Models of sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is one of the most serious life-threatening complications of sepsis. The pathogenesis of S-AKI is complex and there is no effective specific treatment. Therefore, it is crucial to choose suitable preclinical models that are highly similar to human S-AKI to study the pathogenesis and drug treatment. In this review, we summarized recent advances in the development models of S-AKI, providing reference for the reasonable selection of experimental models as basic research and drug development of S-AKI.

Pulmonary microbial spectrum in late-stage SARS-CoV-2 infection: a case series.

Coronavirus disease 2019 (COVID-19), a kind of respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), primarily spreads through the respiratory tract from human to human. Its extensive and rapid spread has led to a global pandemic, causing great harm to human health and economic development all over the world. Current known evidence indicates that SARS-CoV-2 has evolved accumulating multiple mutations, with altered infectivity and viral replication capacity. A better understanding of the complications of COVID-19 and its relationship with underlying diseases is crucial for the prevention and treatment of SARS-CoV-2. This case series reviewed case data of our 4 recent patients with severe or critical COVID-19, including treatment plan, status of pulmonary infection and their microbiology workup with metagenomic next-generation sequencing with bronchoalveolar lavage fluid. This report shed light on the significance of rapid and accurate clinical diagnosis and treatment on COVID-19.

Impact of prior SARS-CoV-2 infection on college students' hesitancy to receive additional COVID-19 vaccine booster doses: A study from Taizhou, China.

Purpose: This study aimed to examine the impact of a history of SARS-CoV-2 infection on the hesitancy of college students to receive additional COVID-19 vaccine booster doses. Methods: A population-based self-administered online survey was conducted in July 2024 in Taizhou, China. A total of 792 respondents were included in this study. Logistic regression was conducted to identify factors associated with college students' hesitation to receive booster doses of the COVID-19 vaccine. Results: Of 792 respondents, 32.2 % hesitated to receive additional doses of the COVID-19 vaccine booster. Furthermore, 23.5 % of the respondents reported an increase in hesitancy to receiving additional COVID-19 vaccine booster doses compared to before they were infected with SARS-CoV-2. In the regression analyses, college students who had a secondary infection were more hesitant to receive additional COVID-19 vaccine booster doses (OR = 0.481, 95 % CI: (0.299-0.774), P = 0.003). Moreover, students with secondary infections who were male (OR = 0.417, 95 % CI: 0.221-0.784, P = 0.007), with lower than a bachelor's degree (OR = 0.471, 95 % CI: 0.272-0.815, P = 0.007), in non-medical majors (OR = 0.460, 95 % CI: 0.248-0.856, P = 0.014), and sophomores or below (OR = 0.483, 95 % CI: 0.286-0.817, P = 0.007) were more hesitant to receive additional COVID-19 vaccine booster doses. Conclusion: A history of SARS-CoV-2 infection affects college students' hesitation to receive additional COVID-19 vaccine booster doses, which was higher in those who experienced secondary infections.

VX-509 attenuates the stemness characteristics of colorectal cancer stem-like cells by regulating the epithelial-mesenchymal transition through Nodal/Smad2/3 signaling.

BACKGROUND: Colorectal cancer stem cells (CCSCs) are heterogeneous cells that can self-renew and undergo multidirectional differentiation in colorectal cancer (CRC) patients. CCSCs are generally accepted to be important sources of CRC and are responsible for the progression, metastasis, and therapeutic resistance of CRC. Therefore, targeting this specific subpopulation has been recognized as a promising strategy for overcoming CRC. AIM: To investigate the effect of VX-509 on CCSCs and elucidate the underlying mechanism. METHODS: CCSCs were enriched from CRC cell lines by in conditioned serum-free medium. Western blot, Aldefluor, transwell and tumorigenesis assays were performed to verify the phenotypic characteristics of the CCSCs. The anticancer efficacy of VX-509 was assessed in HCT116 CCSCs and HT29 CCSCs by performing cell viability analysis, colony formation, sphere formation, flow cytometry, and western blotting assessments in vitro and tumor growth, immunohistochemistry and immunofluorescence assessments in vivo. RESULTS: Compared with parental cells, sphere cells derived from HCT116 and HT29 cells presented increased expression of stem cell transcription factors and stem cell markers and were more potent at promoting migration and tumorigenesis, demonstrating that the CRC sphere cells displayed CSC features. VX-509 inhibited the tumor malignant biological behavior of CRC-stem-like cells, as indicated by their proliferation, migration and clonality in vitro, and suppressed the tumor of CCSC-derived xenograft tumors in vivo. Besides, VX-509 suppressed the CSC characteristics of CRC-stem-like cells and inhibited the progression of epithelial-mesenchymal transition (EMT) signaling in vitro. Nodal was identified as the regulatory factor of VX-509 on CRC stem-like cells through analyses of differentially expressed genes and CSC-related database information. VX-509 markedly downregulated the expression of Nodal and its downstream phosphorylated Smad2/3 to inhibit EMT progression. Moreover, VX-509 reversed the dedifferentiation of CCSCs and inhibited the progression of EMT induced by Nodal overexpression. CONCLUSION: VX-509 prevents the EMT process in CCSCs by inhibiting the transcription and protein expression of Nodal, and inhibits the dedifferentiated self-renewal of CCSCs.

