A simple methodology is developed to realize chiroptical function induced through superstructural chirality of a matrix of helical nanofilaments formed by achiral molecules. In this work, circularly ...polarized luminescence is demonstrated in nanosegregated mesophase comprising only achiral molecules. An achiral molecular mixture of a bent‐core host and a rod‐like guest blended with a fluorescent dye is prepared. Circularly polarized luminescence confirms that the chiral superstructure consisting only of achiral molecules may serve as a chiral super nanospace for inducing chiral emissions from the fluorescent dye that exhibits rod‐like molecular ordering. In other words, the formation of a chiral superstructure by the segregated rod‐like molecules embedded in helical nanofilaments (bent‐core molecules) is confirmed. The results provide a novel strategy for constructing dissymmetric circularly polarized luminescence materials based on achiral molecules, which is potentially applicable in future information and display technologies.
Circularly polarized luminescence (CPL) from a nanosegregated mesophase consisting of only achiral molecules is demonstrated in this work. CPL is induced through self‐assembled chiral aggregates (in chiral super nanospace) formed only by achiral molecules. The results introduce the possibility of developing a novel technique for constructing practical CPL‐active materials.
Background. Although Middle East Respiratory Syndrome coronavirus (MERS-CoV) is characterized by a risk of nosocomial transmission, the detailed mode of transmission and period of virus shedding from ...infected patients are poorly understood. The aims of this study were to investigate the potential role of environmental contamination by MERS-CoV in healthcare settings and to define the period of viable virus shedding from MERS patients. Methods. We investigated environmental contamination from 4 patients in MERS-CoV units of 2 hospitals. MERS-CoV was detected by reverse transcription polymerase chain reaction (PCR) and viable virus was isolated by cultures. Results. Many environmental surfaces of MERS patient rooms, including points frequently touched by patients or healthcare workers, were contaminated by MERS-CoV. Viral RNA was detected up to five days from environmental surfaces following the last positive PCR from patients' respiratory specimens. MERS-CoV RNA was detected in samples from anterooms, medical devices, and air-ventilating equipment. In addition, MERS-CoV was isolated from environmental objects such as bed sheets, bedrails, IV fluid hangers, and X-ray devices. During the late clinical phase of MERS, viable virus could be isolated in 3 of the 4 enrolled patients on day 18 to day 25 after symptom onset. Conclusions. Most of touchable surfaces in MERS units were contaminated by patients and health care workers and the viable virus could shed through respiratory secretion from clinically fully recovered patients. These results emphasize the need for strict environmental surface hygiene practices, and sufficient isolation period based on laboratory results rather than solely on clinical symptoms.
The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel ...SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 10
2
RNA copies close to that of qRT-PCR
.
Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.
Angiogenesis is a dynamic process that involves expansion of a preexisting vascular network that can occur in a number of physiological and pathological settings. Despite its importance, the origin ...of the new angiogenic vasculature is poorly defined. In particular, the primary subtype of endothelial cells (capillary, venous, arterial) driving this process remains undefined.
Endothelial cells were fate-mapped with the use of genetic markers specific to arterial and capillary cells. In addition, we identified a novel venous endothelial marker gene (
) and used it to generate inducible venous endothelium-specific Cre and Dre driver mouse lines. Contributions of these various types of endothelial cells to angiogenesis were examined during normal postnatal development and in disease-specific setting.
Using a comprehensive set of endothelial subtype-specific inducible reporter mice, including tip, arterial, and venous endothelial reporter lines, we showed that venous endothelial cells are the primary endothelial subtype responsible for the expansion of an angiogenic vascular network. During physiological angiogenesis, venous endothelial cells proliferate, migrating against the blood flow and differentiating into tip, capillary, and arterial endothelial cells of the new vasculature. Using intravital 2-photon imaging, we observed venous endothelial cells migrating against the blood flow to form new blood vessels. Venous endothelial cell migration also plays a key role in pathological angiogenesis. This was observed both in formation of arteriovenous malformations in mice with inducible endothelium-specific Smad4 deletion mice and in pathological vessel growth seen in oxygen-induced retinopathy.
