Tumor mutational burden as a predictive biomarker for non-small cell lung cancer treated with immune checkpoint inhibitors of PD-1/PD-L1.

BACKGROUND: The significant clinical benefits of PD-1/PD-L1 immune checkpoint inhibitors (ICIP) in non-small cell lung cancer (NSCLC) have been widely recognized, emphasizing the urgent need for a reliable biomarker. In this study, we find the remarkable capacity of tumor mutational burden (TMB) to serve as an accessible and streamlined indicator. PATIENTS AND METHODS: We designed a retrospective cohort study, consisting of 600 NSCLC patients treated with ICIP. Association between TMB and overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR) has been explored. RESULTS: A strong positive correlation between TMB levels and OS, PFS rates, clinical benefit has been found when TMB > = 16(TMB > = 16 mutations/megabase (mut/Mb)). However, when TMB < 16, increasing TMB values did not exhibit a gradual stepwise increase in OS and PFS rates. The median months of OS in the TMB > = 16 and < 16 are 35.58, and 10.71 months respectively with average 12.39 months (p < 0.0001). The median months of PFS in the TMB > = 16 and < 16 are not-obtained, and 2.79 months respectively with an average of 3.32 months (p < 0.0001). The DCR in the TMB > = 16 and < 16 are 71.4% and 44.2% respectively with an average of 47.7% (p < 0.0001). The ORR in the TMB > = 16 and < 16 are 49.4% and 20.8% respectively with an average of 24.5% (p < 0.0001). CONCLUSION: The TMB > = 16 shows significantly associated with optimal ICIP treatment outcomes, including higher patient survival rates, delayed disease progression, and significant clinical benefits. These results present the potential of TMB as a promising biomarker candidate for NSCLC patients undergoing ICIP treatment.

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release.

BACKGROUND: Several studies have demonstrated neutralizing antibodies to be highly effective against alphavirus infection in animal models, both prophylactically and remedially. In most studies, neutralizing antibodies have been evaluated for their ability to block viral entry in vitro but recent evidence suggests that antibody inhibition through other mechanisms, including viral budding/release, significantly contributes to viral control in vivo for a number of alphaviruses. RESULTS: We describe a BSL-2, cell-based, high-throughput screening system that specifically screens for inhibitors of alphavirus egress using chikungunya virus (CHIKV) and Mayaro virus (MAYV) novel replication competent nano-luciferase (nLuc) reporter viruses. Screening of both polyclonal sera and memory B-cell clones from CHIKV immune individuals using the optimized assay detected several antibodies that display potent anti-budding activity. CONCLUSIONS: We describe an "anti-budding assay" to specifically screen for inhibitors of viral egress using novel CHIKV and MAYV nLuc reporter viruses. This BSL-2 safe, high-throughput system can be utilized to explore neutralizing "anti-budding" antibodies to yield potent candidates for CHIKV and MAYV therapeutics and prophylaxis.

Can mirror reading reverse the flow of time? Evidence from Japanese speakers.

Accumulating evidence over the last two decades has established the causal role of a unidirectional orthography in shaping speakers' mental representations of time. Casasanto and Bottini (Journal of Experimental Psychology: General, 143, 473-479, 2014) extended previous findings by showing that exposure to mirror-reversed orthography of speakers' native language could completely redirect their mental timelines within minutes. However, the question of whether such a causal effect of writing direction on temporal cognition can be identified in speakers whose native languages adopt bidirectional orthographies remains underexplored in the literature. To address this issue, the present study focused on Japanese which uses bidirectional writing systems, one proceeding horizontally from left to right (HLR) and one vertically from top to bottom (VTB). Two experiments were performed, and the tasks asked participants to process standard/mirror orthography prime questions about time arranged horizontally or vertically, followed by horizontal or vertical arrays of pictorial target stimuli about temporal relations. Results demonstrated that Japanese speakers encoded passage of time into a top-to-bottom linear path commensurate with the VTB writing direction, but they did not align their mental representations of time with the HLR writing orientation. Accordingly, exposure to mirror-reversed bidirectional orthographies redirected Japanese speakers' vertical but not horizontal space-time mappings. Theoretical implications concerning the causal effects of bidirectional orthographies and the generalizability of the representational flexibility of time maintained by Casasanto and Bottini (Journal of Experimental Psychology: General, 143, 473-479) are discussed.

