Sex Determination via the Second Cervical Vertebra and Odontoid Process: A Case Report and a Review of the Literature.

Determining an individual's sex is crucial in several fields, such as forensic anthropology, archaeology, and medicine. Accurate sex estimation, alongside the estimation of age at death, stature, and ancestry, is of paramount importance for creating a biological profile. This profile helps narrow the potential pool of missing persons and aids identification. Our research focuses on the second cervical vertebra and odontoid process, which is particularly valuable due to their high sexual dimorphism. This brief research is structured as follows: we provide an overview of morphometric analysis of the second cervical vertebra for accurate sex estimation in forensic anthropology. We then delve into a case report to explore sexual dimorphism of the C2 vertebrae. Moreover, we discuss some of these studies that showed a significant correlation between the dimensions of the second cervical vertebrae and height, suggesting that the C2 can be used as a reliable indicator for stature estimation. The high accuracy rate of sex estimation using the second cervical vertebrae suggests that this method is a valuable tool for forensic anthropologists. Its practical application can significantly contribute to identifying and profiling individuals in a forensic context, thereby aiding in the identification process.

From Jane Doe to Sofia: DNA Extraction Protocol from Bones and Teeth without Liquid Nitrogen for Identifying Skeletal Remains.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

Intracellular tension sensor reveals mechanical anisotropy of the actin cytoskeleton.

The filamentous actin (F-actin) cytoskeleton is a composite material consisting of cortical actin and bundled F-actin stress fibers, which together mediate the mechanical behaviors of the cell, from cell division to cell migration. However, as mechanical forces are typically measured upon transmission to the extracellular matrix, the internal distribution of forces within the cytoskeleton is unknown. Likewise, how distinct F-actin architectures contribute to the generation and transmission of mechanical forces is unclear. Therefore, we have developed a molecular tension sensor that embeds into the F-actin cytoskeleton. Using this sensor, we measure tension within stress fibers and cortical actin, as the cell is subject to uniaxial stretch. We find that the mechanical response, as measured by FRET, depends on the direction of applied stretch relative to the cell's axis of alignment. When the cell is aligned parallel to the direction of the stretch, stress fibers and cortical actin both accumulate tension. By contrast, when aligned perpendicular to the direction of stretch, stress fibers relax tension while the cortex accumulates tension, indicating mechanical anisotropy within the cytoskeleton. We further show that myosin inhibition regulates this anisotropy. Thus, the mechanical anisotropy of the cell and the coordination between distinct F-actin architectures vary and depend upon applied load.

F-actin architecture determines constraints on myosin thick filament motion.

Active stresses are generated and transmitted throughout diverse F-actin architectures within the cell cytoskeleton, and drive essential behaviors of the cell, from cell division to migration. However, while the impact of F-actin architecture on the transmission of stress is well studied, the role of architecture on the ab initio generation of stresses remains less understood. Here, we assemble F-actin networks in vitro, whose architectures are varied from branched to bundled through F-actin nucleation via Arp2/3 and the formin mDia1. Within these architectures, we track the motions of embedded myosin thick filaments and connect them to the extent of F-actin network deformation. While mDia1-nucleated networks facilitate the accumulation of stress and drive contractility through enhanced actomyosin sliding, branched networks prevent stress accumulation through the inhibited processivity of thick filaments. The reduction in processivity is due to a decrease in translational and rotational motions constrained by the local density and geometry of F-actin.

Histological and immunohistochemical study of brain damage in traumatic brain injuries in children, depending on the survival period.

Numerous studies showed that, at present, traumatic brain injury (TBI) is one of the main causes of death in young adults, but also a main cause of disabilities at all ages. For these reasons, TBI are continuously investigated. In our study, we evaluated the histopathological (HP) and immunohistochemical (IHC) changes that occurred in the brain in underage patients after a severe TBI depending on the survival period. We histopathologically and immunohistochemically analyzed a number of 22 cases of children, deceased in Dolj County, Romania, following some severe TBI, undergoing autopsy within the Institute of Forensic Medicine in Craiova between 2015-2020. Patients were divided into three groups depending on the survival period, namely: (i) patients who died during the first 24 hours of the accident; (ii) patients who died after seven days of survival; (iii) patients who died after 15 days of survival. Microscopic examinations of the brain fragments, collected during the necropsy examination, showed that the traumatic agent caused primary injuries in all brain structures (cerebral parenchyma, meninges, blood vessels). However, HP injuries ranged in size and intensity from one area to another of the brain. In patients with a longer survival period, there was observed the presence of smaller primary injuries and larger secondary injuries. There was also observed a growth in the number of meningo-cerebral microscopic injuries, depending on the increase of the survival period.