Nur77 Serves as a Potential Prognostic Biomarker That Correlates with Immune Infiltration and May Act as a Good Target for Prostate adenocarcinoma.

Prostate adenocarcinoma (PRAD) is the most frequent malignancy, and is the second leading cause of death due to cancer in men. Thus, new prognostic biomarkers and drug targets for PRAD are urgently needed. As we know, nuclear receptor Nur77 is important in cancer development and changes in the tumor microenvironment; whereas, the function of Nur77 in PRAD remains to be elucidated. The TCGA database was used to explore the Nur77 expression and its role in the prognosis of PRAD. It was shown that Nur77 was down regulated in PRAD, and low Nur77 expression was correlated with advanced clinical pathologic characteristics (high grade, histological type, age) and poor prognosis. Furthermore, key genes screening was examined by univariate Cox analysis and Kaplan-Meier survival. Additionally, Nur77 was closely related to immune infiltration and some anti-tumor immune functions. The differentially expressed genes (DEGs) were presented by protein-protein interaction (PPI) network analysis. Therefore, the expression level of Nur77 might help predict the survival of PRAD cases, which presents a new insight and a new target for the treatment of PRAD. In vitro experiments verified that natural product malayoside targeting Nur77 exhibited significant therapeutic effects on PRAD and largely induced cell apoptosis by up-regulating the expression of Nur77 and its mitochondrial localization. Taken together, Nur77 is a prognostic biomarker for patients with PRAD, which may refresh the profound understanding of PRAD individualized treatment.

Programmed Cell Death Ligand 1-Transfected Mouse Bone Marrow Mesenchymal Stem Cells as Targeted Therapy for Rheumatoid Arthritis.

Programmed cell death 1 ligand (PD-L1) and its receptor (PD-1) are key molecules for immunoregulation and immunotherapy. PD-L1 binding PD-1 is an effective way to regulate T or B cell immunity in autoimmune diseases such as rheumatoid arthritis (RA). In our study, we overexpressed PD-L1 by constructing a recombinant of PD-L1-lentiviral vector, which was subsequently used to transfect mouse bone marrow mesenchymal stem cells (MBMMSCs) and significantly suppressed the development of collagen-induced arthritis (CIA) in DBA/1j mice. In addition, PD-L1-transfected MBMMSCs (PD-L1-MBMMSCs) ameliorated joint damage, reduced proinflammatory cytokine expression, and inhibited T and B cell activation. Furthermore, PD-L1-MBMMSCs decreased the number of dendritic cells and increased the numbers of regulatory T cells and regulatory B cells in joints of CIA mice. In conclusion, our results provided a potential therapeutic strategy for RA treatment with PD-L1-MBMMSC-targeted therapy.

Malayoside, a cardenolide glycoside extracted from Antiaris toxicaria Lesch, induces apoptosis in human non-small lung cancer cells via MAPK-Nur77 signaling pathway.

Lung cancer is the leading cause of cancer deaths in the world. Non-small cell lung cancer (NSCLC), with poor prognosis and resistance to chemoradiotherapy, is the most common histological type of lung cancer. Therefore, it is necessary to develop new and more effective treatment strategy for NSCLC. Nur77, an orphan member of the nuclear receptor superfamily, induces apoptosis in cancer cells including NSCLC cells, by high expression and translocation to mitochondria. Small molecules trigger expression and mitochondrial localization of Nur77 may be an ideal anti-cancer drug candidate. Here, we report malayoside, a cardiac glycoside in the extract of Antiaris toxicaria Lesch., had different sensitivities to NSCLC cells. Malayoside induced apoptosis in NCI-H460 cells. Meanwhile, malayoside induced Nur77 expression and mitochondrial localization, and its induction of apoptosis was Nur77-dependent. To investigate the molecular mechanism of malayoside inducing Nur77 and apoptosis, we found that malayoside activated MAPK signaling pathway, including both ERK and p38 phosphorylation. The suppression of MAPK signaling activation inhibited the expression of Nur77 and apoptosis induced by malayoside. Our studies in nude mice showed that malayside potently inhibited the growth of tumor cells in vivo. Furthermore, the anti-cancer effect of malayosidwas in vivo was also related to the elevated expression of Nur77, p-ERK, and p-p38 proteins. Our results suggest that malayoside possesses an anti-NSCLC activity in vitro and in vivo mainly via activation of MAPK-Nur77 signaling pathway, indicating that malayoside is a promising chemotherapeutic candidate for NSCLC.

