Search Results (974)

Microsatellite instability (MSI) testing is frequently used to screen patients for the early detection of Lynch syndrome, the most common hereditary colorectal cancer syndrome. MSI testing compares microsatellite repeat lengths in tumor DNA with those in matched normal tissue from the same patient. Therefore, precise sample identification is critical for obtaining reliable test results. The Penta-C and Penta-D pentanucleotide markers are widely used for sample identification in MSI testing. We investigated instability, defined as allelic mismatches or shifts, discordant fragment sizes, or the appearance of alleles in tumor DNA that were absent in the corresponding normal DNA, in the Penta-C and Penta-D loci across 2609 paired colorectal tumor and matched normal tissue or blood DNA samples. The allele sizes of both markers did not match in 0.3% of microsatellite-stable (MSS) and 12.3% of microsatellite instability-high (MSI-H) patients (p < 0.001, difference in proportions, 12.0% (95% CI, 8.9–15.1%)). Non-matching allele sizes in 12.3% of the MSI-H tumors suggest that other repeat markers may also be unstable and not suitable for sample identification in these tumors. Full article

Background: The Pech ethnic group, comprising approximately 6024 individuals in northeastern Honduras, represents one of the country’s smallest indigenous communities with a rich cultural heritage extending to pre-Columbian times. Despite their historical significance, no population genetic studies have been conducted on this group, and population-specific databases are essential for accurate forensic applications. Methods: Allele frequencies for 23 autosomal short tandem repeat (STR) loci were determined in 100 unrelated Pech individuals (58 females, 42 males) from communities in the departments of Olancho, Colón, and Gracias a Dios. DNA was extracted from blood samples collected on FTA cards and amplified using the PowerPlex Fusion 6C System. Statistical parameters were calculated using Genepop v4.2 and Arlequin v5.3.2.2. Results: All loci exhibited substantial polymorphism. No statistically significant deviations from Hardy–Weinberg equilibrium were detected after Bonferroni correction (α = 0.0022). Expected heterozygosity ranged from 0.4033 (TH01) to 0.8563 (FGA). The combined power of discrimination exceeded 99.9999%, and the combined chance of exclusion was 99.9999%. Conclusions: This study presents the first genetic characterization of the Pech population, providing essential reference data for forensic identification, paternity testing, and population genetics research. The dataset fills a critical gap in the Honduran forensic genetic infrastructure and contributes to understanding indigenous Central American genetic diversity, enabling accurate forensic analyses for individuals of Pech ancestry in compliance with CODIS and ESS standards. Full article

Acid sphingomyelinase deficiency (ASMD) is currently treatable with olipudase alfa, increasing the need for early newborn screening (NBS). We conducted a two-center pilot cohort study to characterize dried blood spot (DBS) acid sphingomyelinase (ASM) activity in Japanese neonates in the neonatal intensive care unit (NICU). ASM activity was measured by flow injection-tandem mass spectrometry in 244 NICU-admitted neonates (gestational age 25–41 weeks; birth weight 773–4201 g); longitudinal paired samples were available in 34 neonates with birth weight < 2000 g and concurrent hematology in 43 neonates. The mean ASM activity was 3.7 ± 1.2 μmol/h/L (95% confidence interval, 3.54–3.84; range, 1.7–11.6), with a right-skewed distribution. ASM activity correlated positively with birth weight (r = 0.184, p = 0.0039), gestational age (r = 0.219, p = 0.0006), and lymphocyte count (ρ = 0.394, p = 0.0089) and negatively with hematocrit (ρ = −0.372, p = 0.014). In neonates with a birth weight < 2000 g, ASM increased significantly on repeat sampling (mean difference, 1.60 μmol/h/L; p < 0.0001; Cohen’s d = 0.912). These findings support NICU-specific reference ranges, hematology-informed interpretations, repeat testing after maturation, and the use of second-tier biomarkers for ASMD NBS implementation in Japan. Full article

Background/Objectives: Identifying the true perpetrator among monozygotic (MZ) twins has long posed a challenge in forensic genetics and for police investigations because MZ twins are likely identical in their short tandem repeat (STR) profile. In this study, we propose a workflow to address this issue, anchored in a case study involving MZ twins. Methods: The workflow includes shotgun sequencing of reference samples from one twin and trace samples, followed by a targeted amplicon-based approach (AmpliSeq™ Custom Panel) to validate the identified variants. Results: Biological traces from the crime scene, identified as blood, were available for analysis along with reference material from one twin, collected as a buccal swab. The samples underwent shotgun sequencing, and the resulting reads were aligned to the human reference genome. The sequencing yielded 2.91 and 2.85 billion mapped positions (corresponding to a breadth of coverage of 91% and 89%), with mean depths of coverage of 39.7× and 25.8× for the trace and reference samples, respectively. To minimise the risk of detecting somatic variants in the two different tissues, a stringent heterozygosity balance threshold (Hb: 0.4–0.6) was applied, and 2,047,077 single nucleotide variants (SNVs) were identified, of which 28 showed inconsistencies between the trace and reference samples. These candidate variants were subjected to validation using an AmpliSeq™ Custom Panel. Conclusions: No detectable SNV differences were observed between the reference and trace samples, and it was not possible to determine whether the trace originated from the reference twin or from his monozygotic co-twin. Full article

