Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.

Attenuated West Nile virus mutant NS1130-132QQA/175A/207A exhibits virus-induced ultrastructural changes and accumulation of protein in the endoplasmic reticulum.

We have previously shown that ablation of the three N-linked glycosylation sites in the West Nile virus NS1 protein completely attenuates mouse neuroinvasiveness (≥1,000,000 PFU). Here, we compared the replication of the NS1130-132QQA/175A/207A mutant to that of the parental NY99 strain in monkey kidney Vero cells. The results suggest that the mechanism of attenuation is a lack of NS1 glycosylation, which blocks efficient replication, maturation, and NS1 secretion from the endoplasmic reticulum and results in changes to the virus-induced ultrastructure.

Impact of QseBC system in c-di-GMP-dependent quorum sensing regulatory network in a clinical isolate SSU of Aeromonas hydrophila.

Our earlier studies showed that AhyRI- (AI-1) and LuxS-based (AI-2) quorum sensing (QS) systems were positive and negative regulators of virulence, respectively, in a diarrheal isolate SSU of Aeromonas hydrophila. Recently, we demonstrated that deletion of the QseBC two-component signal transduction system (AI-3 QS in enterohemorrhagic Escherichia coli) also led to an attenuation of A. hydrophila in a septicemic mouse model of infection, and that interplay exists between AI-1, AI-2, and the second-messenger cyclic-di-guanosine monophosphate (c-di-GMP) in modulating bacterial virulence. To further explore a network connection between all of the three QS systems in A. hydrophila SSU and their cross talk with c-di-GMP, we overproduced a protein with a GGDEF domain, which increases c-di-GMP levels in bacteria, and studied phenotypes and transcriptional profiling of genes involved in biofilm formation and motility of the wild-type (WT) A. hydrophila and its ΔqseB mutant. Over-expression of the GGDEF domain-encoding gene (aha0701h) resulted in a significantly reduced motility of the WT A. hydrophila similar to that of the ΔqseB mutant. While enhanced protease production was noted in WT A. hydrophila that had increased c-di-GMP, no enzymatic activity was detected in the ΔqseB mutant overexpressing the aha0701h gene. Likewise, denser biofilm formation was noted for WT bacteria when c-di-GMP was overproduced compared to its respective control; however, overproduction of c-di-GMP in the ΔqseB mutant led to reduced biofilm formation, a finding similar to that noted for the parental A. hydrophila strain. These effects on bacterial motility and biofilm formation in the ΔqseB mutant or the mutant with increased c-di-GMP were correlated with altered levels of fleN and vpsT genes. While we noted transcript levels of qseB and qseC genes to be increased in the ahyRI mutant, down-regulation of the ahyR and ahyI genes was observed in the ΔqseB mutant, which correlated with decreased protease activity. Finally, an enhanced virulence of WT A. hydrophila with increased c-di-GMP was noted in a mouse model when compared to findings in the parental strain with vector alone. Overall, we conclude that cross talk between AI-1 and QseBC systems exists in A. hydrophila SSU, and c-di-GMP modulation on QseBC system is dependent on the expression of the AI-1 system.

Revealing the nexus between tourism development and CO2 emissions in Asia: does asymmetry matter?

Asia is one of the fastest-growing regions in international tourism, economic growth, and CO2 emissions around the globe. However, the relationships between tourism and CO2 emissions are little and unclearly identified. The purpose of the study is to explore the asymmetric nexus between tourism and CO2 emissions in a panel of five high emitters Asian countries covering the period of 1995-2019 by using panel-NARDL-AMG. The empirical results reveal that a positive shock in tourism arrivals increases CO2 emissions, while a negative shock also increases CO2 emissions in the long run. Moreover, a negative shock has a greater effect on CO2 emissions than a positive shock in Asia in the long run. Tourism receipts are expected to maintain the robustness of CO2 emissions in the long run in Asian economies. The authorities should develop and design green international tourism activities in the Asian economies.

Group B streptococcus exploits lipid rafts and phosphoinositide 3-kinase/Akt signaling pathway to invade human endometrial cells.

OBJECTIVE: We sought to determine the role lipid rafts and phosphoinositide 3-kinase (PI3K) in invasiveness of group B streptococci (GBS) to endometrial cells. STUDY DESIGN: Antibiotic protection assay and electron microscopy were used to evaluate the invasion of GBS to human endometrial Ishikawa cells cholesterol-depleted by using methyl-beta-cyclodextrin or treated with PI3K inhibitors: wortmannin or LY294002. Immunoblotting analysis of Akt phosphorylation and cellular imaging of GFP-Akt-PH probe were used to assess PI3Ks activation in infected cells. RESULTS: Infected Ishikawa cells streptococci are associated to membrane ruffles with morphological features of undergoing internalization. GBS remained attached but completely failed to invade to cholesterol-depleted human endometrial cells or displayed decreased invasiveness in the presence of PI3K inhibitors. Cholesterol depletion resulted in loss of membrane ruffling and dispersion of raft-associated molecules: monosialoganglioside GM1 and PI3K. CONCLUSION: This work provides the evidence that lipid rafts and raft-associated PI3K are implicated in GBS invasion to human endometrial cells.

