Applied Biosystems Research Articles

Abstract 4132149: Elevated Levels of Plasma Mitochondrial DNA Damage-Associated Molecular Patterns (DAMPS) in Humans and Dogs with Heart Failure and Reduced Ejection Fraction

Background: Heart failure (HF) with reduced ejection fraction (HFrEF) is associated with increased inflammatory response that contributes to progressive worsening of the HF state. Potential endogenous triggers of this process include damage-associated molecular patterns (DAMPs). DAMPs originate from within cells following tissue stress or injury and activate pattern recognition receptors of the immune system, triggering an inflammatory response. Mitochondria are the major cell organelles that release DAMPs into the circulation. Chronic HF is associated with multi-organ mitochondrial dysfunction and cell injury and death. Purpose: The present study sought to quantify differences in mitochondrial DNA (mtDNA) DAMPs in the setting of HFrEF in humans and dogs compared to normal humans and dogs. Methods: Plasma samples were obtained from 9 HFrEF patients with left ventricular ejection fraction (LVEF) ≤35% and 9 age-matched healthy subjects, and from 6 dogs with coronary microembolization-induced HF (LVEF ≤35%) and 6 normal dogs. DNA fragments were isolated from 1 ml of plasma using a commercially available kit. Quantitative PCR and specifically designed primers were used to measure the amount of DNA in plasma from COX1 (cytochrome c oxidase subunit 1), ND1 (NADH dehydrogenase subunit 1) and ND6 (NADH dehydrogenase subunit 6) using an Applied Biosystems 7500 Fast-PCR unit. The relative abundances of plasma mtDNA DAMPs were expressed as threshold cycles (C T ). Results: Compared to normal human subjects, mtDNA DAMPs levels of COX1 , ND1 and ND6 in plasma of HFrEF patients was 4-fold, 9-fold and 8-fold greater, respectively. Similarly, compared to normal dogs, mtDNA DAMPs levels of COX1 , ND1 and ND6 in plasma of HFrEF dogs was increased 48-fold, 12-fold and 4-fold, respectively. Conclusions: These findings indicate a marked increase of mtDNA DAMPs in plasma of humans and dogs with HFrEF. The increase of mtDNA DAMPs is consistent with the elevated systemic inflammatory state of HFrEF. The results underscore mitochondrial abnormalities as integral players in the progressive worsening of HFrEF.

GJA4 gene inhibition may represent a potential target for novel antithrombotic therapies

Abstract The GJA4 gene encodes a protein called connexin 37, a component of gap junctions in vascular endothelial cells, crucial in regulating endothelial function and influencing inflammation, platelet adhesion and thrombus formation. This gene deletion in mice diminished thrombus formation in vitro in human blood. In these circumstances, connexin inhibition with pharmacological agents may represent potential avenues for developing novel antithrombotic agents. Aim To investigate whether the GJA4 rs618675 T>C variant is a risk factor for progressing atherosclerosis and cardiovascular (CV) events in an asymptomatic free of apparent CAD from a Portuguese population. Methods A prospective study was performed with 1421 individuals without apparent CV disease (73.6% male, aged 52.2±8.3 years) followed during an extended period of 7.2±5.1 years). GJA4 rs618675 T>C variant was genotyped by TaqMan real-time PCR technique (Applied Biosystems). Conventional, anthropometric, biochemical and clinical risk factors were studied. Bivariate analysis and multivariate Cox regression adjusted for age, gender, traditional risk factors, biochemical markers, and the genotypes under study was performed to determine which variables were, significantly and independently, associated with CV events. Kaplan-Meier's estimate assessed the prognosis. Results Bivariate analysis showed that 50.6% of wild genotype (TT) carriers had CV events vs. 66.2% without events. Of the risk genotype (CC) carriers, 10.1% had CV events, and 3.4% did not. After the Kaplan-Meier analysis, the TT carriers had 90% CV event-free survival time and the CC risk carriers had only 64% of event-free survival. Finally, after adjustment, CC genotype remained in the equation with an HR of 3.1 (p=0.003) and CT heterozygous with HR of 1.6 (p=0.043), together with Hypertension (HR 3.0; p<0.0001), Smoking habits (HR 2.3; p=0.001) and Diabetes (HR 1.9; p=0.015). Conclusion: The CC risk genotype of the GJA4 rs618675 represented an independent risk factor for progressing atherosclerosis and CV events in a Portuguese Population. Thrombus formation often begins with the adhesion of platelets to the endothelium, and connexin 37 may influence this process. The inhibition of connexin 37 emerges as a promising candidate for future cardiovascular prevention as well as for the development of new antithrombotic drugs.

Does the LPA genotype have a role in coronary artery disease risk beyond Lp(a) values?