Background: The Garifunas are a distinctive Afro-indigenous community of Honduras, originating from the historical admixture of Island Carib, Arawak, and West African peoples in the seventeenth-century Caribbean. With an estimated 43,111 individuals residing primarily along the northern Atlantic coast. Their dual ancestral composition yields a genetic profile that differs meaningfully from those of other Honduran reference populations, consistent with pairwise FST comparisons with previously published Lenca and Tawahka datasets generated on the identical platform; yet no population-specific short tandem repeat (STR) reference dataset had previously been established. Methods: We genotyped 23 autosomal STR loci using the PowerPlex Fusion 6C System (Promega Corporation) in 100 unrelated Garifuna individuals (70 females, 30 males) sampled from three coastal settlements in the department of Atlántida: Triunfo de la Cruz, Ensenada, and Corozal. DNA was extracted from blood collected on FTA cards, and statistical parameters were computed using Genepop v4.2 and Arlequin v5.3.2.2. Results: A total of 217 distinct alleles were identified, with 5 to 19 alleles per locus (mean 9.43 ± 3.54). Expected heterozygosity (He) ranged from 0.6392 (D13S317) to 0.9010 (SE33), with a population mean of 0.7893. No locus deviated from Hardy–Weinberg equilibrium after Bonferroni correction (α = 0.0022). The combined random match probability was approximately 1.9 × 10⁻²⁶, and the combined chance of exclusion reached 99.99999993%. Conclusions: This study provides the first Honduran Garifuna population-specific autosomal STR reference database for precise forensic likelihood ratio estimates, kinship assessments, and population genetic studies. The Garifuna’s high diversity—consistent with their West African and Amerindian ancestry—indicates the risk of systematic bias when non-specific databases are used. Full article

Nitrogen is an essential element for plant growth, and low nitrogen stress significantly restricts crop yield. Therefore, cultivating crop varieties that are tolerant to low nitrogen is crucial for agricultural production. The AT-hook motif nuclear localization protein (AHL) family is vital for plant stress resistance. To investigate the potential regulatory mechanisms of the AHL family in maize under low nitrogen stress, 35 ZmAHL genes were identified from the maize genome using bioinformatics methods. The results indicated that these genes encode proteins with lengths ranging from 203 to 573 amino acids, with relative molecular weights between 20.68 and 59.68 kDa, and they are unevenly distributed across 10 chromosomes. Most proteins encoded by these genes are alkaline hydrophilic proteins, primarily localized in the nucleus. Family expansion occurred through tandem and fragment repeats, which exhibited evolutionary conservation with rice homologous genes. Transcriptome analysis revealed that the majority of ZmAHL genes in drought-tolerant maize inbred lines were significantly up-regulated under drought and low nitrogen stress, with the ZmAHL10 gene displaying the most pronounced response to low nitrogen conditions. Experiments involving transgenic Arabidopsis thaliana further confirmed that the growth status, nitrogen uptake, and photosynthetic pigment content of ZmAHL10 overexpression strains under low nitrogen conditions were superior to those of the wild type, while the mutant exhibited significant growth inhibition. Overall, this study delineated the fundamental characteristics of the maize ZmAHL gene family and established that ZmAHL10 enhances low nitrogen tolerance in plants by improving nitrogen absorption capacity and maintaining the stability of the photosynthetic system. This research provides candidate genes and a theoretical foundation for the molecular breeding of maize with enhanced low nitrogen tolerance. Full article

