Short-Term Cryopreservation and Thawing have Minimal Effects on Plasmodium Falciparum Ex Vivo Invasion Profile

Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. Our findings indicate that natural P. falciparum infections mostly occur as polyclonal infections. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

Cell-Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Airborne Transmission in Ferret Model

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 – that expresses serine proteases – prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

Cell Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Ferret Airborne Transmission

Airborne transmission of seasonal and pandemic influenza viruses is responsible for their epidemiological success and public health burden in humans. Efficient airborne transmission of H1N1 influenza virus relies on receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examine the role of HA pH of fusion on transmissibility of a cell culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decrease the acid stability of the virus from a pH of 5.5 (WT) to 5.8 (mutant). In addition, we observed that this mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, we demonstrate that the NA segment noncoding regions can impact the pH of fusion of reassortant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein that facilitates serine protease-mediated entry into human airway cells. We report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents MBCS mutations. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

“We Are Back”: Reverse Culture Shock Among Saudi Scholars After Doctoral Study Abroad

The experiences of individuals returning to the most conservative countries from abroad are not being recorded. The present study explores how Saudi scholars working in the higher education sector readjust and reconnect to their workplace after completing their doctoral scholarships abroad. The study has adopted a narrative approach and used the transformational learning theory to account for reverse culture shock. Six assistant professors (three males and three females) from three Saudi universities were recruited and they underwent 30- to 50-min-long semi-structured in-depth interviews. The data were analyzed through thematic analysis and the developed themes included emotional adaptation to home culture, adaptation to their work in their home culture, adaptation of families to home culture, and reentry coping mechanisms. The results depicted how the participants readjusted to their context after extended study abroad. They returned with new identities shaped by their life and education abroad and by their exposure at university to people from different cultural backgrounds. They had also become used to a more comfortable lifestyle in their host countries. The study concludes that there is a need to prepare and organize programs that could assist Saudi new returnees to readjust and reconnect to their context again. Moreover, it would be useful in helping universities prioritize their staff’s well-being and design rehabilitative courses for new returnees helping them integrate into their workplace.

Automate Classroom and The Culture Adaptation

The 4.0 industrial revolution has stimulated advance technology and automation, which is in turn encouraging the redesign of elementary school teaching programmes. The curriculum update leads to learning automation as consequence of digitalization. Teaching methods are increasingly shifting towards automate dsystem in digital classrooms. The purpose of this research is to show the prototype of an automation classroom, and how to adapt classroom automation into sustainability culture. This paper discusses the major impact of education reformation and the culture adaptation on elementary schools. This paper employed the qualitative research by analyzed the development of augmentation process and the cultural sustainability in the future education. The findings suggest that the latest development of automated classrooms are creating a positive impact on the competitiveness of schools. By updating the curriculum wisely, it is possible to integrate education with technology and benefit the children being taught. Keywords: automate classroom, education revolution, culture adaptation, cultural sustainability