Abstract Introduction High lipoprotein(a) [Lp(a)] concentrations are one of the most important genetically determined risk factors for coronary artery disease (CAD). Many functional single nucleotide polymorphisms (SNPs) of the LPA gene have pronounced effects on Lp(a) concentrations. Studies show that rs3798220 polymorphism is strongly associated with Lp(a) concentration and also with risk of CAD. Goal: To evaluate if LPA rs3798220 T>C is associated with CAD beyond its effect on Lp(a) levels. Methods We performed a case-control study with 3,157 individuals (1,721 CAD patents and 1,436 controls). The LPA rs3798220 T>C was genotyped with the TaqMan PCR assay (Applied Biosystems 7300 Real-Time). This variant has a minor allele frequency (MAF) <2%; hence, the risk homozygous CC is a rare genotype, and we used the heterozygous CT in our analysis. Laboratory analysis was performed, and Lp(a) biochemical levels were measured in the cases and controls. Chi-squared tests were used to determine differences by genotype in CAD association (in cases and controls). We also reported a multivariate logistic regression to test if this LPA variant is still independently associated with CAD after adjustment to Lp(a) levels and traditional risk factors for CAD. Results Wild-type genotype TT was increased in the group without CAD, whereas the genotype TC was higher in patients with CAD (p<0.0001). After multivariate logistic regression, adjusted for traditional risk factors, the TC genotype remained in the equation as an independent risk factor for CAD (OR=2.36; 95%CI: 1.54–3.61; p<0.0001). When the multivariate regression analysis is adjusted for traditional risk factors plus Lp(a) levels, the TC genotype remains in the equation as independently associated with CAD despite being substantially attenuated (OR=1.67; 95%CI: 1.07–2.60; p=0.025). Conclusion LPA rs3798220 T>C may be an independent risk factor for CAD beyond the effect of rising Lp(a) levels. Perhaps testing for the genetic determinants of Lp(a) relative to direct measurement of Lp(a) level could provide additional prognostic utility.

Diabetes Mellitus influence in Lipoprotein(a) gene expression and association to coronary artery disease

Abstract Introduction Lp(a) and type 2 diabetes mellitus are both known and established risk factors for the development of coronary artery disease (CAD). Lp(a) levels are 75% to 95% heritable and predominately determined by single-nucleotide variants at the LPA gene; rs3798220 is one of the most studied variants and accounts for a significant portion of Lp(a) levels. Whether diabetes could influence the LPA rs3798220 account for Lp(a) levels is unknown. Aim Evaluate whether there is a different association between polymorphism LPA rs3798220 T>C and Lp(a) levels and their association with CAD in diabetic and non-diabetic populations. Methods 3,209 subjects (mean age 59.3±8.9 years, 76.3% male were selected from the data set of the Research Center. The LPA rs3798220 T>C was genotyped with the TaqMan PCR assay (Applied Biosystems 7300 Real-Time). This genetic variant has a minor allele frequency (MAF) <2%; hence, the risk homozygous CC is a rare genotype, and we used the heterozygous CT in our analysis. Lp(a) biochemical levels were measured in all participants. From the 3,209 participants, 810 had diabetes, and 2399 were no diabetics. For each group, chi-squared tests were used to determine the association of CAD prevalence by genotype, and t-tests were used to evaluate differences in CAD prevalence by Lp(a) levels. Results In non-diabetic subjects, LPA TC was significantly associated with CAD in non-diabetic subjects compared with TT (p<0.0001). Similarly, Lp(a) was higher in the CAD population (37.2±39.7) compared to the healthy population (25.6±30.0) (p<0.0001). In diabetic group, Lp(a) was higher in the CAD population (38.5±41.4) compared to the population without CAD (23.9±29.2) (p<0.0001). Nevertheless, there was no difference in genotype between CAD and non-CAD patients (p=0.710). Conclusion The LPA rs3798220 T>C variant in non-diabetic group can explain high Lp(a) levels and their association with CAD. However, in patients with diabetes, Lp(a) is elevated in CAD patients besides the fact that no significant genotype differences were displayed. Non-genetic influences like behavioural factors or even epigenetic changes may probably explain high Lp(a) levels in this population.