Mitochondrial-Associated Protein LRPPRC is Related With Poor Prognosis Potentially and Exerts as an Oncogene Via Maintaining Mitochondrial Function in Pancreatic Cancer.

Background: The mitochondrial-associated protein leucine-rich pentatricopeptide repeat-containing (LRPPRC) exerts multiple functions involved in physiological processes, including mitochondrial gene translation, cell cycle progression, and tumorigenesis. Previously, LRPPRC was reported to regulate mitophagy by interacting with Bcl-2 and Beclin-1 and thus modifying the activation of PI3KCIII and autophagy. Considering that LRPPRC was found to be negatively associated with survival rate, we hypothesize that LRPPRC may be involved in pancreatic cancer progression via its regulation of autophagy. Methods: Real-time quantitative polymerase chain reaction was performed to detect the expression of LRPPRC in 90 paired pancreatic cancer and adjacent tissues and five pancreatic cancer cell lines. Mitochondrial reactive oxidative species level and function were measured. Mitophagy was measured by performing to detect LC3 levels. Results: By performing a real-time quantitative polymerase chain reaction, the association of LRPPRC with the prognosis of pancreatic cancer was established, and pancreatic cancer tissues had significantly higher LRPPRC expression than adjacent tissues. LRPPRC was negatively associated with the overall survival rate. LRPPRC was also upregulated in pancreatic cancer cell lines. Knockdown of LRPPRC promoted reactive oxidative species accumulation, decreased mitochondrial membrane potential, promoted autophagy/mitophagy, and induced mitochondrial dysfunction. Subsequently, knockdown of LRPPRC inhibited malignant behaviors in PANC-1 cells, including proliferation, migration, invasion, tumor formation, and chemoresistance to gemcitabine. Finally, by inhibiting autophagy/mitophagy using 3-MA, the inhibitory effect of LRPPRC knockdown on proliferation was reversed. Conclusion: Taken together, our results indicate that LRPPRC may act as an oncogene via maintaining mitochondrial homeostasis and could be used as a predictive marker for patient prognosis in pancreatic cancer.

Double-component diazeniumdiolate derivatives as anti-cancer agents.

In this study, we synthesized a series of double-component O2-aryl diazeniumdiolate (DDNO) derivatives, of which each molecule can release up to four nitric oxide molecules. These compounds showed cytotoxic activities to cancer cells, such as human leukemia, breast cancer and lung cancer. Among them, compound 1 (DDNO-1) showed the highest specific activity to human leukemia cells. It induced cell apopotosis and arrest cell cycle of G2/M phase. The JNK and p38 protein kinases were activated by compound 1 to induce cancer cell apoptosis. Compound 1 also increased pro-apoptotic Bax level, which is a same function compared to a reported NO donor, JS-K. More interestingly, it decreased the level of an anti-apoptotic member Bcl-2, which is an opposite effect compared to JS-K. Compound 1 could be developed as a new anti-cancer agent since it increases the Bax/Bcl-2 ratio to overcome the drug resistance.

A meta-analysis: The diagnostic values of long non-coding RNA as a biomarker for gastric cancer.

