.5, EG.5, and JN.1, became the predominant strain in early 2022. Studies indicate that Omicron replicates less efficiently in lung tissue compared to the ancestral strain. However, the infectivity of Omicron in the gastrointestinal tract is not fully defined, despite the fact that 70% of COVID-19 patients experience digestive disease symptoms. Here, using primary human colonoids, we found that, regardless of individual variability, Omicron infects colon cells similarly or less effectively than the ancestral strain or the Delta variant. The variant induced limited type III interferon expression and showed no significant impact on epithelial integrity. Further experiments revealed inefficient cell-to-cell spread and spike protein cleavage in the Omicron spike protein, possibly contributing to its lower infectious particle levels. The findings highlight the variant-specific replication differences in human colonoids, providing insights into the enteric tropism of Omicron and its relevance to long COVID symptoms.

Low thermal contact resistance boron nitride nanosheets composites enabled by interfacial arc-like phonon bridge.

Two-dimensional materials with ultrahigh in-plane thermal conductivity are ideal for heat spreader applications but cause significant thermal contact resistance in complex interfaces, limiting their use as thermal interface materials. In this study, we present an interfacial phonon bridge strategy to reduce the thermal contact resistance of boron nitride nanosheets-based composites. By using a low-molecular-weight polymer, we are able to manipulate the alignment of boron nitride nanosheets through sequential stacking and cutting, ultimately achieving flexible thin films with a layer of arc-like structure superimposed on perpendicularly aligned ones. Our results suggest that arc-like structure can act as a phonon bridge to lower the contact resistance by 70% through reducing phonon back-reflection and enhancing phonon coupling efficiency at the boundary. The resulting composites exhibit ultralow thermal contact resistance of 0.059 in2 KW-1, demonstrating effective cooling of fast-charging batteries at a thickness 2-5 times thinner than commercial products.

Palladium-Catalyzed Enantioselective [4+2] Cycloaddition of 4-Vinylbenzodioxinones with Barbiturate-Derived Alkenes: Con-struction of Chiral Spirobarbiturate-Chromanes.

In this paper, Pd-catalyzed [4+2] decarboxylative cycloaddition of 4-vinylbenzodioxinones with barbiturate-derived alkenes has been developed, leading to various spirobarbiturate-chromane derivatives in high yields with excellent diastereo- and enantioselectivities. The scale-up reaction and further derivation of the product were demonstrated. A plausible reaction mechanism was also proposed.

Calpain-2 mediates SARS-CoV-2 entry via regulating ACE2 levels.

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, much effort has been dedicated to identifying effective antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of calpain inhibitors show excellent antiviral activities against SARS-CoV-2 by targeting the viral main protease (Mpro), which plays an essential role in processing viral polyproteins. In this study, we found that calpain inhibitors potently inhibited the infection of a chimeric vesicular stomatitis virus (VSV) encoding the SARS-CoV-2 spike protein but not Mpro. In contrast, calpain inhibitors did not exhibit antiviral activities toward the wild-type VSV with its native glycoprotein. Genetic knockout of calpain-2 by CRISPR/Cas9 conferred resistance of the host cells to the chimeric VSV-SARS-CoV-2 virus and a clinical isolate of wild-type SARS-CoV-2. Mechanistically, calpain-2 facilitates SARS-CoV-2 spike protein-mediated cell attachment by positively regulating the cell surface levels of ACE2. These results highlight an Mpro-independent pathway targeted by calpain inhibitors for efficient viral inhibition. We also identify calpain-2 as a novel host factor and a potential therapeutic target responsible for SARS-CoV-2 infection at the entry step. IMPORTANCE: Many efforts in small-molecule screens have been made to counter SARS-CoV-2 infection by targeting the viral main protease, the major element that processes viral proteins after translation. Here, we discovered that calpain inhibitors further block SARS-CoV-2 infection in a main protease-independent manner. We identified the host cysteine protease calpain-2 as an important positive regulator of the cell surface levels of SARS-CoV-2 cellular receptor ACE2 and, thus, a facilitator of viral infection. By either pharmacological inhibition or genetic knockout of calpain-2, the SARS-CoV-2 binding to host cells is blocked and viral infection is decreased. Our findings highlight a novel mechanism of ACE2 regulation, which presents a potential new therapeutic target. Since calpain inhibitors also potently interfere with the viral main protease, our data also provide a mechanistic understanding of the potential use of calpain inhibitors as dual inhibitors (entry and replication) in the clinical setting of COVID-19 diseases. Our findings bring mechanistic insights into the cellular process of SARS-CoV-2 entry and offer a novel explanation to the mechanism of activities of calpain inhibitors.

A Viral Protein 4-Based Trivalent Nanoparticle Vaccine Elicited High and Broad Immune Responses and Protective Immunity against the Predominant Rotaviruses.

The current live rotavirus (RV) vaccines show reduced effectiveness in developing countries, calling for vaccine strategies with improved efficacy and safety. We generated pseudovirus nanoparticles (PVNPs) that display multiple ectodomains of RV viral protein 4 (VP4), named S-VP4e, as a nonreplicating RV vaccine candidate. The RV spike protein VP4s that bind host receptors and facilitate viral entry are excellent targets for vaccination. In this study, we developed scalable methods to produce three S-VP4e PVNPs, each displaying the VP4e antigens from one of the three predominant P[8], P[4], and P[6] human RVs (HRVs). These PVNPs were recognized by selected neutralizing VP4-specific monoclonal antibodies, bound glycan receptors, attached to permissive HT-29 cells, and underwent cleavage by trypsin between VP8* and VP5*. 3D PVNP models were constructed to understand their structural features. A trivalent PVNP vaccine containing the three S-VP4e PVNPs elicited high and well-balanced VP4e-specific antibody titers in mice directed against the three predominant HRV P types. The resulting antisera neutralized the three HRV prototypes at high titers; greater than 4-fold higher than the neutralizing responses induced by a trivalent vaccine consisting of the S60-VP8* PVNPs. Finally, the trivalent S-VP4e PVNP vaccine provided 90-100% protection against diarrhea caused by HRV challenge. Our data supports the trivalent S-VP4e PVNPs as a promising nonreplicating HRV vaccine candidate for parenteral delivery to circumvent the suboptimal immunization issues of all present live HRV vaccines. The established PVNP-permissive cell and PVNP-glycan binding assays will be instrumental for further investigating HRV-host cell interactions and neutralizing effects of VP4-specific antibodies and antivirals.