The role and prognostic value of the interleukin 2 gene at multiple myeloma

Aim of the study. To determine the relationship of the polymorphic locus of the IL2 genetic marker (T-330G) with the risk of complicated course of multiple myeloma.Materials and methods. The molecular analysis of the IL2 (T-330G) gene was carried out in the laboratory of Molecular Genetics, cytogenetics and FISH at the Republican Specialized Scientific and Practical Medical Center of Hematology of the Republic of Uzbekistan (Tashkent city). 101 patients aged 34 to 72 years with a reliably established diagnosis of multiple myeloma were examined. IL2 polymorphism (T-330G) was genotyped by real-time PCR using primers from Litech (Russia) using an Applied Biosystems thermal cycler (USA).Results and discussion. An increased risk of complicated course of multiple myeloma compared with healthy individuals was found to be 1.6 times higher among carriers of the mutant G allele (χ2=4.3; p=0.05). Statistically significant differences between unfavorable loci in the group of patients with combined complications of multiple myeloma compared with the control were revealed.Conclusions. The risk of developing multiple myeloma compared with healthy individuals is 1.6 times higher among carriers of the mutant G allele (χ2=4.3; p=0.05) according to the polymorphism of the IL2 gene (T-330G). Along with this, a statistically significant relationship was established between unfavorable loci (G and G/G) with an increase of 2.5 (χ2=9.4; p=0.01) and 3.5 times (χ2=5.3; p=0.03) the risk of developing multiple myeloma complicated by plasmacytoma in combination with nephropathy.

Association of the rs12617656 C/T genetic variant of the DPP4 gene with in-stent restenosis in Mexican population: a cohort study.

We evaluated whether five (rs12617336, rs12617656, rs1558957, rs3788979, and rs17574) single nucleotide polymorphisms located in the DPP4 gene that have not been sufficiently explored, are candidates to be risk markers of in-stent restenosis. The genotypes were determined in 190 patients (60 patients with restenosis and 130 without restenosis) using 5'exonuclease TaqMan assays in accordance with the manufacturer's instructions (Applied Biosystems, Foster City, USA). The results showed that the CC genotype of the rs12617656 C/T SNP was associated with the risk of development restenosis after coronary stent under the co-dominant, and recessive models (odds ratios [OR] = 3.32, p = 0.0009, and OR = 4.96, p = 0.0008, respectively). In addition, our data showed that the genetic distribution of the rs12617336, rs1558957, rs3788979, and rs17574 SNPs were similar between patients with and without restenosis after coronary stenting. On the other hand, the haplotype analysis showed one haplotype (CTC) associated with restenosis susceptibility (p = 0.006). Our study demonstrates that the rs12617656 genetic variant of the DPP4 gene is associated with the risk of developing in-stent restenosis in our population.

B-219 Pseudo-exclusion From Paternity due to Maternal Uniparental Disomy of Chromosome 2

Abstract Background Uniparental disomy (UPD) is the abnormal situation in which both members of a chromosome pair exclusively comprise material of one of the parents. The incidence of uniparental disomy (UPD) of any chromosome is estimated in 1:3500 live births; this rare condition may be relevant not only for medical genetics, but also for parental testing. Methods We describe a case of trio paternity test using 55 informative STR loci, 30 of these were performed whith GlobalFiler Express KIT - Thermo Fisher (GFE) and Investigator HDPlex - Qiagen (IHDP); capillary electrophoresis using 3500XL - Applied BioSystems; analyses using Genemapper IDX 4.0 - Applied BioSystems. The Y haplotype and 8 additional autosomal STRs were performed by a reference laboratory, which assists us in complex cases. Genotyping with a 1,6 million marker SNP-array (GDA-cyto, Illumina) was analyzed using NxClinical software(Bionano). Results Inconsistencies were observed in four loci, namely TPOX, D2S441, D2S1338 and D2S1360. All four markers mapped on chromosome 2 and were compatible with the mother´s genotype, raising the suspicion of a maternal UPD2. SNP-array analyse showed 4 large blocks with absence of heterozygosity on chromosome 2 (AOH - arr[GRCh38] 2p25.3p24.1(10587_22202427)x2 hmz, 2p21p11.2(43674352_90357502)x2 hmz, 2q22.1q31.1(141240699_170583494)x2 hmz, 2q36.3q37.3(227133255_242119896)x2 hmz), comprising a total of 112,9 Mb. No large AOH blocks were observed in any other chromosome. Conclusions The results indicate the presence of a UPD2. As the result of an unusually high-number of crossing-overs (n=6), several heterozygous and homozygous blocks were present on chromosome 2 (see figure 1). Rare cases of pseudo-exclusions of either maternity or paternity due to UPD have been reported. Together with the case reported here, they highlight the necessity to consider inconsistencies of markers that map into a single chromosome as likely resulting from UPD.

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R & D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

Expression of HMGB1, TGF-β1, BIRC3, ADAM17, CDKN1A, and FTO in Relation to Left Ventricular Remodeling in Patients Six Months after the First Myocardial Infarction: A Prospective Study.