Severe Thrombocytopenic Purpura Associated with COVID-19 in a Pediatric Patient.

Purpose: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to cause a diverse spectrum of clinical manifestations ranging from mild, flu-like symptoms to severe progressive pneumonia, acute respiratory distress syndrome with or without other extrapulmonary impairment. Hematological changes such as lymphopenia, neutrophilia, and anemia as the disease progresses, are frequently found in COVID-19. Thrombocytopenia may be drug-induced or can occur secondary to sepsis, disseminated intravascular coagulation or bone marrow suppression. Immune thrombocytopenic purpura (ITP) is frequently observed in children aged 2-5 years and in 60% of cases may proceed an upper respiratory tract infection. The present paper aimed to raise awareness of ITP as a possible pediatric presentation of coronavirus disease. Patients and Methods: We present the case of previously healthy, eight-year-old female patient, who developed an immune thrombocytopenia flare, also known as immune thrombocytopenic purpura (ITP), in the context of COVID-19, with diffuse petechiae and ecchymosis on her body, face and oral mucosa, and a nadir platelet count of 0×103/µL. Results: Platelet count recovery was observed after seven days of combined treatment with intravenous immunoglobulin (IVIG) and corticosteroids. Conclusion: The growing body of literature regarding the clinical and laboratory manifestations of COVID-19 infection in children, has reported thrombocytopenia in relation to unfavorable disease progression or multisystem inflammatory syndrome (MIS-C). Clinicians must be aware that ITP may appear both in mild and severe COVID-19, at any time during its course, and can be associated with a higher bleeding risk, thus its diagnostic may be critical.

SARS-CoV-2: An Overview of the Genetic Profile and Vaccine Effectiveness of the Five Variants of Concern.

With the onset of the COVID-19 pandemic, enormous efforts have been made to understand the genus SARS-CoV-2. Due to the high rate of global transmission, mutations in the viral genome were inevitable. A full understanding of the viral genome and its possible changes represents one of the crucial aspects of pandemic management. Structural protein S plays an important role in the pathogenicity of SARS-CoV-2, mutations occurring at this level leading to viral forms with increased affinity for ACE2 receptors, higher transmissibility and infectivity, resistance to neutralizing antibodies and immune escape, increasing the risk of infection and disease severity. Thus, five variants of concern are currently being discussed, Alpha, Beta, Gamma, Delta and Omicron. In the present review, a comprehensive summary of the following critical aspects regarding SARS-CoV-2 has been made: (i) the genomic characteristics of SARS-CoV-2; (ii) the pathological mechanism of transmission, penetration into the cell and action on specific receptors; (iii) mutations in the SARS-CoV-2 genome; and (iv) possible implications of mutations in diagnosis, treatment, and vaccination.

Autopsy Findings and Inflammatory Markers in SARS-CoV-2: A Single-Center Experience.

Purpose: The systemic inflammatory response related to COVID-19 can be easily investigated in living patients. Unfortunately, not every biomarker is suitable for postmortem analysis since several factors may interfere. The aim of this study was to summarize key histopathological findings within each organ system due to COVID-19 and to assess if serological inexpensive and widely available biomarkers such as CRP, IL-6, fibrinogen and d-Dimers, associated with adverse outcomes in COVID-19, can be implemented in a post-mortem assessment. Patients and Methods: A total of 60 subjects divided in 2 groups were included. All subjects died outside a hospital setting and therefore did not receive specific or symptomatic therapies that could have modulated the inflammatory response. The first group included 45 subjects in which mandatory autopsy was performed in order to establish the cause of death and macroscopic examination of the lungs was highly suggestive of SARS-CoV-2 infection. As controls (Group 2), 20 subjects who died from polytrauma in high velocity car accidents and suicide were selected. Bronchial fluids collected during the autopsy procedure were used for the RT-PCR diagnosis of SARS-CoV-2 and serum samples were sent for analysis of IL-6, CRP, d-Dimers and fibrinogen. Results: Compared with the control group, the subjects of the COVID-19 group were older (59±19.5 vs.38±19.15 years, p=0.0002) and had more underlying comorbidities such as hypertension (60% vs 35%, p=0.06) or were overweight (53.3% vs 30%, p=0.08). The levels of CRP, IL-6, fibrinogen and d-Dimers in postmortem plasma samples were significantly higher in COVID-19 subjects than in control group (p< 0.0001). Moreover, the level of IL-6 was significantly higher in overweight patients (r=0.52, P<0.001). In all COVID-19 subjects, the histological examination revealed features corresponding to the exudative and/or proliferative phases of diffuse alveolar damage. Large pulmonary emboli were observed in 7 cases. Gross cardiac enlargement with left ventricular hypertrophy was observed in 19 cases. The most frequent pathological finding of the central nervous system was acute/early-subacute infarction. Conclusion: Due to the complexity of the inflammatory response, we postulate that a combination of biomarkers, rather than a single laboratory parameter, might be more effective in obtaining a reliable postmortem COVID-19 diagnosis.