Background/Objectives: Preeclampsia (PE) is the primary cause of maternal and perinatal morbidity and mortality on a global scale. It is driven by a multifactorial aetiology, in which genetic factors involved in blood pressure regulation, including the endothelial nitric oxide synthase (eNOS) gene, play an important role. This study aimed to investigate the association between eNOS gene polymorphisms and the development and severity of PE in an Algerian cohort. Methods: A total of 305 Algerian women, comprising 124 patients with PE and 181 healthy controls, were genotyped for two polymorphisms: intron 4 variable number tandem repeat (VNTR) (4a/4b) and the −786 T>C promoter variant. Genotyping was performed using standard polymerase chain reaction (PCR) for VNTR and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) for the −786 T>C variant. Results: Our results revealed no significant difference in the allelic or genotypic frequencies of the −786 T>C polymorphism between the cases and controls (p > 0.05). Interestingly, the frequency of the protective “4b” allele was significantly lower in cases than in controls (odds ratio (OR) = 0.400 [0.278–0.575]; p < 0.0001). However, the “4a” allele and the 4a/4a genotype were significantly associated with an increased risk of preeclampsia (OR = 2.50 [1.74–3.59], p < 0.0001; and OR = 6.20 [2.85-13.50], p < 0.0001, respectively). Furthermore, they were correlated with disease severity (allelic: OR = 2.76 [1.60–4.75], p = 0.0002; and genotypic: OR = 4.64 [1.83-11.78], p = 0.0003). Conclusions: These findings support the potential role of the eNOS VNTR 4a/4b polymorphism in both the risk and severity of preeclampsia in the Algerian population. Full article

Postural control relies on the integration of visual, vestibular, and somatosensory inputs under biomechanical constraints. Conventional Romberg testing provides limited quantitative insight, particularly regarding directional control and sensory dependence. Wearable inertial measurement units (IMUs) enable portable, multidimensional assessment of postural sway. Thirty healthy adults (15 females, 15 males) completed a modified Romberg protocol with systematic manipulation of stance (normal, tandem), visual condition (eyes open, eyes closed), and arm position (arms at sides, arms forward), including both left and right leading foot during tandem stance. Whole-body kinematics were recorded using a full-body IMU system comprising 17 wireless sensors. Center-of-mass (CoM) trajectories were derived from a 23-segment biomechanical model, and linear, spatial, and nonlinear sway metrics were computed. Statistical analyses were conducted using repeated-measures ANOVA, with significance set at p < 0.05. Visual deprivation significantly increased sway path length, mean sway velocity, and sway area across all stance conditions (p < 0.001). Tandem stance elicited greater mediolateral sway than normal stance (p < 0.001). Romberg ratios exceeded unity for all metrics and were significantly higher in tandem stance (p < 0.01). Arm position effects were negligible in normal stance but showed significant Vision × Arm interactions during tandem stance (p < 0.05). Leading foot position had no significant main effects. Combining a modified Romberg protocol with full-body IMU-based CoM analysis enables sensitive characterization of sensory dependence and directional postural control. Tandem stance with visual deprivation increases mediolateral postural demands under reduced base-of-support conditions, providing a more challenging context for evaluating directional postural control. Full article

Scavenger Receptors (SRs) comprise a structurally diverse group of pattern recognition receptors (PRRs) involved in sensing non-self (microbial-associated molecular patterns) or altered-self ligands. CD5 and CD6 are class I SRs (SR-I) preferentially expressed by lymphoid cells and characterized by the presence of several tandem scavenger receptor cysteine-rich (SRCR) domain repeats. Both receptors interact with diverse microbial structures, including tegumental antigens from Echinococcus granulosus sensu lato (s.l.), the cestode parasite responsible for cystic echinococcosis (CE). This is notable as very few PRRs are currently known to detect parasitic helminths and because the infusion of recombinant soluble CD5 and CD6 proteins has shown prophylactic effects in murine secondary CE. Herein, the role of CD5 and CD6 in early immune responses to E. granulosus s.l. tegumental antigens (PSEx) was analyzed using CD5 (Cd5^−/−) and CD6 (Cd6^−/−)-deficient mice. Peritoneal B cells and macrophages from wild-type mice displayed specific and dose-dependent PSEx binding, which was impaired in those from Cd5^−/− and Cd6^−/− mice, supporting direct and/or indirect roles in parasite recognition. Additionally, in vivo exposure of peritoneal exudate cells (PECs) from Cd5^−/− and Cd6^−/− mice to PSEx showed altered activation profiles, including changes in CD80/CD86 expression, impaired early production of natural polyreactive antibodies, and cytokine shift from a Th1/Th17 to a Th2 profile. These findings strongly support the involvement of CD5 and CD6 receptors in the early immune recognition of E. granulosus s.l. antigens by PECs and influence immune responses critical for host resistance, highlighting their relevance in host–parasite interactions. Full article