Background: After myocardial infarction (MI), adverse left ventricular (LV) remodeling may occur. This is followed by LV hypertrophy and eventually heart failure. The remodeling process is complex and goes through multiple phases. The aim of this study was to investigate the expression of HMGB1, TGF-β1, BIRC3, ADAM17, CDKN1A, and FTO, each involved in a specific step of LV remodeling, in association with the change in the echocardiographic parameters of LV structure and function used to assess the LV remodeling process in the peripheral blood mononuclear cells (PBMCs) of patients six months after the first MI. The expression of selected genes was also determined in PBMCs of controls. Methods: The study group consisted of 99 MI patients, who were prospectively followed-up for 6 months, and 25 controls. Cardiac parameters, measured via conventional 2D echocardiography, were evaluated at two time points: 3-5 days and 6 months after MI. The mRNA expression six-months-post-MI was detected using TaqMan® technology (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Results:HMGB1 mRNA was significantly higher in patients with adverse LV remodeling six-months-post-MI than in patients without adverse LV remodeling (p = 0.04). HMGB1 mRNA was significantly upregulated in patients with dilated LV end-diastolic diameter (LVEDD) (p = 0.03); dilated LV end-diastolic volume index (LVEDVi) (p = 0.03); severely dilated LV end-systolic volume index (LVESVi) (p = 0.006); impaired LV ejection fraction (LVEF) (p = 0.01); and LV enlargement (p = 0.03). It was also significantly upregulated in PBMCs from patients compared to controls (p = 0.005). TGF-β1 and BIRC3 mRNA were significantly lower in patients compared to controls (p = 0.02 and p = 0.05, respectively). Conclusions: Our results suggest that HMGB1 is involved in adverse LV remodeling six-months-post-MI, even on the mRNA level. Further research and validation are needed.

B-189 Alinity m You-Create Lab-Developed Test for hepatitis delta virus

Abstract Background Lab developed tests (LDTs) play an important role in the realm of infectious diseases. Current molecular diagnostic platforms offer fully automated high-throughput performance capable of running LDTs. The Alinity m platform enables users to run LDTs concurrently with other FDA cleared or approved IVD assays with its Alinity m You-Create feature. Here, we investigated the performance of an LDT assay targeting hepatitis delta virus (HDV) using commercially available PCR reagents. Methods Alinity m You-Create LDT for HDV was evaluated using Applied Biosystems TaqMan Fast Virus 1-Step Multiplex Master Mix, No ROX, (ThermoFisher, Cat: 5555532). Sample extraction utilized Alinity m Sample Prep Kit 2. Alinity m You-Create HDV LDT analytical performance for sensitivity, linearity and precision was assessed using panels prepared by diluting the HDV 1st WHO international standard in negative plasma. Alinity m You-Create HDV LDT clinical performance was assessed by comparing 45 HDV positive samples previously tested on the m2000 HDV LDT. Assay specificity was assessed testing 97 HDV negative, HBV and/or HCV positive (by serology or nucleic acid amplification test). Results Alinity m You-Create HDV LDT had 100% detection as low as 2.5 IU/mL with SD ≤0.25 Log IU/mL for panels that were ≥2.5 IU/mL. Coefficient of correlation between Alinity m You-Create HDV LDT and m2000 HDV LDT was 0.969 and the mean bias was -0.16 Log IU/mL. All HDV negative specimens were undetected by Alinity m You-Create HDV LDT. Conclusions This study demonstrated successful implementation of an LDT for HDV on the Alinity m platform using the Alinity m You-Create feature using commercially available PCR reagents. The Alinity m You-Create feature for HDV has not been approved by FDA for use in the detection or diagnosis for HDV and its safety and effectiveness has not been established.

Developmental validation of NeoTyper autosomal STR kit

BackgroundShort Tandem Repeats (STRs) are pivotal to efficient human DNA profiling. Including mini-STRs can further improve the assay's efficiency. The FBI's (Federal Bureau of Investigation) expanded Combined DNA Index System (CODIS) list contains twenty STRs for human identification. The NeoTyper Autosomal kit developed by Neom Scientific Solutions Pvt. Ltd. enables the amplification of 28 loci, which include markers from the expanded CODIS list, are National DNA Index System (NDIS) recommended markers, and comply with the NDIS recommended allelic range. In addition, the kit also includes 8 more markers including Penta D and Penta E. The NeoTyper Autosomal kit STR markers includes 14 mini-STR which makes it very effective for the human identification of the degraded samples like unidentified body remains.This is a developmental validation study for NeoTyper, based on the listed Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. MethodologyWe extracted the human genomic DNA from blood and FFPE (Formalin-Fixed Paraffin-Embedded) samples, and performed PCR amplification for the 28 loci using the developed kit, NeoTyper, which features a six-dye chemistry. The amplified products were run on 3500xL Genetic Analyzer. We used GeneMapper ID-X v.1.4 (Applied Biosystems) to analyse peak height and allele calling. Developmental validation parameters, including genetic characterization, analysis of sensitivity, female-male mixture, precision, and accuracy (repeatability and reproducibility), were investigated and analysed. Furthermore, intra- and inter-laboratory comparisons, as well as mini-STR analysis and validation were done. ResultsThe developed NeoTyper Autosomal kit demonstrated 100 % sensitivity up to a DNA concentration of 62.5 pg, as measured by the number of alleles called. The assay also demonstrated good precision (measured in terms of standard deviation, SD ranging between 0.60 and 0.77) and reproducibility, with alleles called at 100 % accuracy. Furthermore, precision was high (measured as SD ranging from 0.50 to 0.66), with a 100 % accuracy. In 90.9 % of the samples (10 out of 11 female-male sample varied ratio), the mixture analysis revealed 100 % of the alleles being called. A comparative analysis of mini-STRs in GlobalFiler and NeoTyper Autosomal kit revealed a 100 % accuracy of the NeoTyper mini-STRs. ConclusionIn conclusion, the NeoTyper Autosomal kit developed by Neom Scientific Solutions Pvt. Ltd. enables the amplification of 28 loci, which includes markers from expanded CODIS list; are NDIS accepted markers and comply with their recommended allelic range. The integration of mini-STRs in the kit demonstrates its efficacy and reliability, particularly when addressing the difficulties posed by degraded samples. This developmental validation study demonstrates consistent performance with high sensitivity, precision and accuracy (repeatability and reproducibility) which is indicative of an advanced and efficient system for human identification.

