STR Profiles Research Articles

To Assess the Role of microRNA-451 in the Progression and Metastasis of Colorectal Cancer.

The study aimed to determine the expression of miR451 in colorectal cancer (CRC) subjects with CRC cells, and the role of miR451 in colorectal cancer cells. In October 2020, ATC purchased CRC and normal mucosal cell lines of CRC and implanted them in DMEM with 10% fetal serum. The suitability of the HT29 cell line is verified using the STR profile. In an incubator with 5% CO2, enlarged cells were placed at 37 °C. TCGA data was used to select the top 120 patients with a high voice and the lowest 120 patients with a low voice. Cells were collected and coated with Annexin V and PE according to the manufacturer's instructions after 24.0 h. After that, the cells were separated. Cells were also tested using flow cytometry. HCT-120 cells were transplanted into a concentration of 5×105/ml cells in 6-source plates. HCT120 cells in the experimental group were combined with miR451 mimics, miR451 inhibitors, or miR451 miR + SMAD4B for 12 h at 37 °C, and cells were collected 24 h later at 37 °C. The sample was injected with 5 ml of Annexin VFITC and PE. Compared with normal colorectal mucosal cells, CRC cell lines decreased miR451 expression levels (fetal human cells (FHC) and HCoEpiC). Then, the HCT120 cells were transfected with miR451 inhibitors, and 72 h after transfection, say of miR451 was normal. There was a significant decrease in cell function in the miR451mimic groups, but an increase when the miR451 was blocked. The proliferation of cancer cells was prevented and chemotherapy was effective when miR451 was overexpressed. The SMAD4 gene provides instructions for making a protein involved in transmitting chemical signals from the cell surface to the nucleus. The SMAD4B expression was tested by RT-qPCR and Western blotting after 72.0 h of transmission. The mRNA and protein expression of SMAD4B decreased significantly when miR451 was significantly higher than when inhibited, as revealed in the results of this study. Seventy-two hours after transplantation, mRNA levels and SMAD4B proteins were measured in HCT120 cells. In addition, the researchers in this study investigated whether miR451 was associated with SMAD4B-directed control of CRC growth and migration. It was found that SMAD4B is highly expressed in both CRC and para-cancer tissues while using the TCGA database to detect SMAD4B expression. Patients with CRC with SMAD4B have a severe prognosis. MiR451 is sensitive to depressive disorders by targeting SMAD4B, according to these studies. We found that miR451 inhibited cell growth and migration, made CRC cells more readily available in chemotherapy, and did so by targeting SMAD4B. The findings suggest that miR451 and its genetic predisposition, SMAD4B, may help predict the prognosis and course of cancer patients. Treatments that target the miR451/SMAD4B axis may be helpful to people with CRC.

Paternity pseudo-exclusion caused by tetragametic chimerism in a gestational surrogacy case

Chimerism is a condition when an organism is composed of two or more populations of genetically distinct cells. Chimerism often produces curious results of medical and genetic investigations and could be a major cause of false negative conclusions in parentage testing. Here we describe a paternity pseudo-exclusion due to tetragametic chimerism in a gestational surrogacy case originated in a fertility clinic. Initial analysis using a buccal swab from the child and a peripheral blood sample from the father showed paternity exclusion at 6 STR loci. To find out the reason for observed paternal discrepancy father's semen sample used for IVF and samples from other tissues were genotyped. Buccal swabs, semen, hair follicles, nail clippings and cerumen showed identical mixed autosomal STR profiles originated from two genetically different cell lines and for all the 24 informative loci contain paternal obligate alleles. Results of Y-STR profiling of all paternal sample types showed a DNA profile originated from a single man. The mixed profiles obtained for different tissue types suggest that two genetically different cell lines contributed to formation of both the endoderm and the ectoderm of the father. The mesoderm seems to be monoclonal having originated from a genetically homogenous cell line as evidenced by the STR profile of peripheral blood. Such allelic pattern for various tissues suggests that the clonal origin happened at the very early stage of embryonic development. Approaches to minimise the rate of false exclusions in DNA parentage tests due to chimerism are discussed.