Molecular Testing of SARS-CoV-2 Infection from Blood Samples in Disseminated Intravascular Coagulation (DIC) and Elevated D-Dimer Levels.

BACKGROUND: In 2020, the SARS-CoV-2 virus spread worldwide and infected more that 10 million people, causing more than 500,000 deaths worldwide. The infection has systemic effects on the respiratory and cardiovascular systems; thus, patients can present a variety of symptoms from asymptomatic to rapid deaths. In this paper, we present the first case of post-mortem SARS-CoV-2 molecular testing in Western part of Romania in a deceased with disseminated intravascular coagulation (DIC) and elevated D-dimer levels. METHODS: During the autopsy which took place at the Institute of Forensic Medicine from Timisoara, Romania, blood sample was collected in a vacutainer with EDTA and sent to the Laboratory of Forensic Genetics from Victor Babes University of Medicine and Pharmacy, Timisoara, Romania. Viral RNA extraction was performed automated on the Maxwell 48 RSC Extraction System (Promega, USA) using the Maxwell RSC Viral Total Nucleic Acid Purification kit (Promega, USA). After RNA extraction, the samples were amplified on a 7500 real-time PCR (Applied Biosystems, USA) using the genesig® Real-Time PCR Assay (Primer Design, UK). RESULTS: The molecular testing showed a cycle threshold value of 23.4 (1.2 x 106 copies/mL), indicating increased viral loads, which correlated with the laboratory analysis results, especially with D-dimer levels. CONCLUSIONS: In cases of coagulopathy of SARS-CoV-2, patients in hospitals should be monitored closely for thrombosis development. Thus D-dimer can be used as prognostic marker in monitoring the evolution of SARS-CoV-2 infected patients.

Circulating Cell-Free Plasma DNA in Noninvasive Prenatal Paternity Testing with Short Tandem Repeats (STRs).

BACKGROUND: Paternity relationship can be established using STR markers in a minimally invasive manner during the prenatal period in the early weeks of pregnancy or in advanced pregnancy using circulating cell-free DNA (ccf DNA) drawn from the mother. The aim of our presentation is to demonstrate the advantages of ccf plasma DNA in establishing the paternity of an unborn child. Between mother and the alleged father (AF) of the fetus, an avuncular relationship as uncle-niece exists. METHODS: As biological samples, saliva was collected with buccal swabs from the mother and AF. For the fetus, we separated plasma from drawn blood from the mother, and further, we isolated ccf DNA from the mother's plasma sample. The DNA samples were quantified on a 7500 ABI Real-Time PCR using Investigator Quantiplex Pro Kit (Qiagen, Germany). Genotyping of the DNA samples was performed on a ProFlex PCR System (Thermo Scientific, USA) using the multiplex STR markers from Global Filer PCR Amplification Kit (Thermo Scientific, USA). Further, PCR products were run on capillary electrophoresis on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA). RESULTS: The AF was confirmed as the biological father of the child, with a probability of paternity (PP) = 99.99999% and a cumulative paternity index (CPI) = 8.300 x 103. CONCLUSIONS: In the case of advanced pregnancies from sexual assaults or incestuous relationships, the use of ccf DNA to establish the genetic profile of the fetus represents an advantage for establishing the paternity relationship between the fetus and AF. The method proves its efficiency as it has the advantage of speed of probation through forensic genetic expertise.

Co-morbidities in the multiple victims of the silent killer in carbon monoxide poisoning.

