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Abstract Apoptosis plays a pivotal role in pathogen elimination and maintaining homeostasis. However, viruses have evolved strategies to evade apoptosis, enabling their persistence within the host. Z-DNA binding protein 1 (ZBP1) is a potent innate immune sensor that detects cytoplasmic nucleic acids and activates the innate immune response to clear pathogens. When apoptosis is inhibited by viral invasion, ZBP1 can be activated to compensate for the effect of apoptosis by triggering an innate immune response. This review examined the mechanisms of apoptosis inhibition and ZBP1 activation during viral invasion. The authors outlined the mechanisms of ZBP1-induced type I interferon, pyroptosis and necroptosis, as well as the crosstalk between ZBP1 and the cGAS-STING signalling pathway. Furthermore, ZBP1 can reverse the suppression of apoptotic signals induced by viruses. Intriguingly, a positive feedback loop exists in the ZBP1 signalling pathway, which intensifies the innate immune response while triggering a cytokine storm, leading to tissue and organ damage. The prudent use of ZBP1, which is a double-edged sword, has significant clinical implications for treating infections and inflammation.

Apoptosis plays a pivotal role in pathogen elimination and maintaining homeostasis. However, viruses have evolved strategies to evade apoptosis, enabling their persistence within the host. Z-DNA binding protein 1 (ZBP1) is a potent innate immune sensor that detects cytoplasmic nucleic acids and activates the innate immune response to clear pathogens. When apoptosis is inhibited by viral invasion, ZBP1 can be activated to compensate for the effect of apoptosis by triggering an innate immune response. This review examined the mechanisms of apoptosis inhibition and ZBP1 activation during viral invasion. The authors outlined the mechanisms of ZBP1-induced type I interferon, pyroptosis and necroptosis, as well as the crosstalk between ZBP1 and the cGAS-STING signalling pathway. Furthermore, ZBP1 can reverse the suppression of apoptotic signals induced by viruses. Intriguingly, a positive feedback loop exists in the ZBP1 signalling pathway, which intensifies the innate immune response while triggering a cytokine storm, leading to tissue and organ damage. The prudent use of ZBP1, which is a double-edged sword, has significant clinical implications for treating infections and inflammation. [ABSTRACT FROM AUTHOR]

[Display omitted] Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the subcellular structural remodeling to invade multiple organs and cell types. Here, we unveil how SARS-CoV-2 boosts and utilizes filopodia to enter the target cells by real-time imaging. Using SARS-CoV-2 single virus-like particle (VLP) tracking in live cells and sparse deconvolution algorithm, we uncover that VLPs utilize filopodia to reach the entry site in two patterns, "surfing" and "grabbing", which avoid the virus from randomly searching on the plasma membrane. Moreover, combining mechanical simulation, we elucidate that the formation of virus-induced filopodia and the retraction speed of filopodia depend on cytoskeleton dynamics and friction resistance at the substrate surface caused by loading-virus gravity, respectively. Further, we discover that the entry process of SARS-CoV-2 via filopodia depends on Cdc42 activity and actin-associated proteins fascin, formin, and Arp2/3. Together, our results highlight that the spatial–temporal regulation of actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry and potentiates it as an antiviral target. [ABSTRACT FROM AUTHOR]

COVID-19 has become one of the few leading causes of death and has evolved into a pandemic that disrupts everyone's routine, and balanced way of life worldwide, and will continue to do so. To bring an end to this pandemic, scientists had put their all effort into discovering the vaccine for SARS-CoV-2 infection. For their dedication, now, we have a handful of COVID-19 vaccines. Worldwide, millions of people are at risk due to the current pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Despite the lack of clinically authorized antiviral medications and vaccines for COVID-19, clinical trials of many recognized antiviral agents, their combination, and vaccine development in patients with confirmed COVID-19 are still ongoing. This discovery gave us a chance to get immune to this disease worldwide and end the pandemic. However, the unexpected capacity of mutation of the SARS-CoV-2 virus makes it difficult, like the recent SAS-CoV-2 Omicron variant. Therefore, there is a great necessity to spread the vaccination programs and prevent the spread of this dreadful epidemic by identifying and isolating afflicted patients. Furthermore, several COVID-19 tests are thought to be expensive, time-consuming, and require the use of adequately qualified persons to be carried out efficiently. In addition, we also conversed about how the various COVID-19 testing methods can be implemented for the first time in a developing country and their cost-effectiveness, accuracy, human resources requirements, and laboratory facilities. [ABSTRACT FROM AUTHOR]