Characterisation of cell lines derived from prostate cancer patients with localised disease.

Prostate cancer is a broad-spectrum disease, spanning from indolent to a highly aggressive lethal malignancy. Prostate cancer cell lines are essential tools to understanding the basic features of this malignancy, as well as in identifying novel therapeutic strategies. However, most cell lines routinely used in prostate cancer research are derived from metastatic disease and may not fully elucidate the molecular events underlying the early stages of cancer development and progression. Thus, there is a need for new cell lines derived from localised disease to better span the disease spectrum. Prostatic tissue from the primary site, and adjacent non-cancerous tissue was obtained from four patients with localised disease undergoing radical prostatectomy. Epithelial cell outgrowths were immortalised with human papillomavirus type 16 (HPV16) E6 and E7 to establish monoclonal cell lines. Chromosomal ploidy was imaged and STR profiles were determined. Cell morphology, colony formation and cell proliferation characteristics were assessed. Androgen receptor (AR) expression and AR-responsiveness to androgen treatment were analysed by immunofluorescence and RT-qPCR, respectively. RNA-seq analysis was performed to identify prostate lineage markers and expression of prostate cancer tumorigenesis-related genes. Two benign cell lines derived from non-cancer cells (AQ0420 and AQ0396) and two tumour tissue derived cancer cell lines (AQ0411 and AQ0415) were immortalised from four patients with localised prostatic adenocarcinoma. The cell lines presented an epithelial morphology and a slow to moderate proliferative rate. None of the cell lines formed anchorage independent colonies or displayed AR-responsiveness. Comparative RNA-seq expression analysis confirmed the prostatic lineage of the four cell lines, with a distinct gene expression profile from that of the metastatic prostate cancer cell lines, PC-3 and LNCaP. Comprehensive characterization of these cell lines may provide new in vitro tools that could bridge the current knowledge gap between benign, early-stage and metastatic disease.

A new tumorgraft panel to accelerate precision medicine in prostate cancer.

Despite the significant advances in the management of advanced prostate cancer (PCa), metastatic PCa is currently considered incurable. For further investigations in precision treatment, the development of preclinical models representing the complex prostate tumor heterogeneity are mandatory. Accordingly, we aimed to establish a resource of patient-derived xenograft (PDX) models that exemplify each phase of this multistage disease for accurate and rapid evaluation of candidate therapies. Fresh tumor samples along with normal corresponding tissues were obtained directly from patients at surgery. To ensure that the established models reproduce the main features of patient's tumor, both PDX tumors at multiple passages and patient's primary tumors, were processed for histological characteristics. STR profile analyses were also performed to confirm patient identity. Finally, the responses of the PDX models to androgen deprivation, PARP inhibitors and chemotherapy were also evaluated. In this study, we described the development and characterization of 5 new PDX models of PCa. Within this collection, hormone-naïve, androgen-sensitive and castration-resistant (CRPC) primary tumors as well as prostate carcinoma with neuroendocrine differentiation (CRPC-NE) were represented. Interestingly, the comprehensive genomic characterization of the models identified recurrent cancer driver alterations in androgen signaling, DNA repair and PI3K, among others. Results were supported by expression patterns highlighting new potential targets among gene drivers and the metabolic pathway. In addition, in vivo results showed heterogeneity of response to androgen deprivation and chemotherapy, like the responses of patients to these treatments. Importantly, the neuroendocrine model has been shown to be responsive to PARP inhibitor. We have developed a biobank of 5 PDX models from hormone-naïve, androgen-sensitive to CRPC primary tumors and CRPC-NE. Increased copy-number alterations and accumulation of mutations within cancer driver genes as well as the metabolism shift are consistent with the increased resistance mechanisms to treatment. The pharmacological characterization suggested that the CRPC-NE could benefit from the PARP inhibitor treatment. Given the difficulties in developing such models, this relevant panel of PDX models of PCa will provide the scientific community with an additional resource for the further development of PDAC research.

