Immunometabolic features of natural killer cells are associated with infection outcomes in critical illness.

Immunosuppression increases the risk of nosocomial infection in patients with chronic critical illness. This exploratory study aimed to determine the immunometabolic signature associated with nosocomial infection during chronic critical illness. We prospectively recruited patients who were admitted to the respiratory care center and who had received mechanical ventilator support for more than 10 days in the intensive care unit. The study subjects were followed for the occurrence of nosocomial infection until 6 weeks after admission, hospital discharge, or death. The cytokine levels in the plasma samples were measured. Single-cell immunometabolic regulome profiling by mass cytometry, which analyzed 16 metabolic regulators in 21 immune subsets, was performed to identify immunometabolic features associated with the risk of nosocomial infection. During the study period, 37 patients were enrolled, and 16 patients (43.2%) developed nosocomial infection. Unsupervised immunologic clustering using multidimensional scaling and logistic regression analyses revealed that expression of nuclear respiratory factor 1 (NRF1) and carnitine palmitoyltransferase 1a (CPT1a), key regulators of mitochondrial biogenesis and fatty acid transport, respectively, in natural killer (NK) cells was significantly associated with nosocomial infection. Downregulated NRF1 and upregulated CPT1a were found in all subsets of NK cells from patients who developed a nosocomial infection. The risk of nosocomial infection is significantly correlated with the predictive score developed by selecting NK cell-specific features using an elastic net algorithm. Findings were further examined in an independent cohort of COVID-19-infected patients, and the results confirm that COVID-19-related mortality is significantly associated with mitochondria biogenesis and fatty acid oxidation pathways in NK cells. In conclusion, this study uncovers that NK cell-specific immunometabolic features are significantly associated with the occurrence and fatal outcomes of infection in critically ill population, and provides mechanistic insights into NK cell-specific immunity against microbial invasion in critical illness.

UFL1 ablation in T cells suppresses PD-1 UFMylation to enhance anti-tumor immunity.

UFMylation is an emerging ubiquitin-like post-translational modification that regulates various biological processes. Dysregulation of the UFMylation pathway leads to human diseases, including cancers. However, the physiological role of UFMylation in T cells remains unclear. Here, we report that mice with conditional knockout (cKO) Ufl1, a UFMylation E3 ligase, in T cells exhibit effective tumor control. Single-cell RNA sequencing analysis shows that tumor-infiltrating cytotoxic CD8+ T cells are increased in Ufl1 cKO mice. Mechanistically, UFL1 promotes PD-1 UFMylation to antagonize PD-1 ubiquitination and degradation. Furthermore, AMPK phosphorylates UFL1 at Thr536, disrupting PD-1 UFMylation to trigger its degradation. Of note, UFL1 ablation in T cells reduces PD-1 UFMylation, subsequently destabilizing PD-1 and enhancing CD8+ T cell activation. Thus, Ufl1 cKO mice bearing tumors have a better response to anti-CTLA-4 immunotherapy. Collectively, our findings uncover a crucial role of UFMylation in T cells and highlight UFL1 as a potential target for cancer treatment.

Spatial multi-omics analyses of the tumor immune microenvironment.

In the past decade, single-cell technologies have revealed the heterogeneity of the tumor-immune microenvironment at the genomic, transcriptomic, and proteomic levels and have furthered our understanding of the mechanisms of tumor development. Single-cell technologies have also been used to identify potential biomarkers. However, spatial information about the tumor-immune microenvironment such as cell locations and cell-cell interactomes is lost in these approaches. Recently, spatial multi-omics technologies have been used to study transcriptomes, proteomes, and metabolomes of tumor-immune microenvironments in several types of cancer, and the data obtained from these methods has been combined with immunohistochemistry and multiparameter analysis to yield markers of cancer progression. Here, we review numerous cutting-edge spatial 'omics techniques, their application to study of the tumor-immune microenvironment, and remaining technical challenges.

Gold nanoparticles (AuNP)-based aptasensor for enteropathogenic Escherichia coli detection.

