The membrane asymmetry regulated by P4-ATPases is crucial for the functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (TgP4-ATPase1-5) and three non-catalytic partner proteins (TgLem1-3) in the intracellular protozoan pathogen, Toxoplasma gondii. However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii. Both proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoite stage. The genetic knockout of P4-ATPase1 and conditional depletion of Lem1 in tachyzoites severely disrupt the asexual reproduction and translocation of PtdSer across the plasma membrane. Moreover, the phenotypic analysis of individual mutants revealed a requirement of lipid flipping for the motility, egress and invasion of tachyzoites. Not least, the proximity-dependent biotinylation and reciprocal immunoprecipitation assays demonstrated the physical interaction of P4-ATPase1 and Lem1. Our findings disclose the mechanism and significance of PtdSer flipping during the lytic cycle and identify the P4-ATPase1-Lem1 heterocomplex as a potential drug target in T. gondii.

Triosephosphate isomerase deficiency (TPI Df) is a rare, aggressive genetic disease that typically affects young children and currently has no established treatment. TPI Df is characterized by hemolytic anemia, progressive neuromuscular degeneration, and a markedly reduced lifespan. The disease has predominately been studied using invertebrate and in vitro models, which lack key aspects of the human disease. While other groups have generated mammalian Tpi1 mutant strains, specifically with the mouse mus musculus, these do not recapitulate key characteristic phenotypes of the human disease. Reported here is the generation of a novel murine model of TPI Df. CRISPR-Cas9 was utilized to engineer the most common human disease-causing mutation, Tpi1 E105D , and Tpi1 null mice were also isolated as a frame-shifting deletion. Tpi1 E105D/null mice experience a markedly shortened lifespan, postural abnormalities consistent with extensive neuromuscular dysfunction, hemolytic anemia, pathological changes in spleen, and decreased body weight. There is a ∼95% reduction in TPI protein levels in Tpi1 E105D/null animals compared to wild-type littermates, consistent with decreased TPI protein stability, a known cause of TPI Df. This work illustrates the capability of Tpi1 E105D/null mice to serve as a mammalian model of human TPI Df. This work will allow for advancement in the study of TPI Df within a model with physiology similar to humans. The development of the model reported here will enable mechanistic studies of disease pathogenesis and, importantly, efficacy testing in a mammalian system for emerging TPI Df treatments.

UNASSIGNED: Partial necrosis of skin flaps is still a substantial problem in plastic and reconstructive surgery. In this study, the role of miR-590-3p in adipose-derived stem cells (ADSCs) transplantation in improving the survival of skin flap in a mouse model was delved into.
UNASSIGNED: An abdominal perforator flap model was established in mice. The histopathological examination of mice skin tissues after ADSCs transplantation was implemented using Hematoxylin & eosin (H&E) staining. Immunohistochemistry (IHC) or immunofluorescence (IF) staining was utilized to assess the PCNA or CD31 levels. The concentrations of VEGFA in the culture medium were quantified using a VEGFA ELISA kit.
UNASSIGNED: The damage of tissue in the skin flap was dramatically relieved by ADSCs transplantation. MiR-590-3p overexpression notably suppressed, while miR-590-3p knockdown facilitated skin flap survival by regulating PCNA, VCAM-1, and VEGFA levels. MiR-590-3p targeted VEGFA to regulate its expression. The knockdown of VEGFA significantly inhibited, while overexpression of VEGFA notably promoted the survival of skin flap.
UNASSIGNED: ADSCs transplantation promotes skin flap survival by boosting angiogenesis. The miR-590-3p/VEGFA axis modulates skin flap angiogenesis and survival in ADSCs. These results reveal that interfering with miR-590-3p in ADSCs could potentially be a novel therapeutic target for the improvement of skin flap survival.

UNASSIGNED: The stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.
UNASSIGNED: A mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.
UNASSIGNED: The TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2\',5\' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.
UNASSIGNED: Macrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.
UNASSIGNED: Liver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

Coronavirus disease 2019 (COVID-19) is regarded as a challenge in health system. Several studies have assessed the immune-related aspect of this disorder to identify the host-related factors that affect the course of COVID-19. microRNAs (miRNAs) as potent regulators of immune responses have gained much attention in this regard. Recent studies have shown aberrant expression of miRNAs in COVID-19 in association with disease course. Differentially expressed miRNAs have been enriched in pathways related with inflammation and antiviral immune response. miRNAs have also been regarded as potential therapeutic targets in COVID-19, particularly for management of pathological consequences of COVID-19. In the current review, we summarize the data about dysregulation of miRNAs in COVID-19.