Persistence of touch DNA on commonly encountered substrates in different storage conditions

The persistence of touch DNA deposited after realistic handling of items typically encountered in forensic investigations has been the subject of few studies. Understanding the long-term persistence of touch DNA on different substrates in varying conditions can be central to the effective triage of samples for further processing. As the time between an alleged incident and collection of evidence may vary from a few days to years after an alleged event, this study assessed three different common substrates for the persistence of touch DNA over a time span up to 9 months. These substrates included fabric, steel, and rubber, each of which were handled in a way to imitate what may happen during a criminal act. The three substrates were exposed to two different environments for up to 9 months: inside a dark cupboard with no traffic to act as a control and an outside semi-exposed environment. Ten replicates from each of the 3 substrates were tested at 5 time points to create 300 samples. All samples were processed using a standard operating workflow to provide genotype data after exposure to different environments. It was found that the fabric samples produced informative STR profiles (defined here as 12 or more alleles) up to the 9 month timepoint for either environment. The rubber and steel substrates for the inside condition produced informative STR profiles up to the 9 month timepoint, but only generated informative STR profiles for the outside condition up to 3 and 6 months, respectively. These data add to our understanding of the external factors that affect DNA persistence.

Bone finds and their medicolegal examination: a study from Hesse, Germany

Bones found by chance can be of great criminal or historical interest. The nature of their appraisal depends on the individual case, the locally effective legislation and the available resources. To assess whether a find is relevant with respect to criminal investigation, the circumstances of the find and the results of the forensic examination carried out by trained personnel must be considered. The aim of this study was to obtain an overview of the circumstances and nature of the finds as well as the results of the subsequent expert opinions by evaluating bone finds from the federal state of Hesse, Germany. For this purpose, over a 10-year period from 2011 to 2020, all bone finds examined at the Institutes of Legal Medicine in Gießen and Frankfurt am Main, Germany, were evaluated retrospectively with regard to the locations and circumstances of the finds, their nature (human or non-human), the postmortem interval, possible traces of violent impact and the results of further examinations. Of the 288 bone finds evaluated, 38.2% were found in forests, meadows and parks. In 50.7%, the finds contained human bones, of which 37.0% had a forensically relevant postmortem interval of 50 years or less. Evidence of trauma was described in 77.4% of the human bone cases: postmortem damage in 78.8%, peri-mortem injury in 9.7% and ante-mortem injury in 11.5%. DNA examinations were performed in 40.4% of the human bone finds. They yielded STR profiles in 81.3%, leading to a definite identification in 35.4%. Among the non-human bones sent in, the most common were bones from pigs (23.4%), deer (18.1%), cattle (16.4%), roe deer (11.7%) and sheep (11.7%). The macroscopic examination is the first step of the forensic-osteological evaluation and sets the course for further examinations or investigations. DNA examinations are of great importance for the reliable identification of human bones. They were responsible for 70.8% of successful identifications.

Identification, forensic evidences and effect of the most used lip cosmetics on the human STR profiling at Kingdom of Saudi Arabia