BACKGROUND: Diarrhea is a major cause of severe gastrointestinal illness in the infant especially in many developing countries. Although this molecular technique has been accepted as standard technique to detect Diarrhea-causing EPEC, the practical aspect of this technique for in-site rapid screening purposes is still facing a major challenge. In this study, we characterized EPEC specific aptamers and applied it as an AuNP-based aptasensor for point of care (POC) diagnosis purpose. METHODS: As many as six selected DNA aptamers was screened using target bacteria and the bound aptamer was measured by qPCR technique. Moreover, Kd value for each optimal bound aptamer was measured by using the same technique. Colorimetry assay was applied to test specificity and LOD of AuNP-based aptasensor. RESULTS: Two DNA aptamers have been successfully obtained to detect Enteropathogenic Escherichia coli K.1.1. DNA aptamer S8-7 exhibited constant dissociation (Kd) value of 17.08 nM, while DNA aptamer S10-5 exhibited Kd value of 34.14 nM. AuNP-based aptasensor showed high selectivity and specificity for EPEC K.1.1 with a limit of detection (LOD) value of 105 CFU/mL. Truncation study on DNA aptamer S8-7 showed that elimination of primer binding sequence only slightly increased both performance of detection and LOD value of AuNP-based aptasensor. CONCLUSION: Further study is necessary to improve AuNP-aptasensor performance such as through mutagenesis approach on targeted DNA aptamers before AuNP-based aptasensor can be applied as a biosensor in point of care (POC) diagnosis.

Inhibition of enteropathogenic Escherichia coli biofilm formation by DNA aptamer.

Enteropathogenic Escherichia coli (EPEC) is a bioagent that causes diarrhea through the formation of biofilm. The recalcitrant of EPEC to the current conventional antibiotic treatment has grown a big concern in a way to find effective alternative inhibitors. Aptamers have been demonstrated to show the ability to kill the pathogenic bacteria through inhibition of biofilm formation. Therefore, this study aimed to investigate antibiofilm activities of six types of aptamers against EPEC K1.1 which was isolated from patients with diarrhea. Environmental conditions such as temperatures and pH which impacted on biofilm formation of EPEC K1.1 and also biofilm inhibition of aptamer on EPEC K1.1 were performed by counting the crystal violet formation in 96-well polystyrene microplates at OD570. The motility examination combined with qPCR were applied to prove the mechanism of aptamers inhibition on biofilm by targeting essential genes that involve biofilm formation. The result showed that by applying cut off value at 0.399, aptamer SELEX 10 Colony 5 exhibited the highest biofilm inhibition against EPEC K1.1 with an absorbance value of 0.126. Further analysis showed that this aptamer also was able to reduce the motility diameter of EPEC K1.1. The effect of this aptamer on EPEC K1.1 motility was confirmed by qPCR where the mRNA level of motB, csgA and lsrA gene reduced significantly compared to the untreated group. Aptamer SELEX 10 Colony 5 was able to inhibit biofilm formation through interfering the motility ability of EPEC K1.1 and also by reducing the mRNA level of biofilm formation-related genes. This study provides evidences that aptamer is effective and promising for both antibiofilm of EPEC K1.1 and alternative treatment of diarrhea.

Modified allele-specific PCR improves HER2 Ile655Val detection by reducing genotyping errors

Background: A reliable method to detect gene polymorphisms must be established to eliminate genotyping errors due to false PCR amplification. In the previous study, we have developed AS-PCR (Allele Specific-Polymerase Chain Reaction) to detect HER2 Ile655Val gene polymorphism with good specificity and sensitivity, yet it produces some errors. This study is aimed to eliminate the source of genotyping errors mainly by betaine treatment and PCR program modification. Methods: Genotyping errors produced by AS-PCR was qualitatively and quantitatively evaluated using two genomic DNA that each contained AA genotype and GG genotype of HER2 Gene. Betaine treatment or PCR program modification was tested to eliminate the occurrence of genotyping errors during AS-PCR amplification. Results: The types of genotyping errors exhibited by HER2 Ile655Val AS-PCR are diverse, ranging from LDO (Locus Drop Out), preferential amplification to ADO (Allele Drop Out). The rate of genotyping errors was from 10% to 50% depending on the amount and ratio of DNA template and the annealing temperature of PCR. In the mixed DNA template model, the betaine treatment has shown to reduce ADO only in preferentially amplified GG genotype amplicon. Alternatively, reducing the template of the heterozygous DNA by half ( -0.5 ng of DNA template) for such case has effectively recovered the AS-PCR from ADO. Furthermore, increasing the denaturation temperature to 96 °C with an annealing time of 40 s at the first 10 cycles of AS-PCR has succeeded in eliminating severe preferential amplification of AA genotype amplicon by preventing the DNA template with GG genotype from forming into a G-quadruplex structure. The guideline offered in this study has been successfully applied for clinical samples of breast cancer that show preferential amplification. Conclusion: Betaine and the modifying AS-PCR program can reduce significantly genotyping errors making AS-PCR for HER2 Ile655Val detection more reliable to be used as a molecular tool for genotyping purpose (AU)

Correction: Budiarto, Bugi Ratno and Desriani. Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study. Sci. Pharm. 2016, 84, 103-111.