Silencing of circular RNA ANRIL attenuates oxygen-glucose deprivation and reoxygenation-induced injury in human brain microvascular endothelial cells by sponging miR-622.

BACKGROUND: Circular RNA (circRNA) is highly expressed in the brain tissue, but its molecular mechanism in cerebral ischemia-reperfusion remains unclear. Here, we explored the role and underlying mechanisms of circRNA antisense non-coding RNA in the INK4 locus (circ_ANRIL) in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell injury. RESULTS: The expression of circ_ANRIL in OGD/R-induced human brain microvascular endothelial cells (HBMECs) was significantly up-regulated, while that of miR-622 was significantly down-regulated. Overexpression of circ_ANRIL significantly inhibited the proliferation of OGD/R-induced HBMECs and aggravated OGD/R-induced cell apoptosis. Moreover, circ_ANRIL overexpression further increased the secretion of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 in OGD/R-treated HBMECs. The results of bioinformatics analysis and luciferase reporter assay indicated that circ_ANRIL served as an miR-622 sponge to negatively regulate the expression of miR-622 in OGD/R-treated HBMECs. Additionally, circ_ANRIL silencing exerted anti-apoptotic and anti-inflammatory effects by positively regulating the expression of miR-622. Furthermore, inhibition of OGD/R-induced activation of the nuclear factor (NF)-κB pathway by circ_ANRIL silencing was significantly reversed by treatment with miR-622 inhibitor. CONCLUSIONS: Knockdown of circ_ANRIL improved OGD/R-induced cell damage, apoptosis, and inflammatory responses by inhibiting the NF-κB pathway through sponging miR-622.

Silencing of circular RNA ANRIL attenuates oxygen-glucose deprivation and reoxygenation-induced injury in human brain microvascular endothelial cells by sponging miR-622

BACKGROUND: Circular RNA (circRNA) is highly expressed in the brain tissue, but its molecular mechanism in cerebral ischemia-reperfusion remains unclear. Here, we explored the role and underlying mechanisms of circRNA antisense non-coding RNA in the INK4 locus (circ_ANRIL) in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell injury. RESULTS: The expression of circ_ANRIL in OGD/R-induced human brain microvascular endothelial cells (HBMECs) was significantly up-regulated, while that of miR-622 was significantly down-regulated. Overexpression of circ_ANRIL significantly inhibited the proliferation of OGD/R-induced HBMECs and aggravated OGD/R-induced cell apoptosis. Moreover, circ_ANRIL overexpression further increased the secretion of interleukin (IL)-1ß, IL-6, tumor necrosis factor-a, and monocyte chemoattractant protein-1 in OGD/R-treated HBMECs. The results of bioinformatics analysis and luciferase reporter assay indicated that circ_ANRIL served as an miR-622 sponge to negatively regulate the expression of miR-622 in OGD/R-treated HBMECs. Additionally, circ_ANRIL silencing exerted anti-apoptotic and anti-inflammatory effects by positively regulating the expression of miR-622. Furthermore, inhibition of OGD/R-induced activation of the nuclear factor (NF)-kB pathway by circ_ANRIL silencing was significantly reversed by treatment with miR-622 inhibitor. CONCLUSIONS: Knockdown of circ_ANRIL improved OGD/R-induced cell damage, apoptosis, and inflammatory responses by inhibiting the NF-κB pathway through sponging miR-622.

Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...(AU)

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...(AU)

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...

Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...