Salivary gland morphology and virus transmission during long-term cytopathologic West Nile virus infection in Culex mosquitoes.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

Ultrastructural study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus (Diptera: Culicidae).

The ultrastructural features of West Nile virus (WNV) replication and dissemination in orally infected Culex pipiens quinquefasciatus Say were analyzed over a 25-d infection period. To investigate the effects of virus replication on membrane induction, cellular organization, and cell viability in midgut and salivary gland tissues, midguts were dissected on days 3, 7, 14, and 21, and salivary glands were collected on days 7, 14, 21, and 25 postinfection (d.p.i.) for examination by transmission electron microscopy (TEM). Whole mosquito heads were embedded for TEM analysis 14 d.p.i. to localize WNV particles and to investigate the effects of replication on nervous tissues of the brain. Membrane proliferation was induced by WNV in the midgut epithelium, midgut muscles, and salivary glands, although extensive endoplasmic reticulum swelling was a unique feature of salivary gland infection. TEM revealed WNV-induced pathology in salivary glands at 14, 21, and 25 d.p.i., and we hypothesize that long-term virus infection of this tissue results in severe cellular degeneration and apoptotic-like cell death. This finding indicates that the efficiency of WNV transmission may decrease with mosquito age postinfection.

Photokinetic Drug Delivery: Light-Enhanced Permeation in an In Vitro Eye Model.

PURPOSE: To investigate light-enhanced molecular movement as a potential technology for drug delivery. To do this, we developed an in vitro eye model while representing similar concentration gradient conditions and compositions found in the eye. METHODS: The eye model unit was fabricated by inserting a cross-linked type I collagen membrane in a spectrophotometer cuvette with 1% hyaluronic acid as the drug recipient medium. Photokinetic delivery was studied by illuminating 1 mg/mL methotrexate (MTX) placed in the drug donor compartment on top of the membrane, with noncoherent 450 nm light at 8.2 mW from an LED source pulsed at 25 cycles per second, placed in contact with the solution. A modified UV-visual spectrophotometer was employed to rapidly determine the concentration of MTX, at progressive 1 mm distances away from the membrane, within the viscous recipient medium of the model eye after 1 h. RESULTS: A defined, progressive concentration gradient was observed within the nonagitated drug recipient media, diminishing with greater distances from the membrane. Transport of MTX through the membrane was significantly enhanced (ranging from 2 to 3 times, P < 0.05 to P ≤ 0.001) by photokinetic methods compared with control conditions by determining drug concentrations at 4 defined distances from the membrane. According to scanning electron microscopy images, no structural damage or shunts were created on the surface of the cross-linked gelatin membrane. CONCLUSION: The application of pulsed noncoherent visible light significantly enhances the permeation of MTX through a cross-linked collagen membrane and hyaluronic acid recipient medium without causing structural damage to the membrane.

The conjunction effect: new evidence for robustness.

Five studies investigated the conjunction effect (or conjunction fallacy), in which participants report that the conjunction of two events is more rather than less likely than one of the events alone. There was no evidence that feedback or monetary reinforcement for correct answers affected students' performance on conjunction problems. Under some circumstances the context in which the conjunction problem was presented (after questions emphasizing logic or questions emphasizing opinions) affected occurrence of the effect. Location of the conjunction among the statements being rated had a significant effect. The effect occurred with or without a framing description and whether the conjunction consisted of two or three simple statements. However, statements representing the conjunction of three simple statements were (appropriately) judged less likely than those representing the conjunction of two simple statements. The substantial incidence of the effect, even without the descriptive frame and even when incentive and feedback were provided for correct answers, argues for its robustness.

Tumoral calcinosis--a pathogenetic overview: a histological and ultrastructural study with a report of two new cases, one in infancy.

Tumoral calcinosis occurs as a well-defined pathologic entity in 3 heterologous groups of diseases--hyperphosphatemic familial tumoral calcinosis, normophosphatemic tumoral calcinosis, and secondary tumoral calcinosis. The histological lesion is stereotypic developing from the concurrence of a juxta-articular injury with an elevated calcium-phosphorus product. The reparative response to injury is histiocytic featuring synovial metaplasia forming bursa-like structures that create the characteristic compartmentalization of the lesion. Histiocytic-derived osteoclastogenesis occurs as a response to the calcifying process initiated in the mitochondria of necrotic histiocytes forming the bursa-like structures. These calcifications, propelled by a gamut of conditions elevating serum phosphorus, facilitate the further nucleation of hydroxyapatite in mitochondria, matrical lipidic debris located in the cytoplasm and lysosomes of osteoclasts and in the locular contents, and on collagen and other extracellular matrix materials. The lesions enlarge because of new locule formation and failure to reduce the calcified burden by the compartment lining histiocytes and dysmorphic osteoclasts that are unable to solubilize the hydroxyapatite. The histological landmarks of tumoral calcinosis may be lost when its development becomes quiescent. The classic calcifying classifications are inadequate for tumoral calcinosis requiring creation of a new category for this entity.