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B-NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure-activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B-NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Emergence of new variant of SARS-CoV-2, namely omicron, has posed a global concern because of its high rate of transmissibility and mutations in its genome. Researchers worldwide are trying to understand the evolution and emergence of such variants to understand the mutational cascade events.
We have considered all omicron genomes (n = 302 genomes) available till 2nd December 2021 in the public repository of GISAID along with representatives of variants of concern (VOC), i.e., alpha, beta, gamma, delta, and omicron; variant of interest (VOI) mu and lambda; and variant under monitoring (VUM). Whole genome-based phylogeny and mutational analysis were performed to understand the evolution of SARS CoV-2 leading to emergence of omicron variant.
Whole genome-based phylogeny depicted two phylogroups (PG-I and PG-II) forming variant specific clades except for gamma and VUM GH. Mutational analysis detected 18,261 mutations in the omicron variant, majority of which were non-synonymous mutations in spike (A67, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H), followed by RNA dependent RNA polymerase (rdrp) (A1892T, I189V, P314L, K38R, T492I, V57V), ORF6 (M19M) and nucleocapsid protein (RG203KR).
Delta and omicron have evolutionary diverged into distinct phylogroups and do not share a common ancestry. While, omicron shares common ancestry with VOI lambda and its evolution is mainly derived by the non-synonymous mutations.

Once inhaled, SARS-CoV-2 particles enter respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). Understanding the nature of ACE2 within airway tissue has become a recent focus particularly in light of the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells and the air-liquid interface (ALI) model of differentiation. Using ALI tissue, three distinct transcript variants of ACE2 were identified. One transcript encodes the documented full-length ACE2 protein. The other two transcripts are unique truncated isoforms, that until recently had only been predicted to exist via sequence analysis software. Quantitative PCR revealed that all three transcript variants are expressed throughout differentiation of airway mucociliary epithelia. Immunofluorescence analysis of individual ACE2 protein isoforms exogenously expressed in cell-lines revealed similar abilities to localize in the plasma membrane and interact with the SARS CoV 2 spike receptor binding domain. Immunohistochemistry on differentiated ALI tissue using antibodies to either the N-term or C-term of ACE2 revealed both overlapping and distinct signals in cells, most notably only the ACE2 C-term antibody displayed plasma-membrane localization. We also demonstrate that ACE2 protein shedding is different in ALI Tissue compared to ACE2-transfected cell lines, and that ACE2 is released from both the apical and basal surfaces of ALI tissue. Together, our data highlights various facets of ACE2 transcripts and protein in airway mucociliary tissue that may represent variables which impact an individual\'s susceptibility to SARS-CoV-2 infection, or the severity of Covid-19.

The outbreak of the COVID-19 pandemic has cost five million lives to date, and was caused by a positive-sense RNA virus named SARS-CoV2. The lack of drugs specific to SARS-CoV2, leads us to search for an effective and specific therapeutic approach. Small interfering RNA (siRNA) is able to activate the RNA interference (RNAi) pathway to silence the specific targeted gene and inhibit the viral replication, and it has not yet attracted enough attention as a SARS-CoV2 antiviral agent. It could be a potential weapon to combat this pandemic until the completion of full scale, effective mass vaccination. For this study, specific siRNAs were designed using a web-based bioinformatics tool (siDirect2.0) against 14 target sequences. These might have a high probability of silencing the essential proteins of SARS-CoV2. such as: 3CLpro/Mpro/nsp5, nsp7, Rd-Rp/nsp12, ZD, NTPase/HEL or nsp13, PLpro/nsp3, envelope protein (E), spike glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), ORF8, ORF3a, nsp2, and its respective 5\' and 3\'-UTR. Among these potential drug targets, the majority of them contain highly conserved sequences; the rest are chosen on the basis of their role in viral replication and survival. The traditional vaccine development technology using SARS-CoV2 protein takes 6-8 months; meanwhile the virus undergoes several mutations in the candidate protein chosen for vaccine development. By the time the protein-based vaccine reaches the market, the virus would have undergone several mutations, such that the antibodies against the viral sequence may not be effective in restricting the newly mutated viruses. However, siRNA technology can make sequences based on real time viral mutation status. This has the potential for suppressing SARS-CoV2 viral replication, through RNAi technology.