Empowering adolescents living with perinatally-acquired HIV: tailored CD4+ count assessment for optimized care, the EDCTP READY-study.

The elevated rate of AIDS-related mortality in Sub-Saharan Africa among adolescents living with HIV (ALHIV) is influenced by various factors, notably immunosuppression, within a framework of limited therapeutic alternatives. We aimed to enhance the management of pediatric HIV by assessing the immune response and associated factors in perinatally-infected ALHIV on antiretroviral therapy (ART) in Cameroon. A cohort study was conducted from 2018-2020 among 271 ART-experienced ALHIV in Cameroon. Sociodemographic data, immunological (CD4), and virological (plasma viral load, PVL) responses were measured at enrolment (T0), 6-months (T1), and 12-months (T2) using PIMA CD4 (Abbott/Pantech (Pty) Ltd) and Abbott Applied Biosystem platform (Real-Time PCR m2000RT) respectively. Immunological failure (IF) was defined as absolute CD4 < 250 cells/mm3, and Virological failure (VF) as PVL ≥ 1,000 copies/ml. A linear mixed-effects model with R version 4.4.1 was used to estimate both fixed and random effects, with significance set at p < 0.05. Of the 271 perinatally-infected ALHIV enrolled over three phases, females were predominant (55.7, 55.1, and 56.0%); median age was 14 (IQR: 12-17); majority of the participants were followed-up in urban areas (77.5, 74.5, and 78.6%); and the age distribution favored older adolescents (48.7, 61.2, and 58.5%). Most participants achieved clinical success (93.1, 89.7, 88.9%), predominantly on first-line ART (80.8, 66.2, and 53.0%), with good adherence (64.2, 58.9, and 64.5%). Most participants had secondary education (67.2, 70.1, and 67.5%). Median CD4+ counts fluctuated overtime, with values of 563 (IQR: 249.0-845.0), 502 (IQR: 319.0-783.5), and 628 (IQR: 427.5-817.5), respectively. Of note, being male was linked to a reduction in CD4+ count compared to females, [-200.63 (-379.32 to -21.95), p = 0.028]. Similarly, late adolescence was associated with lower CD4+ counts compared to early adolescence, [-181.08 (-301.08 to -61.09), p = 0.003]. Moreover, participants experiencing VF showed significantly lower CD4+ counts compared to those with undetectable viral loads, [-353.08 (-465.81 to -240.36), p < 0.001]. Additionally, there was a marginally significant interaction between male gender and secondary educational level, [209.78 (-6.94-426.51), p = 0.058]. Among perinatally-infected ALHIV, age, gender, educational level, and virological status are key factors influencing their immune health and treatment outcomes. Prioritizing targeted interventions and close monitoring within these subgroups is crucial for optimal management, employing holistic care strategies that consider not only medical interventions but also psychosocial support and education.

First Report of Stem Blight of Mungbean (Vigna radiata) Caused by Diaporthe longicolla in the United States.