Background/Objectives: The aztreonam/avibactam combination represents a promising therapeutic option for severe infections caused by multidrug-resistant Gram-negative pathogens, particularly in critically ill patients. Due to marked pharmacokinetic variability and the need to achieve joint pharmacokinetic/pharmacodynamic (PK/PD) targets of both agents, therapeutic drug monitoring (TDM) may play a pivotal role in optimizing treatment. This study aimed to develop and validate two rapid, accurate, and sensitive UHPLC–qTOF MS/MS sequential methods for quantifying aztreonam and avibactam in human plasma, suitable for routine clinical TDM. Methods: Plasma concentrations were determined by means of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC–qTOF MS/MS), operating in positive and negative electrospray ionization modes for aztreonam and avibactam, respectively. Sample preparation consisted of protein precipitation with isotopically labeled internal standards. The method’s validation was performed according to the European Medicines Agency guidelines, by assessing selectivity, linearity, precision, accuracy, recovery, matrix effects, carry-over, and stability. Clinical applicability was evaluated by reprocessing plasma samples, which were already previously collected for routine clinical practice from 20 hospitalized patients undergoing treatment with ceftazidime-avibactam plus aztreonam. Results: The methods showed excellent linearity (R² ≥ 0.999) over ranges of 0.2–100 µg/mL for aztreonam and 0.1–50 µg/mL for avibactam. Lower limits of quantification were 0.2 µg/mL and 0.1 µg/mL, respectively. Intra- and inter-day precision and accuracy met the EMA criteria at all of the quality control levels. Extraction recovery exceeded 90% for both analytes, and matrix effects were effectively compensated by internal standards. Stability testing highlighted the need for careful sample handling, particularly for aztreonam under repeated freeze–thaw conditions. Clinical application revealed substantial inter-individual variability in steady-state concentrations. Conclusions: The validated UHPLC–qTOF MS/MS assays provide robust and sensitive sequential quantification of aztreonam and avibactam in human plasma, supporting TDM-guided dose optimization in clinical practice. Full article

Background: low-quantity or degraded samples are often studied in forensic genetics. Therefore, it is important to efficiently obtain all the available DNA from the biological sample analyzed to provide the most reliable results. This is particularly challenging in bone marrow processing due to its hydrophobic molecular structure, as for other lipid-rich tissues, especially if rancid. In fact, during adipose tissue decomposition, the putrefaction of fatty acids can in some instances give a compact cerous consistency to the lipidic tissue, hardly susceptible to the nucleic acid extraction mechanisms. According to environmental circumstances, this condition is notably observable in submerged bodies or in putrefied bone marrow. Thus, this study is focused on developing an optimized nucleic acids extraction protocol for putrefied bone marrow. Methods: genetic analyses were performed on putrefied yellow bone marrow collected from 20 human femora recovered from bodies in different decomposition stages. The optimized method was developed by integrating additional steps, reagents and time intervals on a silica-based column commercial kit. This strategy was compared in DNA yield to a standard extraction protocol, represented by the same commercial kit, but following the manufacturer’s directions. Both these strategies were tested in nucleic acid isolation efficiency by performing DNA typing, including real-time PCR quantification, Short Tandem Repeats (STR) amplification and fragments analysis steps. The analytical parameters evaluated were allele count, DNA concentration (ng/µL) and Degradation Index (DI). Results: for allele count and DNA concentration parameters, the optimized protocol showed clear and significant qualitative and quantitative improvements compared with the standard protocol, supporting its potential applicability in forensic casework and laying the foundation for future studies. Conclusions: prior to appropriate laboratory internal validation, the optimized protocol can be used for tough lipid-rich tissues processing without the need to purchase a dedicated system and using a same commercial kit routinely adopted for other forensic genetics matrices. Full article

Background: Orthostatic intolerance (OI) is an important factor affecting daily functional capacity in patients with long COVID. Traditionally, most OI symptoms have been attributed to exaggerated sympathetic nervous system activation associated with postural orthostatic tachycardia syndrome (POTS). Disequilibrium, also referred to as postural instability, may contribute to the development of OI in patients with long COVID. Methods: This study evaluated 32 patients with long COVID using neurological examinations and the active 10-min standing test. Disequilibrium was assessed using the Romberg and tandem gait tests. OI was defined as the inability to complete the active 10-min standing test. Results: Seven patients (22%) were diagnosed with OI. None of them had POTS, whereas six (86%) demonstrated disequilibrium, as detected by the Romberg and/or tandem gait test. POTS was observed in eight patients (25%), none of whom had OI. Disequilibrium was observed in nine patients (28%), six of whom (67%) had OI. Multiple regression analysis revealed that disequilibrium was positively associated with OI (r = 0.64, p < 0.001), whereas POTS was inversely associated (r = −0.38, p < 0.05). After 6 weeks of oral minocycline treatment in six patients and 2 weeks of repetitive transcranial magnetic stimulation therapy following minocycline in the other one patient, symptom amelioration was reported in six patients with OI. OI concomitant with disequilibrium recovered in five of the six patients treated and tested, although one patient who experienced symptom recovery failed to undergo the repeated standing test. Conclusions: Disequilibrium, rather than POTS, was the primary determinant of OI in patients with long COVID. Full article