This study aimed to identify the DNA STR profiles that were obtained from the lips with various lip cosmetics involved in lip pencil, lipsticks and lip gloss for a brand – Makeup Forever and lip balms (Labello brand) – have been popularly used by Saudi women at KSA. The study was involved 35 unrelated participants (healthy female donors) aged between 26 and 32. The swabbing of lip cosmetics was done prior to using them as negative control samples, other sterilized swabs were collected from the used lip cosmetics which contained the lip cells for each participant as a study sample. Moreover, the buccal swabs were firmly collected from the cleaned oral cavities for the same donors as reference samples. The air-drying of the collected swabs was done for ten minutes at room temperature and then stored them at − 20 °C before the DNA analysis. The 7500 Real-Time PCR (qRT-PCR) was quantified the extracted DNA. The amplification of 16 STR loci was done using the AmpFlSTR® Identifiler® PCR amplification kit using the Thermocycler ABI 9700 to amplify the extracted DNA. The Applied Bio-systems 3130™ Genetic Analyzer with Gene Mapper® ID-X Software v3.5 was used to analyze the PCR products. The data for quantifying DNA recorded significant decrease in the concentrations of DNA samples ranged from 0.15 to 0.55 ng/µL in comparison to the reference samples, while DNA was not detected in all the negative control samples. Some STR loci showed considerably high inhibition and low heterozygosity loss in the study samples compared to the reference and negative samples. The possibility of extracting DNA samples from lip cosmetics were used in the present study could be useful and successful in some cases due to the effect of the chemical compositions such as heavy pigments, organic components, and aromatic wax on the STR profiles in the lip cosmetics, especially in the lipsticks, lip glosses and lip pencils.

Abstract P3-08-03: Establishment of patient-derived xenograft tumor models from breast cancer patients for quantitative elemental profiles and testing candidate therapies

Abstract Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Triple negative breast cancer (TNBC) is among the most aggressive clinical manifestations of breast cancer (BC), and represents a significant clinical challenge to the effective treatment of early metastatic and treatment-refractory disease because of its poor outcomes. Therefore, there exists a need to develop pre-clinical models that retain the characteristics of the original TNBC tumor—e.g., PDX tumor models—to better understand the mechanisms of drug resistance in metastatic TNBC and to effectively evaluate the effects of anti-cancer drugs on patients with this disease. Furthermore, quantitative changes in cellular element signatures, which indicate levels of enzymatic activity, are emerging new biomarkers for cancer in the clinic, but are of yet systematically understudied in tumor samples from breast cancer patients or/and PDX models of these tumors. Due to limitations in current cellular element signature quantitation profiles, there also exists a need to investigate further. Seven samples of advanced BC patient pleural effusion were obtained from Northwestern Memorial Hospital to establish a PDX tumor model in immunodeficient NSG female mice using breast fat pad xenografting and to develop derived 3D spheroid cultures for anti-cancer drug evaluation. To authenticate, STR profiling against with original patient tumor DNA was conducted. Five developed BC PDX tumor models were shown by pathology to have highly heterogeneous characteristics and the metastatic features of the origin patient tumor. Liver and lung metastases were observed in breast fat pad xenografted PDX tumor mice. 3D tumor spheroid cultures were successfully established from original BC pleural effusion or/and PDX tumor cultures. Using the newly developed, highly sensitive, and reliable Wash-Free Inductively Coupled Plasma Mass Spectrometry (WF ICP-MS) method, we evaluated the inorganic phenotypes from samples of breast cancer patient tumor tissue and of the established PDX tumor to quantify the mobile elements (Na, K, and Ca) and less mobile elements (P, Mg, Mn, Fe, Cu, and Zn) simultaneously within the same sample. The data was collected for further analysis of breast cancer inorganic signatures from normal and tumor cells. Our results suggested that BC 3D spheroid cultures and PDX tumor models could serve as models to further study the mechanisms of MBC and serve as promising tools for in vivo and in vitro quick testing and mechanistic studies of novel antitumor drugs respectively. Identifying quantitative elemental profiles is fundamental to understanding the pathologies of various metal-related cancers and thus opens up new opportunities for disease management and therapeutic intervention Citation Format: Wenan Qiang, Haimei Chen, Yi Yang, Andrew Crawford, Demirkan B. Gursel, Jian-Jun Wei, Thomas O’Halloran, Massimo Cristofanilli. Establishment of patient-derived xenograft tumor models from breast cancer patients for quantitative elemental profiles and testing candidate therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-03.