Mungbean (Vigna radiata) is primarily grown in Asia and directly consumed by humans. U.S. consumers embraced mungbean as a plant-based protein in vegan eggs and meat substitutes. New cultivars are being developed for American farmers because of the crop's tolerance to heat and drought, and its adaptability to current farming infrastructure. Mungbean's short season complements various cropping systems such as intercropping, alternative cropping, and green manure. With rotations and inclusion with soybean systems, there is a concern about the overlap of common pathogens for soybean and mungbean. During August 2022 when mungbeans reached full maturity (growth stage R6), reddish-brown and necrotic stem lesions with linear rows of black pycnidia were observed on Berken and OK2000 cultivars at fields located in Hancock County, IA and Story County, IA in the United States. Pycnidia measured 0.5-0.6mm in length. Disease incidence was approximately 10% of plants in Hancock County, IA and less than 3% of plants in Story County, IA. Pycnidia from 16 plants were excised and immersed in a 0.5% NaOCl solution for 1 min, rinsed with autoclaved distilled water, and placed onto potato dextrose agar (PDA). Eighteen isolates were hyphal tipped and grown on PDA and were stored at 25°C. Isolates were then visually identified by culture and conidia morphology (Hobbs et al. 1985, Santos et al. 2011). Colonies were cream to white, dense, and floccose. Large black stromata were formed in a concentric pattern or scattered; alpha conidia were ellipsoidal. Template DNA for PCR amplification of the internal transcribed spacer region of the nuclear ribosomal DNA operon (ITS) and the beta-tubulin gene (TUB) was extracted from 18 isolates by scraping mycelia with a sterile pipette tip and transferring it into 50 ul of PrepMan Ultra Sample Reagent (Applied Biosystems, Foster City, California, USA). Fungal primers were ITS1 and ITS4 (White et al. 1990) and Bt-2F/Bt-2R (Udayanga et al. 2014). Sequences of isolates obtained from fields in both counties were identical, providing no species diversity. GenBank accession numbers for the ITS region were PP105598 and PP105599; PP108254 and PP108255 for TUB sequences. BLAST results showed the ITS 550/550 base pairs with type specimen D. longicolla ATCC 60326 GB NR_144924 and the TUB 446/446 base pairs with type specimen D. longicolla ATCC 60325 GB KJ610883. Thus, the isolates were identified as Diaporthe longicolla (Hobbs) J.M. Santos, Vrandecic & A.J.L. Phillips based on morphology and molecular characters (Santo et al 2011; Udayanga et al. 2014). To confirm the pathogenicity of the D. longicolla isolates, twenty mungbean plants (cv. Berken and OK2000) were grown in the greenhouse at 85% RH and 16hr light for 20 days. Inoculum was prepared by placing sterile toothpicks on 1/3 PDA with a single representative isolate from each field location for 21 days (Ghimire et al. 2019). Mungbean plants were grown in a 10cm-by-10cm pot containing a greenhouse professional growing mix (Sungrow, Agawam, Massachusetts, USA) and grown for 30 days post emergence. After 12 days of growing, a 3mm segment of the infested toothpick was inserted into a stem wound below the first trifoliate and sealed with parafilm. A sterile toothpick was inserted into the control plants. After 14 days, red lesions extended downward 1 to 3 cm from the inoculation site, and white mycelial was present in the wound. At 21 days red lesions spanned 3 to 9 cm upward and downward from the inoculation site. Pycnidia were present on collapsed stem tissue, and leaves became chlorotic. Damage was limited to 2mm from the mock-inoculation site, with no discoloration in the control plants. Symptomatic tissues were plated and compared to the original isolates. Alpha conidia were ellipsoidal with the base end rounded. To our knowledge, this is the first report of Diaporthe longicolla causing disease on mungbean within the U.S. and worldwide. The presence of this disease in two locations suggests the potential for Diaporthe longicolla to be a serious disease of mungbean in the future.

Individual Identification and Assessment of Genetic Diversity Using Microsatellite Markers in Racing Pigeons Raised in Turkiye

The implementation of a swift and economical molecular genetic approach, ensuring both efficacy and cost-effectiveness and facilitating population certification, is of utmost significance for breeders and the conservation of Turkiye's native pigeon biodiversity. In this study, we aimed to examine the genetic structure of racing pigeons (Columba livia domestica) raised in Turkiye using a genetic marker panel consisting of eight short tandem repeat (STR) loci. For this purpose, DNA was isolated from the shed feathers of 216 pigeons. Genomic DNA was amplified using the multiplex allele-specific PCR and subsequent capillary electrophoresis with ABI PRISM 3130XL Genetic Analyzer. Next, PCR products were analyzed in the GeneMapper Software program (Applied Biosystems). For parent testing, paternity index (PI), combined paternity index (CPI), and cumulative probability of paternity (CPP) were calculated. Furthermore, population genetic diversity was evaluated using heterozygosity (He), polymorphism information content (PIC), and Hardy–Weinberg equilibrium (HWE) testing. Results revealed that the total number of alleles is 81 and the number of alleles per locus varies between 4 and 19. The similarity rate between parent and offspring was calculated as 99.99% and above. Since no pedigree information was given when the samples were analyzed, obtaining this similarity ratio demonstrates the reliability of the panel. He values range from 0.362 to 0.919, and the PIC values range from 0.295 to 0.909. Loci PG-1, PG-2, and PG-3 show significant genetic diversity, with moderate to high PIC values reflecting varied allele frequencies in the population. Consequently, the set of seven STR markers (+ one sex marker) can be applied to identify and confirm parentage on a regular basis, thereby facilitating efficient breeding programs and ensuring genetic diversity conservation. This panel enables efficient pedigree analysis and gender determination, optimizing cost-effectiveness. The methodology presented in this study is ideal for pedigree analysis and breed certification in the Turkish pigeon breeding industry. Consequently, we affirm that the study data carries considerable national importance.