Carbon monoxide (CO) remains an insidious and silent killer due to its physical and chemical properties; its lethal effects are encountered in cases of household accidents, occupational hazards or suicide. Deaths due to CO poisoning were studied retrospectively in the period 2000-2018 at the Institute of Forensic Medicine, Timisoara, Romania. These cases represent 1.75% of all the autopsies and 0.63% of all violent deaths. There have been cases of single deaths and cases with multiple victims - concomitant deaths. The analysis of lethal CO intoxication cases that occurred in different circumstances (incomplete burning with CO accumulation, fires - associated with burns, death in the fountain - due to fossil fuel pump failure, suicide due to exhaust gases) was based on the examination of 298 autopsy files. In this type of poisoning, the forensic examination of the body is marked by the non-specific character of most of the macroscopic and microscopic changes. Although inconstant, these types of changes (e.g., red discoloration of livor mortis) raise the suspicion of death by CO poisoning; the essential contribution to establishing cause of death resides in the determination of carboxyhemoglobin (COHb) concentration by spectroscopy. In all cases, the cerebral and cardio-pulmonary modification and their contribution to the cause of death were studied. Co-morbidities interfere with the cause of death in cases with average COHb concentrations, in the 20-50% range, where CO blood levels alone are not reason enough to explain the onset of death.

Importance of Autosomal and Y-STR Markers in Establishing Sibling Relationship by DNA Genotyping.

BACKGROUND: The aim of the present study was the identification of an unknown person found in an advanced decomposed state using DNA samples provided by two alleged brothers as reference samples. To obtain an increased reliability of the test, we used autosomal and Y-STR markers. METHODS: Tissue fragments were obtained for the DNA isolation during the autopsy examination from the unidentified person. DNA was isolated from the reference samples obtained from buccal swabs of the two alleged brothers. The DNA was isolated from the biological samples using PureLink Genomic DNA (Invitrogen, USA). The quantification of the DNA samples was done on an ABI 7500 real-time PCR system with HID Analysis software v1.2 incorporated. For DNA amplification we used the multiplex PCR kit AmpFlSTR Identifiler Plus Kit for autosomal STR markers and AmpFlSTR Y-filer PCR Amplification Kit for the Y-STR markers. Further, we separated the DNA products on an ABI 3500 genetic analyzer. Gene Mapper ID-X version 1.4 software was used to visualize the DNA fragments. Data interpretation was done using the Kinship Examination of GenoProof-3 (qualitype, Dresden, Germany). RESULTS: We obtained genetic profiles for the three alleged brothers on autosomal and Y-STR markers and, thus, could establish a full sibling relationship between them. CONCLUSIONS: Since the introduction of DNA in human identification, it represents a useful tool in establishing sibling relationship from different biological samples.

DNA-Based Identification of a Carbonized Victim by Kinship Analysis.

BACKGROUND: Our study will present the DNA identification of a carbonized victim using the DNA genotyping and by comparing the victim's DNA genotype with his parents' genotypes. METHODS: Blood obtained from the heart chambers was used for the identification of the carbonized body's genotype. Biological samples were also obtained by buccal swabs from his biological parents. We used an ABI 7500 real-time PCR system for quantification and a ProFlex PCR System to amplify. PCR products were separated on an ABI 3500 genetic analyser and identified using GeneMapper ID-X vers. 1.4 software. RESULTS: We obtained the three DNA genotypes (mother, father, and carbonized victim). Using maternity and paternity DNA testing we established that the victim's genetic profile matched the DNA profiles of his biological parents. The probability of maternity (PM) and probability of paternity (PP) were of 99.99999% for each of the parents. CONCLUSIONS: Body fluids (blood, saliva) represent a better source for DNA compared to hard tissue, and its processing times are shorter than those for bone or teeth.

Day surgery in Romania.