Castration is widely used in goat production to improve meat quality and manage reproduction, yet conventional surgical methods raise significant animal welfare concerns. Immunocastration targeting gonadotropin-releasing hormone (GnRH) offers a promising, welfare-friendly alternative, but its efficacy in slow-growing indigenous breeds remains poorly defined. In this study, we developed a novel tandem-repeat GnRH(30) recombinant vaccine and evaluated its effects on growth performance, reproductive function, and meat quality in male Leizhou goats. Eighteen six-month-old bucks were assigned to an immunocastration group (IM), surgical castration group (SC), or intact control group (IC). Vaccinated goats produced sustained anti-GnRH antibodies and exhibited significantly suppressed testosterone levels comparable to surgical castrates. Immunocastration induced marked testicular atrophy, disrupted spermatogenesis, reduced semen volume and sperm motility, and increased sperm abnormalities. Importantly, early growth performance and final body weight were not significantly affected. Compared with intact males, both IM and SC goats showed improved meat quality traits, including reduced drip loss and shear force, accompanied by increased intermuscular fat deposition, with overall patterns in the IM group closely resembling those of surgical castration. Overall, these findings indicate that the GnRH(30) vaccine can effectively suppress spermatogenesis and improve meat quality without affecting growth, providing an effective technical approach for castration management in indigenous goats. Full article

Atypical allelic patterns with additional alleles at multicopy Y-short tandem repeats (Y-STRs), such as DYS385a/b and DYF387S1, mainly arise from duplication or gene conversion events occurring in the palindromic regions of the Y chromosome where these markers are located. Although rarely encountered in forensic genetics, these allelic patterns require accurate deconvolution of the single allelic components to ensure correct genotype interpretation. Capillary electrophoresis (CE), the standard method for STR typing, infers multiallelic Y-STR genotypes through intra-locus and intra-color peak height ratios. However, this approach may be insufficient when the pattern includes isoalleles (alleles identical in length but differing in sequence), potentially leading to an underestimation of the number of alleles and therefore the true allelic configuration. Massively parallel sequencing (MPS) technique, through amplicon sequence-based analysis, provides additional information that can overcome ambiguities and interpretative errors arising from CE analysis of complex Y-STR patterns. In this study, analysis of raw MPS sequence data enabled the identification, in two male-derived DNA samples, of complex tri-allelic patterns at DYS385a/b and DYF387S1 loci, classifiable as type 2-B and 2-C, respectively. Furthermore, in a third male-derived DNA sample, a previously undescribed tetra-allelic pattern was detected at DYF387S1, characterized by an isoallele with double read counts. Full article

Background/Objectives: To identify US military unknowns, the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory has historically relied upon mitochondrial DNA and Y-chromosomal short tandem repeat testing. Where no appropriate family reference sample (FRS) is available or skeletal samples are degraded, autosomal single nucleotide polymorphism (SNP) testing with next-generation sequencing could assist. Methods: A method utilizing hybridization capture enrichment of a 95,000 (95K) SNP panel, amenable to FRS and extremely challenging samples, was validated. The Parabon Fx Forensic Analysis Platform was used for analysis and extended kinship inference. Skeletal samples (n = 65) and associated FRS (n = 64) were selected for a performance evaluation and case-type sample study. Results: Considering FRS with ≥7 ng DNA input into library preparation, 94% yielded ≥66,320 SNPs at ≥5X coverage. SNP recovery for skeletal samples at ≥1X coverage ranged from 5 to 94,197 SNPs, averaging 40,770 SNPs. When skeletal samples resulted in ≥13,000 SNPs, the most likely relationship category was consistent with the expected relationship. A log10 likelihood ratio of ≥4 and a posterior probability of ≥99.99% were established as thresholds for strong statistical support, and 87% of inferences met these thresholds while 13% were considered inconclusive. Pairwise kinship inference between unrelated individuals yielded an unrelated result in 85% of comparisons, 66% with strong statistical support. There were 170 instances of false positive 4th degree relationship inferences with strong statistical support. All false positives involved skeletal samples from individuals of admixed ancestry. Conclusions: With this approach, autosomal SNP testing can result in reliable kinship inferences between related individuals out to 3rd, and in some cases 4th, degree relationships, increasing the scope of eligible FRS to aid in identifications. Full article