Long non-coding RNAs (lncRNAs) have been identified as novel biomarkers for the diagnosis, staging and prognosis for gastric cancer. However, various studies have reported a series of significances based on different diagnostic values. Therefore, the current study performed a systematic review and meta-analysis to evaluate the diagnostic accuracy of lncRNAs for gastric cancer, and to discuss lncRNA types and sources of heterogeneity. The Cochrane Central Register of Controlled Trials, MEDLINE, PubMed, EMBASE, the Chinese Biomedical Literature Database, the China Academic Journals Full-text Database and the Chinese Scientific Journals Database were systematically searched for potential studies. Studies were included if they were associated with lncRNAs, gastric cancer and reported diagnostic outcomes. Analysis of diagnostic values was used to summarize the overall test performance of lncRNAs. Ten studies were included in this meta-analysis. The ranges of the diagnostic value of lncRNAs for gastric cancer were as follows: Sensitivity was 0.45-0.83, and pooled sensitivity was 0.63; specificity was 0.60-0.93, and pooled specificity was 0.75; positive likelihood ratio was 1.80-6.92, and pooled positive likelihood ratio was 2.51; negative likelihood ratio was 0.23-0.67, and pooled negative likelihood ratio was 0.50; diagnostic odds ratio was 3.33-13.75, and pooled diagnostic odds ratio was 5.47. An overall area under the curve value of the summary receiver operating characteristic curve was 0.7550. LncRNAs did not have a high accuracy for identifying gastric cancer at present, but may be a useful screening tool for diagnosing gastric cancer due to their correlation with gastric cancer biological features. LncRNAs are potential biomarkers for gastric cancer if the screening strategy is altered, or they are combined with other biomarkers to diagnose gastric cancer.

Protein Biomarkers in Serum of Patients with Schizophrenia.

This study was devised to identify potential biomarkers of schizophrenia (SP) using proteomics techniques. We obtained 44 serum specimens from patients with SP, 26 specimens from patients with depression, and 40 specimens from healthy controls. Immobilized metal affinity capture protein chips (IMAC30) and surface-enhanced laser desorption-ionization time-of-flight mass spectrometry were used to isolate and obtain mass spectrometric data of differentially expressed serum proteins. The sequences of the peaks discrepant among the study groups were obtained using matrix-assisted laser desorption/ionization mass spectrometry and proteins identified using Mascot database. In the SP group, there were 91 protein peaks that were different from other study groups at the p value of <0.05 and 54 peaks different at the p value of <0.01. Two protein peaks at the mass-to-charge ratio of 1,207.41 and 1,466.78 were markedly different among the study groups, with the lowest expression in specimens from patients with SP. The amino acid sequences were, respectively, Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg (EGDFLAEGGGVR) and Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg (DSGEGDFLAEGGGVR). These proteins were identified as the N-terminal fragments of fibrinogen. In conclusion, these biomarker proteins may be useful for molecular diagnosis of SP.

The tumor suppressor pVHL down-regulates never-in-mitosis A-related kinase 8 via hypoxia-inducible factors to maintain cilia in human renal cancer cells.

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.

[Construction of Recombinant Adenovirus Vector with Human/Mouse Interferon Gene and Its Transfection into Human Umbilical Cord Mesenchymal Stem Cells].

OBJECTIVE: Construction and identification of recombinant adenovirus vector with human/mouse interferon (IFN-gamma) and effectively transfection into human umbilical cord mesenchymal stem cells (HUMSCs). METHODS: IFN-gamma gene of human/mouse were amplified from the plasmid by polymerase chain reaction (PCR) and then inserted into the plasmid pDC316 to generate pDC316-IFN-gamma. After being confirmed by restriction enzyme digestion and DNA sequencing, the DNA encoding IFN-gamma in the new structure were inserted into the vector of recombinant plasmid adenovirus and confirmed by restricition enzyme digestion. Then the human embryonic kidney cell line 293 were transfected with confirmed Ad-IFN-gamma, and the recombinant adenovirus were amplified, and the virus titer were detected using 50% tissue culture infective dose (TCID50) assay. The expression of the green fluorescent protein (GFP) and IFN-gamma were detected by fluorescent microsope and Western blot and ELISA after the recombinant adenovirus transfected HUMSCs. RESULTS: The recombinant adenovirus Ad-hIFN-gamma were constructed successfully, and amplified with titer of 1.6 x 10(10) IU/mL. The titer of Ad-mIFN-gamma was 1.0 x 10(10) IU/mL and the titer of Ad-GFP was 1.0 x 10(9) IU/mL. The green fluorescence proteins could be observed under fluorescent microscope in HUMSCs 24 h after transfection and with a stronger degree after 72 h, and IFN-gamma expression in HUMSCs were confirmed by Western blot and ELISA. CONCLUSION: Construction and identification of recombinant adenovirus vector of human/mouse IFN-ygamma and effectively transfection of HUMSCs were successful.

Selective interaction of heparin with the variable region 3 within surface glycoprotein of laboratory-adapted feline immunodeficiency virus.