Polymorphisms of the collagen genes of the fibrous ring rs1800012, rs2276454, rs1793953, and the VDR gene rs2228570 with intervertebral disc degeneration

Background. Spinal diseases cause significant disability, with genetic factors influencing up to 70 % of cases. This study purposed to examine the association of polymorphisms of COL1A1rs1800012, COL2A1rs2276454, COL2A1rs1793953 (collagen genes), and VDRrs2228570 with L4-L5, L5-S1, C5-C7 with intervertebral disc degeneration among ethnic Ukrainians. Materials and methods. The study included 90 individuals with L5-S1 disc degeneration, 50 — with L4-L5 degeneration, 30 — with C5-C7, and 66 controls without disc degeneration. Applied Biosystems (USA) kits were used for genotyping. Statistical analysis was performed using SNPStats. Results. There was an association between the C/C genotype and L5-S1 disc degeneration in men (odd ratio (OR) was 2.255, 95% confidence interval (CI): 1.089–4.670; χ2 = 4.905; p = 0.027), whereas the C/T genotype may have a protective effect (OR = 0.418, 95% CІ: 0.217–0.802; χ2 = 6.689, p = 0.009). The C/T genotype may also have protective significance for C5-C7 disc degeneration in men: its occurrence was higher among men in the control group compared to women (OR = 3.85, 95% CІ: 1.086–13.648; χ2 = 4.67; p = 0.031). The G/A COL2A1rs2276454 variant may have a protective effect on the L5-S1 disc (OR = 3.50, 95% CІ: 1.26–9.72; χ2 = 6.02; p = 0.015). The pair of alleles COL2A1rs2276454/COL2A1rs1793953 were linked to degenerative changes of the L4-L5 disc in the case group (p = 0.001); COL1A1rs1800012/VDRrs2228570 and COL2A1rs1793953/VDRrs2228570 were linked to degenerative changes in the C5-C7 disc. Conclusions. The C/C VDRrs2228570 genotype in men was associated with L5-S1 intervertebral disc degeneration. The T/C VDRrs2228570 genotype may have a protective significance for men with L5-S1 and C5-C7 degeneration. The COL2A1rs2276454 variant may have a protective effect against the development of L5-S1 degenerative changes in men. The allele pairs COL1A1rs1800012/VDRrs2228570, COL2A1rs1793953/VDRrs2228570, and COL2A1rs2276454/COL2A1rs1793953 were associated with C5-C7 degeneration, while the COL2A1rs2276454/COL2A1rs1793953 pair were associated with L4-L5 degeneration.

Population genetic structure and phylogenetic analysis of Anopheles hyrcanus (Diptera: Culicidae) inferred from DNA sequences of nuclear ITS2 and the mitochondrial COI gene in the northern part of Iran

BackgroundThe Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide.MethodsMosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software.ResultsIn this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity.ConclusionsAn. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

Circulating miR-28-5p is overexpressed in patients with sarcopenia despite long-term remission of Cushing's syndrome: a pilot study.

Patients with Cushing's syndrome (CS) in remission show sustained fatigue, myopathy, and an increased prevalence of sarcopenia. The mechanisms that determine these persistent muscle problems are not well known. We aimed to identify circulating microRNAs (miRNAs) with differential expression that could be potential biomarkers for the diagnosis and/or prognosis in CS. Thirty-six women in sustained remission for 13 ± 7 years (mean ± SD) from CS, with a median age (IQ range) of 51 (45.2-60) years and mean ± SD BMI of 27 ± 4 Kg/m2, and 36 matched healthy controls were investigated. In 7 patients sarcopenia was present according to the European Working Group on Sarcopenia in Older People (EWGSOP) criteria. Small RNA libraries were generated and indexed using a modified Illumina TruSeq small RNA-sequencing protocol. MiRNAs were identified in plasma using bioinformatic analysis, and validation was carried out using RT-qPCR. For the validation, Taqman probes were performed on QuantStudio 5 equipment (Applied Biosystems). In a first discovery group using RNA-sequencing, plasma samples of 18 CS patients and 18 healthy subjects were investigated; circulating miR-28-5p, miR-495-3p and miR-654-5p were upregulated in CS patients as compared with controls (p<0.05). In a validation study of the 3 upregulated miRNAs in 36 patients and 26 controls, no differences were observed by RT-qPCR; however, the expression of circulating miR-28-5p was upregulated in CS patients with sarcopenia as compared with those without (AUC for fold-change in the ROC analysis, 0.798; p=0.0156). The optimized cut-off value for miR-28-5p to identify CS patients with sarcopenia was 3.80, which yielded a sensitivity of 86% and a specificity of 69%. MiR-28-5p, a muscle-specific microRNA involved in myotube proliferation and differentiation in vivo, may serve as an independent non-invasive biomarker for identifying CS patients at high-risk of sarcopenia despite biochemical remission.