Our studies establish that venous endothelial cells are the primary endothelial subtype responsible for normal expansion of vascular networks, formation of arteriovenous malformations, and pathological angiogenesis. These observations highlight the central role of the venous endothelium in normal development and disease pathogenesis.
Endothelial cells differ from other cell types responsible for the formation of the vascular wall in their unusual reliance on glycolysis for most energy needs, which results in extensive production ...of lactate. We find that endothelium‐derived lactate is taken up by pericytes, and contributes substantially to pericyte metabolism including energy generation and amino acid biosynthesis. Endothelial–pericyte proximity is required to facilitate the transport of endothelium‐derived lactate into pericytes. Inhibition of lactate production in the endothelium by deletion of the glucose transporter‐1 (GLUT1) in mice results in loss of pericyte coverage in the retina and brain vasculatures, leading to the blood–brain barrier breakdown and increased permeability. These abnormalities can be largely restored by oral lactate administration. Our studies demonstrate an unexpected link between endothelial and pericyte metabolisms and the role of endothelial lactate production in the maintenance of the blood–brain barrier integrity. In addition, our observations indicate that lactate supplementation could be a useful therapeutic approach for GLUT1 deficiency metabolic syndrome patients.
Synopsis
The contribution of circulating nutrients and metabolic pathways to crosstalk between endothelial cells and neurovascular pericytes remains ill‐defined. Here, lactate secreted by endothelial cells is identified as a principle source of carbons fueling pericyte metabolism and maintaining blood‐brain barrier integrity.
Endothelium‐derived lactate is utilized for energy production and amino acid synthesis by pericytes in the CNS vasculature in mice.
Loss of GLUT1 in endothelial cells reduces lactate secretion and pericyte coverage, increasing blood brain barrier permeability.
MCT5 and MCT12 are lactate transporters responsible for shuttling lactate between endothelial cells and pericytes.
Metabolic shuttling of lactate between endothelial cells and neurovascular pericytes supports CNS vasculature integrity.
With the progression of acute cholecystitis, antimicrobial therapy becomes important for infection control. Current antibiotic recommendations were mostly based on reports of patients with acute ...cholangitis whose bile specimens were sampled from the biliary tract. However, as most infections of acute cholecystitis are limited to the gallbladder, direct sampling from the site increases the probability of identifying the causative pathogen. We investigated 321 positive bile cultures from 931 patients with acute cholecystitis who underwent laparoscopic cholecystectomy between January 2003 and December 2017. The frequency of enterococci declined (P = 0.041), whereas that of Enterobacteriales (P = 0.005), particularly Escherichia (P = 0.008), increased over time. The incidence of ciprofloxacin-resistant Enterobacteriales showed a significant increasing trend (P = 0.031). Vancomycin-resistant E.faecium, carbapenem-resistant Enterobacteriales, and extended-spectrum beta-lactamase-producing Enterobacteriales were recently observed. In grade I and II acute cholecystitis, there were no significant differences in perioperative outcomes in patients with and without early appropriate antimicrobial therapy. In conclusion, the changing incidence of frequently isolated microorganisms and their antibiotic resistance over time would be considered before selecting antibiotics for the treatment of acute cholecystitis. Surgery might be a crucial component of infection control in grade I and II acute cholecystitis.
Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and ...regulation of the "eat‐me" signal that initiates glia‐mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal‐specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well‐known "eat‐me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post‐synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post‐synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post‐synapse elimination. Moreover, we found that phosphatidylserine is used for microglia‐mediated pruning of inhibitory post‐synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat‐me" signal for inhibitory post‐synapse elimination.