Heparan sulfate proteoglycans (HSPG) can act as binding receptors for certain laboratory-adapted (TCA) strains of feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV). Heparin, a soluble heparin sulfate (HS), can inhibit TCA HIV and FIV entry mediated by HSPG interaction in vitro. In the present study, we further determined the selective interaction of heparin with the V3 loop of TCA of FIV. Our current results indicate that heparin selectively inhibits infection by TCA strains, but not for field isolates (FS). Heparin also specifically interferes with TCA surface glycoprotein (SU) binding to CXCR4, by interactions with HSPG binding sites on the V3 loop of the FIV envelope protein. Peptides representing either the N- or C-terminal side of the V3 loop and containing HSPG binding sites were able to compete away the heparin block of TCA SU binding to CXCR4. Heparin does not interfere with the interaction of SU with anti-V3 antibodies that target the CXCR4 binding region or with the interaction between FS FIV and anti-V3 antibodies since FS SU has no HSPG binding sites within the HSPG binding region. Our data show that heparin blocks TCA FIV infection or entry not only through its competition of HSPG on the cell surface interaction with SU, but also by its interference with CXCR4 binding to SU. These studies aid in the design and development of heparin derivatives or analogues that can inhibit steps in virus infection and are informative regarding the HSPG/SU interaction.

MicroRNAs as biomarkers for hepatocellular carcinoma: a diagnostic meta-analysis.

BACKGROUND: Numerous studies reported various microRNAs (miRNAs) could be novel serum biomarkers for hepatocellular carcinoma (HCC). However, the diagnostic ability of different miRNA biomarkers varies among the reports. In this paper, we made a meta-analysis about the diagnostic accuracy of miRNAs for HCC. METHODS: We systematically searched The Cochrane Central Register of Controlled Trials, MEDLINE, Pub Med, EMBASE, the Chinese Biomedical Literature Database, the China Academic Journals Full-text Database, and the Chinese Scientific Journals Database for potential studies. Studies were included if they were related to miRNAs and HCC and reported diagnostic outcomes. Diagnostic values analysis was used to summarize the overall test performance of miRNAs. RESULTS: Eight studies were included in this meta-analysis. The ranges of the diagnostic value of miRNAs for HCC were as follows: sensitivity was 0.72 - 0.98, pooled sensitivity was 0.87; specificity was 0.76 - 1.00, pooled specificity was 0.90; positive likelihood ratio was 3.52 - 97.45, pooled positive likelihood ratio was 8.70; negative likelihood ratio was 0.02 - 0.31, pooled negative likelihood ratio was 0.13; and diagnostic odds ratio was 19.06 - 2,646.00, pooled diagnostic odds ratio was 86.69. CONCLUSIONS: MiRNAs showed high accuracy in identifying HCC, and could be a useful screening tool for diagnosing HCC patients.

Mapping of Receptor Binding Interactions with the FIV surface Glycoprotein (SU); Implications Regarding Immune surveillance and cellular Targets of Infection.

Similar to HIV, FIV uses a two-receptor mechanism to infect CD4(+) T cells, the primary target cells in the cat. The T cell activation marker, CD134, serves as a primary binding receptor similar to the role of CD4 for HIV and facilitates interaction with the entry receptor, CXCR4. Heparan sulfate proteoglycans (HSPG) can also act as binding receptors for certain tissue culture adapted FIV and HIV isolates. In the present study, we employed site-directed mutagenesis to investigate the importance of specific residues on the FIV envelope for CD134 and HSPG interactions. We show that certain mutations that disrupt CD134 interactions facilitate HSPG binding by FIV-PPR. In particular, an E407K mutation at the base of the V3 loop knocks out CD134 binding; enhances HSPG binding; and in combination with additional Env mutations E656K and V817I increases entry into CD134(-), CXCR4(+) target cells by greater than 80-fold over wild type FIV-PPR. The CD134-independent mutant, termed FIV-PPRcr, exhibits a broadened host cell range, but also becomes readily susceptible to CD134-dependent neutralizing monoclonal antibodies. The findings are consistent with the notion that FIV-PPRcr Env has an "open" conformation that readily associates with CXCR4 directly, similar to wild type FIV-PPR Env after CD134 binding. The findings highlight the utility of a two-receptor mechanism that allows FIV V3 residues critical for CXCR4 binding to remain cryptic until reaction occurs with the primary binding receptor, thus thwarting immune surveillance.

Identification of amino acid residues important for heparan sulfate proteoglycan interaction within variable region 3 of the feline immunodeficiency virus surface glycoprotein.