AIM: Well know in USA, Australia and then in western European countries, day surgery is still at the beginning in Romania and eastern European countries. In this paper we want to analyze the evolution and actual situation of day surgery in Romania and in County Emergency Hospital Timisoara (CEHT). MATERIAL AND METHODS: In the implementation of day surgery in our country there were two distinct periods. Between 2007- 2013 the CEHT negotiated its own day surgery baskets with Local Health Insurance Company (LHIC). Starting from 2014 until now, the National Health Insurance Company has established new day surgery baskets which can be negotiated between CEHT and LHIC. RESULTS: Our study shows that day surgery in CEHT has had an undulating evolution - after a rising development at the beginning it stopped for a few years and now it has an ascendant evolution. DISCUSSION: In this context, International Association for Ambulatory Surgery (IAAS) has initiated a series of actions to support implementation and development of day surgery in Romania and Eastern European countries. The first action was the support that the International Association for Ambulatory Surgery gave to the Romanian Society of Ambulatory Surgery in organizing on 15-16 September 2013 in Timisoara the course "Day Surgery - Making it Happen Overcoming Obstacles and Barriers". Discussions after the presentation of local realities in Eastern and Western Europe were particularly creative, being the stand in the accelerated development of day surgery in Romania. CONCLUSIONS: Day surgery and ambulatory surgery have many advantages for patients (increased comfort, lower surgical risk, minimal stress and low anxiety, high satisfaction rate), for hospital (reducing congestion in hospital, enabling it to have a better capacity to deal with serious cases), and for healthcare (increased economic efficiency, cost / patient / surgery is lower than for continuous admissions). Known in our country from 2000, the implementation of day surgery still faces many hardships. KEY WORDS: Day surgery, ambulatory surgery, Short hospitalization.

Molecular DNA Analysis in Forensic Identification.

BACKGROUND: Serological and biochemical identification methods used in forensics have several major disadvantages, such as: long time in processing biological sample and lack of sensitivity and specificity. In the last 30 years, DNA molecular analysis has become an important tool in forensic investigations. DNA profiling is based on the short tandem repeats (STR) and aids in human identification from biological samples. METHODS: Forensic genetics, can provide information on the events which occurred at the crime scene or to supplement other methods of forensic identification. Currently, the methods used in identification are based on polymerase chain reaction (PCR) analyses. This method analyses the autosomal STRs, the Y-chromosome, and the mitochondrial DNA. RESULTS: Correlation of biological samples present at the crime scene with identification, selection, and the probative value factor is therefore the first aspect to be taken into consideration in the forensic genetic analysis. CONCLUSIONS: In the last decade, because of the advances in the field of molecular biology, new biomarkers such as: microRNAs (miR), messenger RNA (mRNA), and DNA methylation have been studied and proposed to be used in the forensic identifications of body fluids.

Post-Mortem Identification of a Fire Carbonized Body by STR Genotyping.

BACKGROUND: Identification of bodies of unknown identity that are victims of exposure to very high temperatures, resulting from fires, plane crashes, and terrorist attacks, represents one of the most difficult sides of forensic genetics, because of the advanced state of decomposition. The aim of this study was the identification of the carbonized cadaver of a fire victim through STR genotyping. METHODS: We used blood samples obtained from the iliac artery during the autopsy examination as biological samples from the unidentified victim. After DNA isolation and quantification, we proceeded to its amplification using the multiplex PCR kit AmpFlSTR Identifiler. The DNA products were separated using an ABI 3500 genetic analyzer. Further analysis of the data was done using Gene Mapper ID-X version 1.4 software. RESULTS: In this case, it was possible to obtain a complete DNA profile from the biological samples. Due to the fact that the amelogenin gene presented two alleles, X and Y, we concluded that the victim was a man. CONCLUSIONS: We conclude that STR profiling of unidentified bodies (carbonized, decomposed) represents a powerful method of human identification in forensic medicine.

Circulating MicroRNAs as Promising Biomarkers in Forensic Body Fluids Identification.

In the last 20 years, DNA molecular analysis has become an important tool in forensic investigations. Currently, it is possible to genotype all types of biological traces or micro-traces containing nucleated cells if they are not entirely destroyed, chemically or bacterial. The DNA profiling is based on the short tandem repeats (STR) and aids in human identification from biological samples, but due to the recent advances in molecular genetics, other biomarkers have been proposed to be used in forensic identifications, such as: messenger RNA(mRNA), microRNA (miRNA), and DNA methylation. MicroRNAs are part of a class of small, non-coding RNAs that contain 19 - 23 nucleotides. MicroRNAs play an important role in the regulation of biochemical mechanisms, cell proliferation and other cellular mechanisms in the human body. The level of microRNAs in blood and other body fluids (urine, saliva, sweat) increases as a consequence of altered pathophysiological mechanisms and tissue insult. Moreover, the stability and specificity of microRNAs make them ideal candidates for circulating biomarkers in forensic bioanalytical procedures. In this review, we want to present a brief overview of biogenesis, functions, and applications of miRNAs in the identification of forensic body fluids.