Diagnosis of hydatidiform moles using circulating gestational trophoblasts isolated from maternal blood

IntroductionIdentifying hydatidiform moles (HMs) is crucial due to the risk of gestational trophoblastic neoplasia. When a HM is suspected on clinical findings, surgical termination is recommended. However, in a substantial fraction of the cases, the conceptus is actually a non-molar miscarriage. If distinction between molar and non-molar gestations could be obtained before termination, surgical intervention could be minimized. MethodsCirculating gestational trophoblasts (cGTs) were isolated from blood from 15 consecutive women suspected of molar pregnancies in gestational week 6–13. The trophoblasts were individually sorted using fluorescence activated cell sorting. STR analysis targeting 24 loci was performed on DNA isolated from maternal and paternal leukocytes, chorionic villi, cGTs, and cfDNA. ResultsWith a gestational age above 10 weeks, cGTs were isolated in 87% of the cases. Two androgenetic HMs, three triploid diandric HMs, and six conceptuses with diploid biparental genome were diagnosed using cGTs. The STR profiles in cGTs were identical to the profiles in DNA from chorionic villi. Eight of the 15 women suspected to have a HM prior to termination had a conceptus with a diploid biparental genome, and thus most likely a non-molar miscarriage. DiscussionGenetic analysis of cGTs is superior to identify HMs, compared to analysis of cfDNA, as it is not hampered by the presence of maternal DNA. cGTs provide information about the full genome in single cells, facilitating estimation of ploidy. This may be a step towards differentiating HMs from non-HMs before termination.

Optimization of InnoXtract™ extraction and purification system for DNA extraction from skeletal samples.

The InnoXtract™ extraction and purification system is a purification method designed for DNA extraction from low-template samples, specifically rootless hair shafts. Its ability to successfully capture highly fragmented DNA suggests its suitability for use with other challenging sample types, including skeletal remains. However, the lysis and digestion parameters required modifications to successfully optimize the method for this sample type. A two-part digestion was developed utilizing a homebrew digestion buffer (0.5M EDTA, 0.05% Tween 20, and 100mM NaCl) and a supplemental lysis with the Hair Digestion Buffer included in the InnoXtract™ kit. Additionally, the magnetic bead volume was modified to improve DNA recovery from these challenging samples. With the altered protocol, the quality and quantity of DNA recovered from InnoXtract™ extracts were comparable to another commercial skeletal extraction method (PrepFiler™ BTA). This modified extraction method successfully purified sufficient amounts of quality DNA from a variety of skeletal samples to produce complete STR profiles. Successful STR typing from surface decomposition, burned, cremated, buried, and embalmed remains indicates the potential of this new method for challenging human identification and missing-person cases.

Generating human STR DNA profiles from blood ingested by leeches

Aquatic crime scenes may include the presence of annelids, such as leeches, which may have ingested human blood and can potentially aid in the identification of a victim and/or suspect. In this research, human blood from one male donor was fed to 35 North American medicinal leeches. These leeches were then euthanized at various periods (0, 1, 2, 4-, 6-, 12-, and 24-hours post-feed). Blood from their midguts (crop) was amplified using three methods. First, the tip of each Copan microFLOQ® Direct Swab was dipped into the midgut of the individual leeches and amplified directly. Second, Copan microFLOQ® Direct Swabs were utilized to sample the crop, concentrating crop blood followed by direct amplification. Third, 4N6 FLOQSwabs® forensic collection devices collected, extracted, and amplified remaining crop blood. The aim was to determine if blood found in the midgut of leeches can be used in revealing human identity. This research can aid in unique forensic cases where annelids might be present at a crime scene. Autosomal STR profiles were generated using PowerPlex Fusion 6 C System and GlobalFiler Express. Y-STR profiles were obtained with PowerPlex® Y23 System and Yfiler® Plus amplification kits. Complete and partial, concordant, and consistent, autosomal, and Y-STR profiles were observed between 420 autosomal and 420 Y-STR profiles. All three methods can be used to generate DNA profiles from blood ingested by leeches when collected within a 24-hour period. The results indicate that blood ingested by annelids can serve as a valuable source of evidence in unique crime scene cases.