Understanding how emerging viruses persist in bat populations is a fundamental step to understand the processes by which viruses are transmitted from reservoir hosts to spillover hosts. Hendra virus, which has caused fatal infections in horses and humans in eastern Australia since 1994, spills over from its natural reservoir hosts, Pteropus bats (colloquially known as flying foxes). It has been suggested that the Hendra virus maintenance mechanism in the bat populations might be implicated with their metapopulation structure. Here, we examine whether a metapopulation consisting of black flying fox (P. alecto) colonies that are smaller than the critical community size can maintain the Hendra virus. By using the Gillespie algorithm, stochastic mathematical models were used to simulate a cycle, in which viral extinction and recolonisation were repeated in a single colony within a metapopulation. Given estimated flying fox immigration rates, the simulation results showed that recolonisation occurred more frequently than extinction, which indicated that infection would not go extinct in the metapopulation. Consequently, this study suggests that a collection of transient epidemics of Hendra virus in numerous colonies of flying foxes in Australia can support the long-term persistence of the virus at the metapopulation level. [ABSTRACT FROM AUTHOR]

Many studies have shown that patients with Coronavirus disease 2019 (COVID-19) have different degrees of liver injury. However, the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invasion into the liver are still not fully understood. This review mainly summarizes the recently published works on the abnormal liver biochemical indicators and the mechanism of viral invasion with liver injury in COVID-19 patients. Generally, SARSCoV-2 infection of the liver was caused by blood circulation or retrograde infection of the digestive tract, which led to the liver injury through direct cytopathic effect induced by virus or immunopathological effect caused by excessive inflammation. Besides these, hypoxia, endothelial injury and drug-induced jury were also the main reasons of liver injury in COVID-19 patients. In the liver function indicators, elevated alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, and lactate dehydrogenase levels with reduced albumin levels were observed in COVID-19 patients. [ABSTRACT FROM AUTHOR]

[Display omitted] A virus that can cause a global pandemic must be highly adaptive to human conditions. Such adaptation is not likely to have emerged suddenly but, instead, may have evolved step by step with each step favored by natural selection. It is thus necessary to develop a theory about the origin in order to guide the search. Here, we propose such a model whereby evolution occurs in both the virus and the hosts (where the evolution is somatic; i.e., in the immune system). The hosts comprise three groups – the wild animal hosts, the nearby human population, and farther-away human populations. The theory suggests that the conditions under which the pandemic has initially evolved are: (i) an abundance of wild animals in the place of origin (PL 0); (ii) a nearby human population of low density; (iii) frequent and long-term animal-human contacts to permit step-by-step evolution; and (iv) a level of herd immunity in the animal and human hosts. In this model, the evolving virus may have regularly spread out of PL 0 although such invasions often fail, leaving sporadic cases of early infections. The place of the first epidemic (PL 1), where humans are immunologically naïve to the virus, is likely a distance away from PL 0. Finally, this current model is only a first attempt and more theoretical models can be expected to guide the search for the origin of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Understanding how emerging viruses persist in bat populations is a fundamental step to understand the processes by which viruses are transmitted from reservoir hosts to spillover hosts. Hendra virus, which has caused fatal infections in horses and humans in eastern Australia since 1994, spills over from its natural reservoir hosts, Pteropus bats (colloquially known as flying foxes). It has been suggested that the Hendra virus maintenance mechanism in the bat populations might be implicated with their metapopulation structure. Here, we examine whether a metapopulation consisting of black flying fox (P. alecto) colonies that are smaller than the critical community size can maintain the Hendra virus. By using the Gillespie algorithm, stochastic mathematical models were used to simulate a cycle, in which viral extinction and recolonisation were repeated in a single colony within a metapopulation. Given estimated flying fox immigration rates, the simulation results showed that recolonisation occurred more frequently than extinction, which indicated that infection would not go extinct in the metapopulation. Consequently, this study suggests that a collection of transient epidemics of Hendra virus in numerous colonies of flying foxes in Australia can support the long-term persistence of the virus at the metapopulation level. It is meaningful to understand how Hendra virus can be continuously circulated in the populations of flying foxes, while the virus disappears after only a series of infections in other kinds of species. The population structure of flying foxes was found to have an important role in the viral persistence. This finding can help design appropriate control of flying foxes to prevent Hendra virus infections in horses and in humans. [ABSTRACT FROM AUTHOR]