Heparan sulfate proteoglycans (HSPGs) act as binding receptors or attachment factors for the viral envelope of many viruses, including strains of HIV and feline immunodeficiency virus (FIV). The FIV gp95 glycoprotein (SU) from laboratory-adapted strains (tissue culture adapted [TCA]) such as FIV-34TF10 can bind to HSPG, whereas SU from field strains (FS) such as FIV-PPR cannot. Previous studies indicate that SU-HSPG interactions occur within the V3 loop. We utilized a series of nested V3 peptides to further map the HSPG binding sites and found that both sides of the predicted V3 loop stem were critical for the binding but not the CXCR4 binding domain near the predicted tip of the V3 loop. Neutralization assays for TCA strain entry using the same set of V3 peptides showed that peptides targeting CXCR4 or HSPG binding sites can block infection, supporting the V3 loop as a critical neutralization target. Site-directed mutagenesis identified two highly conserved arginines, R379 and R389, on the N-terminal side of the V3 stem as critical for the contact between SU and HSPG. Residues K407, K409, K410, and K412 on the C-terminal side of the V3 stem form a second nonconserved domain necessary for HSPG binding, consistent with the observed specificity distinctions with FS FIV. Our findings discriminate structural determinants important for HSPG and CXCR4 binding by FIV SU and thus further define the importance of the V3 loop for virus entry and infection.

Fine definition of the CXCR4-binding region on the V3 loop of feline immunodeficiency virus surface glycoprotein.

The chemokine receptor CXCR4 is shared by primary and laboratory-adapted strains of feline immunodeficiency virus (FIV) for viral entry. Our previous studies implicated a contiguous nine-amino-acid region of the V3 loop of the FIV envelope surface as important in CXCR4 binding and virus entry. The binding is specific for CXCR4 since it can be inhibited by AMD3100, a selective CXCR4 inhibitor. Additional site-directed mutagenesis was used to further reveal the key residues. Binding studies indicated that basic residues R395, K397, R399 as well as N398 are critical for CXCR4 binding. The effect of other amino acid residues on receptor binding depends on the type of amino acid residue substituted. The binding study results were confirmed on human CXCR4-expressing SupT1 cells and correlated with entry efficiency using a virus entry assay. Amino acid residues critical for CXCR4 are not critical for interactions with the primary binding receptor CD134, which has an equivalent role as CD4 for HIV-1 binding. The ELISA results show that W394 and W400 are crucial for the recognition by neutralizing anti-V3 antibodies. Since certain strains of HIV-1 also use CXCR4 as the entry receptor, the findings make the feline model attractive for development of broad-based entry antagonists and for study of the molecular mechanism of receptor/virus interactions.

OrfA downregulates feline immunodeficiency virus primary receptor CD134 on the host cell surface and is important in viral infection.

Feline immunodeficiency virus (FIV) OrfA is an accessory protein that is critical for productive viral replication and infection in T cells. Here, we show that OrfA acts to markedly reduce cell surface expression of the FIV primary binding receptor. Downregulation does not occur at the transcriptional or translational level in that the amounts of CD134 mRNA and protein in total cell lysates are not altered between parental 104-C1 T cells and the same cell line stably expressing OrfA (104-C1-OrfA). Analysis by confocal microscopy revealed significant accumulation of CD134 in the Golgi apparatus of 104-C1 cells expressing OrfA. OrfA does not cause a generalized disruption of membrane trafficking in that surface expression of CD9 is unaffected by OrfA overexpression. Consistent with the above observations, OrfA-negative FIV-34TF10 productively infects CrFK (CD134-negative) and 104-C1-OrfA (CD134 downregulated by OrfA) cells but fails to productively infect either 104-C1 (CD134-positive) cells or GFox (CrFK cells overexpressing CD134) cells. FIV-34TF10 in which the OrfA reading frame is open (OrfArep) productively infects CrFK, GFox, 104-C1, and 104-C1-OrfA cells. We hypothesize that reduced surface expression of the receptor, a hallmark of retrovirus infections, may facilitate an increase in virus release from the infected cell by minimizing receptor interactions with budding virus particles.

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand-binding domain.

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand-binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRalpha LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator-activated receptor gamma (PPARgamma) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line-broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo- or hetero-dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor-interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRalpha LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C-terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line-broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRalpha LBD-antagonist-peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRalpha LBD conformations in favor of a compact state, and that a PPARgamma LBD-agonist complex can prevent the antagonist from enhancing the RXRalpha LBD-coactivator binding interaction.