Loss of Heterozygosity in the Circulating Tumor DNA and CD138+ Bone Marrow Cells in Multiple Myeloma.

Multiple myeloma (MM) is characterized by heterogeneity of tumor cells. The study of tumor cells from blood, bone marrow, plasmacytoma, etc., allows us to identify similarities and differences in tumor lesions of various anatomical localizations. The aim of this study was to compare the loss of heterozygosity (LOH) by tumor cells by assessing STR profiles of different MM lesions. We examined paired samples of plasma circulating tumor DNA (ctDNA) and CD138+ bone marrow cells in MM patients. For patients with plasmacytomas (66% of 38 patients included), the STR profile of plasmacytomas was also studied when biopsy samples were available. Diverse patterns of LOH were found in lesions of different localization for most patients. LOH in plasma ctDNA, bone marrow, and plasmacytoma samples was found for 55%, 71%, and 100% of patients, respectively. One could expect a greater variety of STR profiles in aberrant loci for patients with plasmacytomas. This hypothesis was not confirmed-no difference in the frequency of LOH in MM patients with or without plasmacytomas was found. This indicates the genetic diversity of tumor clones in MM, regardless of the presence of extramedullar lesions. Therefore, we conclude that risk stratification based on molecular tests performed solely on bone marrow samples may not be sufficient for all MM patients, including those without plasmacytomas. Due to genetic heterogeneity of MM tumor cells from various lesions, the high diagnostic value of liquid biopsy approaches becomes obvious.

Detecting STR profiles from degrading menstrual blood samples and their use as possible evidence in forensic investigations

This research explores DNA consistency and attempts to detect STR profiles from the degrading menstrual blood samples (MBS) as reliable forensic evidence. Peripheral (PBS) and MBS of 30 healthy fertile females were taken on the menstrual cycle’s second day. They were obtained at different time periods (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, and 48 h) at 25 °C. DNA evaluation was fulfilled to analyze DNA profiles. A considerable elevation in the median concentrations of DNA between 0 and 14-h intervals were documented, whereas decreased extents were registered between 16 and 48 h. Moreover, complete STR profiles (24/24) for DNA were discovered in all the intervals (0, 2, and 48 h). Periods of 0−8 h demonstrated the maximum extents of DNA materials. Full STR were discovered in all the intervals (0, 2, and 48 h). Eventually, MBS can be utilized as forensic evidence.

Geolocation prediction from STR genotyping: a pilot study in five geographically distinct global populations

Background Traditional CE-based STR profiles are highly useful for the purpose of individualisation. However, they do not give any additional information without the presence of the reference sample for comparison. Aim To assess the usability of STR-based genotypes for the prediction of an individual’s geolocation. Subjects and Methods Genotype data from five geographically distinct populations, i.e. Caucasian, Hispanic, Asian, Estonian, and Bahrainian, were collected from the published literature. Results A significant difference (p < 0.05) in the observed genotypes was found between these populations. D1S1656 and SE33 showed substantial differences in their genotype frequencies across the tested populations. SE33, D12S391, D21S11, D19S433, D18S51, and D1S1656 were found to have the highest occurrence of “unique genotype’s” in different populations. In addition, D12S391 and D13S317 exhibited distinct population-specific “most frequent genotypes.” Conclusions Three different prediction models have been proposed for genotype to geolocation prediction, i.e. (i) use of unique genotypes of a population, (ii) use of the most frequent genotype, and (iii) a combinatorial approach of unique and most frequent genotypes. These models could aid the investigating agencies in cases where no reference sample is available for comparison of the profile.