Since 2015, Fowl adenovirus serotype 4 (FAdV-4) infection has caused serious economic losses to the poultry industry worldwide. We isolated and identified the FAdV-4 strain NP, from infected chickens on a layer farm, using chicken embryo allantoic cavity inoculation, electron microscopy, viral genome sequencing, and regression analysis. To explore the pathogenesis of FAdV-4 infection, we conducted transcriptome sequencing analysis of the liver in chickens infected with FAdV-4, using the Illumina® HiSeq 2000 system. Two days after infection with the FAdV-4 NP strain, 13,576 differentially expressed genes (DEGs) were screened in the liver, among which, 7,480 were up-regulated and 6,096 were down-regulated. Gene ontology (GO) analysis indicated that these genes were involved in 52 biological functions. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that those DEGs were involved in 33 pathways. We then focused on the KEGG pathway of phagosome and found that mRNA levels of the 25 DEGs in that pathway were up-regulated, and seven DEGs were down-regulated. Real-time quantitative polymerase chain reaction (qPCR) confirmed the accuracy and reliability of these findings. Moreover, 24 h after LMH cells were infected with FAdV-4, the mRNA levels of F-actin, Rab7, TUBA, and DVnein were significantly increased. These four genes were all subsequently silenced by RNA interference, and viral replication of FAdV-4 was then significantly down-regulated. These findings demonstrate the isolation and identification of the FAdV-4 NP strain, and the DEGs in KEGG pathway of phagosome were utilized by FAdV-4 to benefit its infection.

Malaria is a threat to human mankind and kills about half a million people every year. On the other hand, COVID‐19 resulted in several hundred thousand deaths since December 2019 and remains without an efficient and safe treatment. The antimalarials chloroquine (CQ) and its analog, hydroxychloroquine (HCQ), have been tested for COVID‐19 treatment, and several conflicting evidence has been obtained. Therefore, the aim of this review was to summarize the evidence regarding action mechanisms of these compounds against Plasmodium and SARS‐CoV‐2 infection, together with cytometry applications. CQ and HCQ act on the renin angiotensin system, with possible implications on the cardiorespiratory system. In this context, flow and image cytometry emerge as powerful technologies to investigate the mechanism of therapeutic candidates, as well as for the identification of the immune response and prognostics of disease severity. Data from the large randomized trials support the conclusion that CQ and HCQ do not provide any clinical improvements in disease severity and progression of SARS‐CoV‐2 patients, as well as they do not present any solid evidence of increased serious side effects. These drugs are safe and effective antimalarials agents, but in SARS‐CoV‐2 patients, they need further studies in the context of clinical trials. © 2020 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Since 2015, Fowl adenovirus serotype 4 (FAdV-4) infection has caused serious economic losses to the poultry industry worldwide. We isolated and identified the FAdV-4 strain NP, from infected chickens on a layer farm, using chicken embryo allantoic cavity inoculation, electron microscopy, viral genome sequencing, and regression analysis. To explore the pathogenesis of FAdV-4 infection, we conducted transcriptome sequencing analysis of the liver in chickens infected with FAdV-4, using the Illumina® HiSeq 2000 system. Two days after infection with the FAdV-4 NP strain, 13,576 differentially expressed genes (DEGs) were screened in the liver, among which, 7,480 were up-regulated and 6,096 were down-regulated. Gene ontology (GO) analysis indicated that these genes were involved in 52 biological functions. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that those DEGs were involved in 33 pathways. We then focused on the KEGG pathway of phagosome and found that mRNA levels of the 25 DEGs in that pathway were up-regulated, and seven DEGs were down-regulated. Real-time quantitative polymerase chain reaction (qPCR) confirmed the accuracy and reliability of these findings. Moreover, 24 h after LMH cells were infected with FAdV-4, the mRNA levels of F-actin, Rab7, TUBA , and DVnein were significantly increased. These four genes were all subsequently silenced by RNA interference, and viral replication of FAdV-4 was then significantly down-regulated. These findings demonstrate the isolation and identification of the FAdV-4 NP strain, and the DEGs in KEGG pathway of phagosome were utilized by FAdV-4 to benefit its infection. [ABSTRACT FROM AUTHOR]

Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo, we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible. IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor. [ABSTRACT FROM AUTHOR]