Volume density and distribution of mitochondria in harbor seal (Phoca vitulina) skeletal muscle.

Recent studies have shown that harbor seals (Phoca vitulina) have an increased skeletal muscle mitochondrial volume density that may be an adaptation for maintaining aerobic metabolism during diving. However, these studies were based on single samples taken from locomotory muscles. In this study, we took multiple samples from a transverse section of the epaxial (primary locomotory) muscles and single samples from the m. pectoralis (secondary locomotory) muscle of five wild harbor seals. Average mitochondrial volume density of the epaxial muscles was 5.6%, which was 36.6% higher than predicted for a terrestrial mammal of similar mass, and most (82.1%) of the mitochondria were interfibrillar, unlike athletic terrestrial mammals. In the epaxial muscles, the total mitochondrial volume density was significantly greater in samples collected from the deep (6.0%) compared with superficial (5.0%) regions. Volume density of mitochondria in the pectoralis muscle was similar (5.2%) to that of the epaxial muscles. Taken together, these adaptations reduce the intracellular distance between mitochondria and oxymyoglobin and increase the mitochondrial diffusion surface area. This, in combination with elevated myoglobin concentrations, potentially increases the rate of oxygen diffusion into mitochondria and prevents diffusion limitation so that aerobic metabolism can be maintained under low oxygen partial pressure that develops during diving.

Loss of cell-adhesion molecule complexes in solid pseudopapillary tumor of pancreas.

Solid pseudopapillary tumor of pancreas (SPT) is a rare neoplasm that occurs most often in young females with the two distinct features, the 'solid-cystic' gross appearance, and the 'solid-pseudopapillary' microscopic pattern. It has been reported that almost all SPT tumors contain a mutation in the beta-catenin gene; however, the histogenetic origin of this tumor remains largely a mystery. E-cadherin is a cell adhesion molecule that links to catenins to form cell adhesion junctions, which is associated with the cytoskeleton formation. In this study, we examined the expression of E-cadherin and beta-catenin from SPT in an attempt to determine the molecular basis for the unusual morphology of this tumor. Nine cases of SPT were retrieved from Surgical Pathologic Archives of three institutions, including one male and eight females. H&E slides of each case were reviewed to confirm the diagnosis. The beta-catenin gene was sequenced in one case. E-cadherin and beta-catenin immunostains, were performed on all nine cases. Sequencing analysis on one case showed a point mutation of the beta-catenin gene, confirming previous findings that almost all SPT tumors contain mutation in the beta-catenin gene. Immunostains showed that, in both solid and pseudopapillary areas, all the tumor cells lost expression of E-cadherin, and beta-catenin nuclear expression was observed in all cases. Our findings suggest that loss of cytoplasmic beta-catenin protein in the cell adhesion complex due to beta-catenin gene mutation, results in instability of the complex, loss of E-cadherin in cell membrane, and eventually dissociation of the tumor cells to form the pseudopapillary pattern.

Basaloid squamous cell carcinoma of the esophagus with or without adenoid cystic features.

CONTEXT: Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare malignant tumor that morphologically could bear some resemblance to adenoid cystic carcinoma (ACC) originating from salivary glands. OBJECTIVE: The purpose of this study is to describe the histologic, immunohistochemical, and ultrastructural findings of BSCCs of the esophagus, with an emphasis on comparing tumors with or without adenoid cystic features. DESIGN: We reviewed 239 cases of primary esophageal carcinoma and detected 12 cases (5%) of BSCC. The light and electron microscopic findings and immunocytochemical localization of various antigens, including cytokeratins (AE1, AE3), carcinoembryonic antigen, epithelial membrane antigen, S100, smooth muscle actin, and p53, were examined in these BSCC cases. RESULTS: Histologically, all BSCCs were composed of solid lobules or nests of basaloid cells with well-demarcated outlines surrounded by a fibrous stroma. Seven of 12 tumors showed areas of ACC-like features, that is, cribriform-like pseudoglandular lumina formation and hyaline material surrounding the tumor nests, whereas the remaining 5 tumors were apparently pure basaloid carcinomas. These 2 groups of tumors were histologically and immunohistochemically identical in many aspects, namely, high-grade nuclei of the tumor cells with frequent mitoses, abundant comedo-type necrosis, focal areas of concomitant squamous differentiation, consistent immunoreactivity for cytokeratins, and poor or absent staining for S100 and smooth muscle actin. Ultrastructurally, the basaloid tumor cells exhibited relatively undifferentiated cellular characteristics and undeveloped cell organelles. CONCLUSION: Basaloid squamous cell carcinomas of the esophagus frequently have an intimate association with ACC-like patterns, but their histologic, immunocytochemical, and ultrastructural features correspond more to poorly differentiated squamous cell carcinoma than to salivary gland ACC. This distinction is important because genuine ACC is much less aggressive than BSCC.