Optimized Recovery of DNA and Subsequent Short Tandem Repeat Profiling of Different Tissues Sampled from Embalmed Human Cadavers

Abstract Introduction: Storage of specimens sampled from human remains for pathological testing, embalming for burial purposes, and for human identification often requires formalin fixation and/or paraffin embedding. Current knowledge in molecular biology techniques and forensic DNA analysis makes it possible to optimize the extraction of amplifiable DNA from formalin-fixed tissues by improving the pre-treatment, optimizing the digestion condition of proteinase K, simplifying the extraction protocol and purifying the extracted DNA with optimized volumes of alcohol. Aim: This research sought to extract amplifiable DNA from thirteen brain, bone marrow and cartilage samples from four formalin embalmed human cadavers. Materials and Methods: Brain, cartilage and bone marrow samples were taken from four different cadavers at autopsy at the Ghana Police Hospital mortuary in Accra, Ghana sixty-two days after embalming. An optimized preparation and DNA extraction protocol was carried out on all the samples. Brain samples were also taken from a non-formalin treated fifth cadaver of known STR profile, and standard DNA extraction performed to serve as positive control. Results: Our optimized protocol yielded detectable quantities of DNA from the samples when quantified with the 7500 Real-Time PCR equipment. The extracted DNA also yielded full STR profiles with varying peak heights for forensic identification purposes. The measured degradation indexes of the DNA samples were greater than 1.0, with peak heights of generated STR profiles above the limits of detection of the 3500 genetic analyzer. Conclusion: Our current study demonstrated an optimized method of DNA extraction from tissues (brain, cartilage and bone marrow) sampled from formalin embalmed human cadavers. The optimized protocol reduced the concentration of formalin fixation residues in extracted DNA from formalin-fixed tissues, thereby improving the amplification efficiency for STR profiling. Brain, bone marrow and cartilages can be a good source of DNA from embalmed and degraded human remains, though for skeletonized human remains together with teeth and long bones.

Eficacia de 3 métodos de extracción de ADN a partir de restos óseos obtenidos de 3 contextos tafonómicos

IntroductionThe extraction techniques of deoxyribonucleic acid (DNA) from old bone samples are based on the use of complex methodologies, which deal with the degradation of the sample, the low amount of DNA and the presence of inhibitors that can be extracted simultaneously. ObjectiveTo compare the efficiency in obtaining DNA from the methods: organic extraction, commercial kit and previous total demineralization, to obtain STR profiles of old bone samples from three taphonomic processes (alkaline, acid and moist mitotic). Materials and methods29 skeletal remains from three different taphonomic contexts were processed: acidic, alkaline and wet-fungal. The amount of DNA obtained from three methodologies was evaluated in a comparative manner: organic extraction method (phenol-chloroform-isoamyl alcohol) (SO), extraction by silica column KIT QIAamp® DNA Investigator of QIAgen® (KC) and the methodology of extraction by previous total demineralization (DP). Finally, the obtaining of the STR profiles from the methodology of greater performance was tested. ResultsThe following quantification values were obtained: i) alkaline medium: 0.068 ± 0.07 ng/μL (SO), 0.021 ± 0.01ng/μL (KC) y 0.073 ± 0.052 ng/μL (DP); ii) acidic medium: 0.098 ± 0.064 ng/μL (SO), 0.041 ± 0.029 ng/μL (KC) y 0.068 ± 0.042 ng/μL; iii) Wet-mitotic medium: 0.25 ± 0.061 ng/μL (SO), 0.04 ± 0.027 ng/μL (KC) y 0.15 ± 0.072 ng/μL (DP). Likewise, using the DNA samples obtained by the SO method, complete profiles were obtained for the wet taphonomic context while the alkaline taphonomic process proved to be the most drastic for DNA degradation, presenting a greater number of incomplete profiles. ConclusionsThe methodology of organic extraction was optimal in obtaining DNA from the three taphonomic processes evaluated. On the other hand, the wet-fungal taphonomic process is the one that produces the least negative impact on the preservation of DNA from skeletal remains.