DNA methylation profiles of cancer-related fatigue associated with markers of inflammation and immunometabolism.

Cancer patients are commonly affected by fatigue. Herein, we sought to examine epigenetic modifications (i.e., DNA methylation) related to fatigue in peripheral blood among patients during and after treatment for head and neck cancer (HNC). Further, we determined whether these modifications were associated with gene expression and inflammatory protein markers, which we have previously linked to fatigue in HNC. This prospective, longitudinal study enrolled eligible patients with data collected at pre-radiotherapy, end of radiotherapy, and six months and one-year post-radiotherapy. Fatigue data were reported by patients using the Multidimensional Fatigue Inventory (MFI)-20. DNA methylation (Illumina MethylationEPIC) and gene expression (Applied Biosystems Clariom S) arrays and assays for seven inflammatory markers (R&D Systems multiplex) were performed. Mixed models and enrichment analyses were applied to establish the associations. A total of 386 methylation loci were associated with fatigue among 145 patients (False Discovery Rate [FDR] < 0.05). Enrichment analyses showed the involvement of genes related to immune and inflammatory responses, insulin and lipid metabolism, neuropsychological disorders, and tumors. We further identified 16 methylation-gene expression pairs (FDR < 0.05), which were linked to immune and inflammatory responses and lipid metabolism. Ninety-one percent (351) of the 386 methylation loci were also significantly associated with inflammatory markers (e.g., interleukin 6, c-reactive protein; FDR < 0.05), which further mediated the association between methylation and fatigue (FDR < 0.05). These data suggest that epigenetic modifications associated with inflammation and immunometabolism, in conjunction with relevant gene expression and protein markers, are potential targets for treating fatigue in HNC patients. The findings also merit future prospective studies in other cancer populations as well as interventional investigations.

Preliminary study of identified novel susceptibility loci for HAPE risk in a Chinese male Han population.

High altitude pulmonary edema (HAPE) is a life-threatening form of non-cardiogenic pulmonary edema. In recent years, association studies have become the main method for identifying HAPE genetic loci. A genome-wide association study (GWAS) of HAPE risk-associated loci was performed in Chinese male Han individuals (164 HAPE cases and 189 healthy controls) by the Precision Medicine Diversity Array Chip with 2,771,835 loci (Applied Biosystems Axiom™). Eight overlapping candidate loci in CCNG2, RP11-445O3.2, NUPL1 and WWOX were finally selected. In silico functional analyses displayed the PPI network, functional enrichment and signal pathways related to CCNG2, NUPL1, WWOX and NRXN1. This study provides data supplements for HAPE susceptibility gene loci and new insights into HAPE susceptibility.

Chromosome 7 Isodisomy in a Child with Silver-Russell Síndrome

Silver-Rusell syndrome is a rare genetic disease. There is evidence that the genetic causes of the disorder are heterogeneous, with predominant alterations in the imprinted regions of chromosomes 11 and 7, in addition to other genomic alterations, such as chromosomal structural aberrations, single nucleotide polymorphisms, copy number variations, and small insertions and deletions. The most prevalent clinical manifestations include prenatal and postnatal growth retardation, dysmorphic features, and feeding difficulties. We present a case of a 4-year-old boy with phenotypic features consistent with Silver-Russell syndrome. The sample was subjected to conventional karyotyping analysis. The analysis was also conducted using the SALSA MLPA Probemix ME032-A1 UDP7-UDP14 and Applied Biosystems CytoScan 750K Suite. MS-MLPA analysis revealed the presence of hypermethylation in the &lt;em&gt;GRB-10&lt;/em&gt; and &lt;em&gt;MEST&lt;/em&gt; genes on chromosome 7. SNP-array analysis revealed a loss of heterozygosity (LOH) at 7q11.22q31.1 (38.7 Mb). The methylation of the genes involved in this epigenetic event, in conjunction with LOH and the clinical characterization of this child, indicates that the origin of the disease is due to an isodisomy of maternal chromosome 7. This report of a child who exhibits the clinical characteristics of SRS and presents a UPD of chromosome 7, most likely originating from the mother, once again demonstrates the involvement of these genes in SRS despite the incomplete understanding of the underlying mechanism. A multidisciplinary strategy has been proposed for the follow-up and treatment of this disease according to its etiology in the proband.