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models.

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

A mouse model to distinguish NLRP6-mediated inflammasome-dependent and -independent functions.

The NOD-like receptor (NLR) family pyrin domain containing 6 (NLRP6) serves as a sensor for microbial dsRNA or lipoteichoic acid (LTA) in intestinal epithelial cells (IECs), and initiating multiple pathways including inflammasome pathway and type I interferon (IFN) pathway, or regulating nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. NLRP6 can exert its function in both inflammasome-dependent and inflammasome-independent manners. However, there is no tool to distinguish the contribution of individual NLRP6-mediated pathway to the physiology and pathology in vivo. Here, we validated that Arg39 and Trp50 residues in the pyrin domain (PYD) of murine NLRP6 are required for ASC recruitment and inflammasome activation, but are not important for the RNA binding and PYD-independent NLRP6 oligomerization. We further generated the Nlrp6R39E&W50E mutant mice, which showed reduced inflammasome activation in either steady state intestine or during viral infection. However, the type I IFN production in cells or intestine tissue from Nlrp6R39E&W50E mutant mice remain normal. Interestingly, NLRP6-mediated inflammasome activation or the IFN-I production seems to play distinct roles in the defense responses against different types of RNA viruses. Our work generated a useful tool to study the inflammasome-dependent role of NLRP6 in vivo, which might help to understand the complexity of multiple pathways mediated by NLRP6 in response to the complicated and dynamic environmental cues in the intestine.

Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus.

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Embracing Large Natural Data: Enhancing Medical Image Analysis via Cross-Domain Fine-Tuning.

With the rapid advancements of Big Data and computer vision, many large-scale natural visual datasets are proposed, such as ImageNet-21K, LAION-400M, and LAION-2B. These large-scale datasets significantly improve the robustness and accuracy of models in the natural vision domain. However, the field of medical images continues to face limitations due to relatively small-scale datasets. In this article, we propose a novel method to enhance medical image analysis across domains by leveraging pre-trained models on large natural datasets. Specifically, a Cross-Domain Transfer Module (CDTM) is proposed to transfer natural vision domain features to the medical image domain, facilitating efficient fine-tuning of models pre-trained on large datasets. In addition, we design a Staged Fine-Tuning (SFT) strategy in conjunction with CDTM to further improve the model performance. Experimental results demonstrate that our method achieves state-of-the-art performance on multiple medical image datasets through efficient fine-tuning of models pre-trained on large natural datasets.

Genome-wide and targeted CRISPR screens identify RNF213 as a mediator of interferon gamma-dependent pathogen restriction in human cells.

To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.

Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.

During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co-localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA-seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi-well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Iterative immunofluorescence Basic Protocol 2: Computational analysis pipeline Support Protocol 1: Generating micropatterned multi-well slides Support Protocol 2: Differentiation of 2D gastruloids.

Influence of Braking Speed on the Friction and Wear Characteristics of High-Speed Railway Braking Materials under High Ambient Humidity Conditions.

The friction and wear tests of high-speed railway braking materials for a variety of braking speeds (600, 400, and 200 rad/min) at 65% and 98% RH RH (RH: relative humidity) were carried out utilizing a friction-testing machine and humidity generator. The research results indicate that braking speeds and ambient humidity have a prominent influence on the friction and wear characteristics of high-speed railway braking materials. At 65% and 98% RH, the lower the braking speed, the lower the wear rate, and the better the wear resistance property of the braking material. Furthermore, at 600 rad/min, the wear rate of the braking material at 98% RH was smaller than that at 65% RH. However, at 200 rad/min, the wear rate of the braking material at 98% RH was greater compared to that at 65% RH. Concretely, at 600 rad/min, compared with 65% RH, the wear rate to the brake disc at 98% RH was reduced by about 9%, and the brake pin decreased by about 6%. However, at 200 rad/min, compared to 65% RH, the wear rate to the brake disc at 98% RH increased by about 39%, and the brake pin increased by about 37%.

Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus.

Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. C. hominis infection induced a strong interferon response from enterocytes, likely driven by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Determination of vitamin K in infant formulas by liquid chromatography-online electrochemical reduction based on porous Pt/Ti electrode-fluorescence detection.

A highly sensitive and selective HPLC method for the determination of vitamin K vitamers including phylloquinone (PK) and menaquinones (MK-4) in infant formulas is described. The K vitamers were quantified with a fluorescence detector after online post-column electrochemical reduction occurring in a laboratory-made electrochemical reactor (ECR) equipped with platinum plated porous titanium (Pt/Ti) electrodes. The morphology of the electrode showed that the grain size of Pt was homogeneous and well plated on the porous Ti substrate, resulting in largely improved electrochemical reduction efficiency due to the large specific surface area. In addition, the operation parameters such as mobile phase/supporting electrolyte and working potential were optimized. The detection limits of PK and MK-4 were 0.81 and 0.78 ng g-1. Infant formula varying in stages were detected, showing PK ranged from 26.4 to 71.2 µg/100 g, while MK-4 was not detected.