Comparative study of Rapid DNA versus conventional methods on compromised bones

Equivalent amounts of compromised bones were used to directly compare STR success of conventional and Rapid DNA methods. Conventional DNA extraction methods, including manual full demineralization and semi-automated PrepFiler BTA/ AutoMate Express (ThermoFisher Scientific), provided insights regarding the DNA quantity and extent of degradation of each compromised bone analyzed with ANDE 6C (ANDE Corp) and RapidHIT ID (ThermoFisher Scientific) Rapid systems. Full demineralization provided higher DNA yields than extraction with the AutoMate Express for quality control (QC) and environmentally challenged bones. The degradation indices ranged from ∼1.8 to 73. Both demineralization and AutoMate Express extracts benefited from additional clean-up with NucleoSpin XS devices, which usually resulted in more alleles being detected than without further clean-up. Complete “CODIS 20″ profiles could be obtained with bone QC1 with all methods. However, among the 14 compromised bones with low DNA content, complete CODIS 20 profiles were detected for 7, 4, and 0 bones analyzed with demineralization, AutoMate Express and ANDE methods, respectively. The RapidHIT ID was the least sensitive method, providing the fewest detectable alleles for the bones tested. Whereas extracted DNA of approximately 0.1 ng can yield complete GlobalFiler STR profiles, at least 30 ng was required for complete FlexPlex 27 profiles using the ANDE 6C Rapid DNA system. In addition to being less sensitive than conventional methods, the tested Rapid DNA approaches were less predictable when attempting to improve STR success and proved to be less reliable in genotyping accuracy.

Forensic evaluation of mitochondrial DNA heteroplasmy in Gujarat population, India

Background Owing to its high copy number and its small size, mtDNA analysis is the most reliable choice when biological materials from crime scenes are degraded or have mixed STR profiles. Aim To examine the occurrence of heteroplasmy along with its frequency and pattern in both HV1 and HV2 regions of the mtDNA among unrelated individuals from India. Subjects and methods Mitochondrial DNA control region [hypervariable region one (HV1) and hypervariable region two (HV2)] were analysed in blood and buccal tissues of 104 unrelated individuals from the Indian state of Gujarat. Results A high frequency of point heteroplasmy (PH) and length heteroplasmy (LH) was revealed. PH was detected in 7.69% of the population, with a higher frequency observed in blood than in buccal samples. However, there were no statistically significant differences in PH between the two tissues (Chi-square = 0.552, p ≥ 0.05). A total of six PH positions were detected: three at HV1, and another three at HV2. The studied population showed 46.15% LH in the HV1 and HV2 regions of both tissues. The LH positions observed in the Gujarat population were the same as those previously reported at HV1 np16184–16193 and HV2 np303–315. Conclusions Our findings suggest that differences in the pattern of heteroplasmy found in different tissues can complicate the forensic analysis, on the other hand, the probability of a match between the questioned and reference samples increases when the heteroplasmy is identical in both tissues. Variability of PH among persons and even within tissues recommends analysing multiple tissue samples before drawing a conclusion in forensic mtDNA analyses.

Retraction: ANGPTL1 Interacts with Integrin α1β1 to Suppress HCC Angiogenesis and Metastasis by Inhibiting JAK2/STAT3 Signaling.

This article (1) has been retracted at the request of the editors. The two cell lines used throughout the study, QGY-7703 and LO2, have been reported to be misidentified (2, 3). The authors performed STR profiling of their cell cultures following publication of the article. The QGY-7703 and LO2 cell lines could not be authenticated, and the QGY-7703 cell line was confirmed to be contaminated with HeLa cells.A copy of this Retraction notice was sent to the last known email addresses for 11 of the 11 authors (1). Yan Li agreed to the retraction; Qian Yan and Xin Yuan Guan did not agree to the retraction; and Lingxi Jiang, Ming Liu, Dandan Yu, Yu Zhang, Shuo Fang, Yan Li, Ying-Hui Zhu, and Yunfei Yuan did not respond.

Expanding the prostate cancer cell line repertoire with ACRJ-PC28, an AR-negative neuroendocrine cell line derived from an African-Caribbean patient.

Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.