The authors wish to make the following corrections to their paper [1]:[...].

Dataset reporting detection of breast cancer-related HER2I655V polymorphism using allele-specific polymerase chain reaction.

The dataset presented in this article is related to the research article entitled "Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study" (B.R. Budiarto, Desriani, 2016) [1] with some modification in primers used and in PCR optimization strategy to eliminate false-positive result that may occur in HER2I655V polymorphism detection. Combining a new set of primers with PCR gradient, The allele-specific PCR well performs to detect all type of breast cancer-originated HER2I655V genotypes. The validation of this method was done using Sanger DNA sequencing, offering an alternative tool for HER2I655V polymorphism detection in another type of cancer.

Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study.

Cancers are the most deadly diseases in the world and their incidences continue to increase over time. Particularly, breast cancer in females places 1(st) rank among other types of cancers in term of cancer cases (23%) and death incidence (14%). Recent findings support the correlation between (Ile)655(Val) SNP in the HER2 gene with breast cancer risk. Moreover, the (Ile)655(Val) HER2 gene polymorphism could be a predictive factor in a neoadjuvant therapy setting. Precise detection of the (Ile)655(Val) HER2 gene SNP in early breast cancer patients will be beneficial in designing the most suitable treatment and in increasing the efficacy of anticancer drugs. Here we develop a rapid and inexpensive method for (Ile)655(Val) SNP detection in the HER2 gene based on allele-specific PCR technology. Two forward primers and one common reverse primer were designed to anneal specifically either on the HER2 gene fragment containing the GG genotype or to the HER2 gene fragment containing the AA genotype where one of these primers had been added with poly-GC at 5' upstream. Moreover, to increase discrimination level, mismatch bases at the SNP site and the 3(rd) base of each forward primers from 3'end were added. To test the performance of the designed primers in discriminating a polymorphism and its annealing temperature, breast cancer specimen-derived genomic DNA (with GG genotype) and pGEM_HER2/AA (with AA genotype) were used as templates in the PCR reaction. The optimal annealing temperature for SNP detection was at 51.5°C as showed by the appearance of a 150 base pair (bp) band as AA genotype (pGEM_HER2/AA template), 116bp band as GG genotype (genomic DNA template), and both types of bands as AG genotype (mix of pGEM_HER2/AA and genomic DNA template). Allelic types of breast cancer patients were also determined using this optimized method compared to sanger sequencing. The 100% accordance was shown for all types of genotypes in both methods. The allele-specific PCR in this study may have application in determining polymorphisms of the breast cancers-originated (Ile)655(Val) HER2 gene.

Hepatitis C virus genotype in blood donors and associated liver disease in Indonesia.

OBJECTIVE: The aim of this study was to investigate the distribution of hepatitis C virus (HCV) genotype and the possible association between genotype and HCV-associated liver disease in Indonesia. METHODS: 32 anti-HCV-positive asymptomatic carriers (AC), 55 chronic hepatitis (CH), 41 liver cirrhosis (LC), and 35 hepatocellular carcinoma (HCC) patients were included in this study. HCV genotyping was performed by phylogenetic analysis of the NS5B and 5'-UTR regions. RESULTS: The HCV subtype 1b (36.5%), based on NS5B region, was the most prevalent, followed by subtypes 3k (15.4%), 2a (14.4%), 1a (12.5%) and 1c (12.5%), and 2e (4.8%). Subtypes 2f, 3a, 3b, and 4a were also found in some of the samples. HCV subtypes 3k (40.0%) and 1a (35.0%) were the two major subtypes in AC. HCV subtype 1b was not found in AC, but it was common in CH (31.3%), LC (50.0%), and HCC (57.1%). CONCLUSION: HCV subtype 1b was prevalent in samples of HCV-associated liver disease patients, including CH, LC and HCC. The percentage of subtype 1b was increased with the disease severity (AC < CH < LC < HCC).