Infectious spleen and kidney necrosis virus disease (ISKNVD) caused significant economic losses to the fishery industry. Epidermal growth factor receptor (EGFR), phosphatidylinositide 3-kinase (PI3K) played an important role in ISKNV invasion. However, the molecular regulatory mechanisms among EGFR, PI3K-Akt, and ISKNV invasion are not clear. In this study, ISKNV infection rapidly induced EGFR activation. While, EGFR activation promoted virus entry, but EGFR inhibitors and specific RNA (siRNA) decreased virus invasion. The PI3K-Akt as downstream signalling of EGFR was activated upon ISKNV infection. Consistent with the trends of EGFR, Akt activation increased ISKNV entry into cells, Akt inhibition by specific inhibitor or siRNA decreased ISKNV invasion. Akt silencing combination with EGFR activation showed that EGFR activation regulation ISKNV invasion is required for activation of the Akt signalling pathway. Those data demonstrated that ISKNV-induced EGFR activation positively regulated virus invasion by PI3K-Akt pathway and provided a better understanding of the mechanism of EGFR-PI3K-Akt involved in ISKNV invasion.

[Display omitted] • Force-induced conformational change regulates receptor-ligand catch bond formation. • Spatial confinement by cell membrane affects kinetics of receptor-ligand interaction. • Biomechanical parameters determines membrane receptors transmembrane signaling. • Membrane receptors dynamics buffer force fluctuations. Mechanical cues dynamically regulate membrane receptors functions to trigger various physiological and pathological processes from viral invasion to immune defense. These cues mainly include various types of dynamic mechanical forces and the spatial confinement of plasma membrane. However, the molecular mechanisms of how they couple with biochemical cues in regulating membrane receptors functions still remain mysterious. Here, we review recent advances in methodologies of single-molecule biomechanical techniques and in novel biomechanical regulatory mechanisms of critical ligand recognition of viral and immune receptors including SARS-CoV-2 spike protein, T cell receptor (TCR) and other co-stimulatory immune receptors. Furthermore, we provide our perspectives of the general principle of how force-dependent kinetics determine the dynamic functions of membrane receptors and of biomechanical-mechanism-driven SARS-CoV-2 neutralizing antibody design and TCR engineering for T-cell-based therapies. [ABSTRACT FROM AUTHOR]

The RNA genome of a resistance-breaking isolate of Lettuce mosaic virus (LMV-E) was engineered to express the jellyfish green fluorescent protein (GFP) or β-glucuronidase (GUS) fused to the helper-component proteinase (HC-Pro) to study LMV invasion and spread in susceptible and resistant lettuce cultivars. Virus accumulation and movement were monitored by either histochemical GUS assays or detection of GFP fluorescence under UV light. The GFP- and GUS-tagged viruses spread systemically in the susceptible lettuce cultivars Trocadero and Vanguard, where they induced attenuated symptoms, compared with the wild-type virus. Accumulation of the GFP-tagged virus was reduced but less affected than in the case of the GUS-tagged virus. Systemic movement of both recombinant viruses was very severely affected in Vanguard 75, a lettuce cultivar nearly isogenic to Vanguard but carrying the resistance gene mo12 . Accumulation of the recombinant viruses in systemically infected leaves was either undetectable (GUS-tag) or erratic, strongly delayed, and inhibited by as much as 90% (GFP-tag). As a consequence, and contrary to the parental virus, the recombinant viruses were not able to overcome the protection afforded by the mo12 gene. Taken together, these results indicate that GUS or GFP tagging of the HC-Pro of LMV has significant negative effects on the biology of the virus, abolishing its resistance-breaking properties and reducing its pathogenicity in susceptible cultivars.

Abstract: Nedd4 binding protein 1 (N4BP1) has been identified as an interacting protein and a substrate of Nedd4 E3 ligase. However, the report about N4BP1's function is limit. In this study, a novel N4BP1 gene (CiN4BP1) was cloned from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiN4BP1 (3022 bp) included an open reading frame (ORF) of 2565 bp, which encoded a putative peptides of 854 amino acids containing one KH domain and one NYN domain. It was close homology (47% identify) to Oryzias latipes N4BP1. And mRNA expression of CiN4BP1 gene showed relatively high level in skin, gill, head kidney and spleen. After grass carp reovirus (GCRV) infection, CiN4BP1 was up-regulated in vivo and in vitro. Furthermore, overexpression of CiN4BP1 in CIK cells inhibited viral gene transcription. These data indicated that CiN4BP1 might play an important role in immune response to viral invasion. [Copyright &y& Elsevier]