SYNOPSIS
Neuronal‐specific deletion of the flippase chaperone Cdc50a leads to exposure of phosphatidylserin on neuronal outer membranes causing specific loss of inhibitory post‐synapses and seizures. Microglial phagocytosis via the phagocytic receptor MERTK promotes inhibitory post‐synapse elimination in Cdc50a cKO brains. Inhibitory post‐synapses in normal juvenile brains also use phosphatidylserine for synapse elimination, suggesting that phosphatidylserine exposure functions as an “eat‐me” signal for microglia‐dependent inhibitory post‐synapse elimination.
Neuronal Cdc50a deletion induces rapid lethality with appearance of audiogenic seizure.
Neuronal Cdc50a deletion causes the specific loss of inhibitory post‐synapses without affecting other synapses.
Ablating microglia or deleting microglial Mertk rescues the loss of inhibitory post‐synapses and seizure behaviors in Cdc50a cKO mice.
Microglial Mertk deletion increases the number of phosphatidylserine‐exposed inhibitory post‐synapses in the wild‐type juvenile brains.
Mouse models with increased neuron‐specific exposure of an apoptotic cell‐defining phospholipid provide insight into the nature of the "eat‐me" signal and its recognition during synapse elimination.
As the SARS-CoV-2 pandemic remains uncontrolled owing to the continuous emergence of variants of concern, there is an immediate need to implement the most effective antiviral treatment strategies, ...especially for risk groups. Here, we evaluated the therapeutic potency of nirmatrelvir, remdesivir and molnupiravir, and their combinations in SARS-CoV-2 infected K18-hACE2 transgenic mice. Systemic treatment of mice with each drug (20 mg/kg) resulted in slightly enhanced antiviral efficacy and yielded an increased life expectancy of only about 20–40% survival. However, combination therapy with nirmatrelvir (20 mg/kg) and molnupiravir (20 mg/kg) in lethally infected mice showed profound inhibition of SARS-CoV-2 replication in both the lung and brain and synergistically improved survival rates up to 80% compared to those with nirmatrelvir (36%, P < 0.001) and molnupiravir (43%, P < 0.001) administered alone. This combination therapy effectively reduced clinical severity score, virus-induced tissue damage, and viral distribution compared to those in animals treated with these monotherapies. Furthermore, all these assessments associated with this combination were also significantly higher than that of mice receiving remdesivir monotherapy (P < 0.001) and the nirmatrelvir (20 mg/kg) and remdesivir (20 mg/kg) combination (P < 0.001), underscored the clinical significance of this combination. By contrast, the nirmatrelvir and remdesivir combination showed less antiviral efficacy, with lower survival compared to nirmatrelvir monotherapy due to the insufficient plasma exposure of the remdesivir, demonstrating the inefficient therapeutic effect of this combination in the mouse model. The combination therapy with nirmatrelvir and molnupiravir contributes to alleviated morbidity and mortality, which can serve as a basis for the design of clinical studies of this combination in the treatment of COVID-19 patients.
In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities ...with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans.
We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses).
We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%).
Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.
Abstract
Memory T cells contribute to rapid viral clearance during re-infection, but the longevity and differentiation of SARS-CoV-2-specific memory T cells remain unclear. Here we conduct ex vivo ...assays to evaluate SARS-CoV-2-specific CD4
+
and CD8
+
T cell responses in COVID-19 convalescent patients up to 317 days post-symptom onset (DPSO), and find that memory T cell responses are maintained during the study period regardless of the severity of COVID-19. In particular, we observe sustained polyfunctionality and proliferation capacity of SARS-CoV-2-specific T cells. Among SARS-CoV-2-specific CD4
+
and CD8
+
T cells detected by activation-induced markers, the proportion of stem cell-like memory T (T
SCM
) cells is increased, peaking at approximately 120 DPSO. Development of T
SCM
cells is confirmed by SARS-CoV-2-specific MHC-I multimer staining. Considering the self-renewal capacity and multipotency of T
SCM
cells, our data suggest that SARS-CoV-2-specific T cells are long-lasting after recovery from COVID-19, thus support the feasibility of effective vaccination programs as a measure for COVID-19 control.
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