Lassa fever (LF) is a viral hemorrhagic disease which affects one-fourth to two million people annually with the fatality rate of about 10,000. It is associated with sensorineural hearing loss (SNHL) usually at the convalescent stage. Recently, cases of SNHL at the acute phase have been reported. This study was done to further investigate the incidence and features of SNHL in acute phase of LF. It is a prospective case-control study of LF patients seen with acute SNHL conducted between July 2007 and April 2009 at Irrua Specialist Teaching Hospital Nigeria. The diagnosis of acute LF was based on the clinical features and detection of IgM antibodies and/or positive Lassa virus-specific reverse transcriptase-polymerase chain reaction using primers S36+ and LVS 339 while SNHL was diagnosed clinically and confirmed with PTA and speech discrimination tests. Patients with other acute febrile illnesses were used as control. Statistical analysis was done using SPSS version 11 and Fisher's exact test while level of significance was set at p < 0.05. Out of the 37 confirmed cases of LF, 5 (13.5%) and none (0%) of the control developed early-onset SNHL ( p = 0.03). Forty percent of the cases studied had negative IgM. The audiograms showed involvement at all frequency groups with pure tone average 65-85 dB and the speech discrimination 20-40%. The overall case fatality rate was 27.0%, and for early SNHL cases 60.0% ( p > 0.05). The incidence of SNHL in LF infection is about 13.5% and could be a reflection of a worse disease process. There is possibility of direct viral invasion aside immunological reaction as a causative mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The interactions between Plum pox virus (PPV), a member of the Potyvirus genus, and Prunus host plants are, up to now, poorly understood. In the current paper, fluorescence stereomicroscopy, in situ hybridisation and immunogold detection were performed in order to evaluate the virus transport and cellular distribution. The behavior of PPV in several susceptible (cv. ‘Moniqui’ and ‘Screara’) and resistant apricot genotypes (cv. ‘Harlayne’, ‘Henderson’, ‘Harcot’, ‘Goldrich’, ‘Stella’ and ‘Stark Early Orange’) were compared. Viral RNA was detected by in situ hybridisation in stem tissues close to the inoculation point, irrespective of the resistance status of the variety. Systemic infection was evidenced by virus immunodetection and by fluorescence detection of a GFP-tagged PPV in distant leaf sections. The signal obtained by in situ hybridisation colocalised with the fluorescence produced by GFP-tagged PPV in the same plant material but did not colocalise with the signal obtained by immunostaining. Intensity of the PPV infection in susceptible apricot cultivars varied depending on genotypes. The behavior of PPV in systemic leaves was clearly distinct between susceptible and resistant cultivars. While PPV was spreading widely around the major and minor veins in susceptible leaves, in the resistant apricot genotypes it was restricted to isolated spots consisting of few cells embedded in the mesophyll tissue. In summary, differences in the ability of PPV to systemically infect susceptible and resistant apricot cultivars were evident but nevertheless, long-distance transport of PPV occured in resistant apricot scions. [Copyright &y& Elsevier]

Abstract In recent years, RNA silencing, usage of small double stranded RNAs of ~21 – 25 base pairs to regulate gene expression, has emerged as a powerful research tool to dissect the role of unknown host cell factors in this 'post-genomic' era. While the molecular mechanism of RNA silencing has not been precisely defined, the revelation that small RNA molecules are equipped with this regulatory function has transformed our thinking on the role of RNA in many facets of biology, illustrating the complexity and the dynamic interplay of cellular regulation. As plants and invertebrates lack the protein-based adaptive immunity that are found in jawed vertebrates, the ability of RNA silencing to shut down gene expression in a sequence-specific manner offers an explanation of how these organisms counteract pathogen invasions into host cells. It has been proposed that this type of RNA-mediated defence mechanism is an ancient form of immunity to offset the transgene-, transposon- and virus-mediated attack. However, whether 1) RNA silencing is a natural immune response in vertebrates to suppress pathogen invasion; or 2) vertebrate cells have evolved to counteract invasion in a 'RNA silencing' independent manner remains to be determined. A number of recent reports have provided tantalizing clues to support the view that RNA silencing functions as a physiological response to regulate viral infection in vertebrate cells. Amongst these, two manuscripts that are published in recent issues of Science and Immunity, respectively, have provided some of the first direct evidences that RNA silencing is an important component of antiviral defence in vertebrate cells. In addition to demonstrating RNA silencing to be critical to vertebrate innate immunity, these studies also highlight the potential of utilising virus-infection systems as models to refine our understanding on the molecular determinants of RNA silencing in vertebrate cells.

A role for microbial invasion leading to inflammation in the amniotic cavity and subsequent pre-term delivery has been well established. For years, the role of viral infections in pregnancy has been minimized and thought of as harmless, with a few exceptions. Recent evidence now encourages us to expand our thinking and realize that viral infections during pregnancy may influence pregnancy more that we thought. [ABSTRACT FROM AUTHOR]