Death by accidental intravenous administration of gasoline.

Herein, we present the case of accidental intravenous injection of gasoline in a 62-year-old male who was admitted to a dialysis center for his regular hemodialysis. Due to previous contact with another SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) positive patient, the hemodialysis was conducted in an isolated room. At the end of the procedure, the nurse, wearing all necessary personal protective equipment (PPE), in the intent to clean the dialysis catheter, applied medical gasoline, instead of 0.9% sodium chloride, intravenously to the patient. Soon afterwards, the patient's clinical condition deteriorated, and cardiopulmonary resuscitation was started. Despite the immediate reaction of the medical staff, after two successful cardiopulmonary reanimation and necessary intensive care measures, the patient suffered respiratory, metabolic, and lactic acidosis, hypotension, and tachyarrhythmia and ultimately died 7 h after the incident. The autopsy was conducted under the order of the district attorney. Main autopsy findings were marked congestion; right pleural and pericardial effusion; brain and lung edema; enlarged heart with left ventricle thickening and mild perivascular fibrosis; nephrosclerosis; tubular thyroidization; and interstitial fibrosis with inflammation. Gasoline presence was indisputably proven by conducted toxicology analysis in lung, bile, and brain samples. Traces of gasoline could be noted in the patient's blood sample in comparison to the blood that did not contain gasoline, but it was not possible to confidently claim that gasoline was present in the blood. Based on relevant findings, we concluded that the death of the patient was violent and that the cause of death was acute intoxication by gasoline.

Bioprospecting of Five Ocimum sp. Cultivars from Croatia: New Potential for Dietary and Dermatological Application with Embryotoxicity Tests.

Ocimum basilicum L. is the most common Ocimum species, and it is used as an ornamental plant and in food condiments. This unique study examined the chemical composition and biological activities of six extracts from five basil cultivars, including their antimicrobial, antidiabetic, antilipidemic, neuroprotective, and anticollagenase activity. Moreover, their toxicological effects were studied using the zebrafish Danio rerio. Volatile components were determined using HS-SPME and GC-MS, while total polyphenols were detected using HPLC and the spectrophotometric Folin-Ciocalteu method. Spectrophotometric assays (DPPH, ABTS, ORAC, FRAP) were performed to determine antioxidant activity, collagenase inhibition, acetylcholinesterase inhibition, and pancreatic lipase inhibition. Antimicrobial activity was determined using the broth microdilution test. The study found that the biological activities of different basil cultivars varied depending on the proportion of active compounds, as determined by chemical analyses. All six basil extracts significantly inhibited α-amylase, while Purple basil extract most significantly inhibited the activity of collagenase, acetylcholinesterase, and pancreatic lipase. Purple basil and Dark Opal basil I extracts exhibited the highest antimicrobial activity, while the Dark Opal basil II extract had the most significant antioxidant potential. The findings in this study suggest that ethanolic basil extracts have the potential to be used as dietary drugs and implemented in antiaging products. This study is unique in its aims to compare the chemical composition and biological activities of basil cultivars from Croatia and to evaluate potential toxicological effects through embryotoxicity tests on zebrafish Danio rerio embryos, and it reports the first evidence of anticollagenase, antidiabetic, and antilipidemic activities for these cultivars.

Intra-Species Variations of Bioactive Compounds of Two Dictyota Species from the Adriatic Sea: Antioxidant, Antimicrobial, Dermatological, Dietary, and Neuroprotective Potential.

The marine environment has a significant impact on life on Earth. Organisms residing in it are vital for the ecosystem but also serve as an inexhaustible source of biologically active compounds. Herein, the biodiversity of two brown seaweeds, Dictyota dichotoma and Dictyota fasciola from the Adriatic Sea, was evaluated. The aim of the study was the determination of differences in compound composition while comparing their activities, including antioxidant, antimicrobial, and enzyme inhibition, in connection to human digestion, dermatology, and neurological disorders. Chemical analysis revealed several terpenoids and steroids as dominant molecules, while fucoxanthin was the main identified pigment in both algae. D. dichotoma had higher protein, carbohydrate, and pigment content. Omega-6 and omega-3 fatty acids were identified, with the highest amount of dihomo-γ-linolenic acid and α-linolenic acid in D. dichotoma. Antimicrobial testing revealed a dose-dependent inhibitory activity of methanolic fraction against Escherichia coli and Staphylococcus aureus. Moderate antioxidant activity was observed for both algae fractions, while the dietary potential was high, especially for the D. fasciola dichloromethane fraction, with inhibition percentages of around 92% for α-amylase and 57% for pancreatic lipase at 0.25 mg/mL. These results suggest that Dictyota species might be a potent source of naturally derived agents for obesity and diabetes.

Biotechnological Enhancement of Probiotics through Co-Cultivation with Algae: Future or a Trend?

The diversity of algal species is a rich source of many different bioactive metabolites. The compounds extracted from algal biomass have various beneficial effects on health. Recently, co-culture systems between microalgae and bacteria have emerged as an interesting solution that can reduce the high contamination risk associated with axenic cultures and, consequently, increase biomass yield and synthesis of active compounds. Probiotic microorganisms also have numerous positive effects on various aspects of health and represent potent co-culture partners. Most studies consider algae as prebiotics that serve as enhancers of probiotics performance. However, the extreme diversity of algal organisms and their ability to produce a plethora of metabolites are leading to new experimental designs in which these organisms are cultivated together to derive maximum benefit from their synergistic interactions. The future success of these studies depends on the precise experimental design of these complex systems. In the last decade, the development of high-throughput approaches has enabled a deeper understanding of global changes in response to interspecies interactions. Several studies have shown that the addition of algae, along with probiotics, can influence the microbiota, and improve gut health and overall yield in fish, shrimp, and mussels aquaculture. In the future, such findings can be further explored and implemented for use as dietary supplements for humans.

The Stereological Analysis and Spatial Distribution of Neurons in the Human Subthalamic Nucleus.

The subthalamic nucleus (STN) is a small, ovoid structure, and an important site of deep brain stimulation (DBS) for the treatment of Parkinson's disease. Although the STN is a clinically important structure, there are many unresolved issues with regard to it. These issues are especially related to the anatomical subdivision, neuronal phenotype, neuronal composition, and spatial distribution. In this study, we have examined the expression pattern of 8 neuronal markers [nNOS, NeuN, parvalbumin (PV), calbindin (CB), calretinin (CR), FOXP2, NKX2.1, and PAX6] in the adult human STN. All of the examined markers, except CB, were present in the STN. To determine the neuronal density, we have performed stereological analysis on Nissl-stained and immunohistochemical slides of positive markers. The stereology data were also used to develop a three-dimensional map of the spatial distribution of neurons within the STN. The nNOS population exhibited the largest neuronal density. The estimated total number of nNOS STN neurons is 281,308 ± 38,967 (± 13.85%). The STN neuronal subpopulations can be divided into two groups: one with a neuronal density of approximately 3,300 neurons/mm3 and the other with a neuronal density of approximately 2,200 neurons/mm3. The largest density of STN neurons was observed along the ventromedial border of the STN and the density gradually decreased toward the dorsolateral border. In this study, we have demonstrated the presence of 7 neuronal markers in the STN, three of which were not previously described in the human STN. The human STN is a collection of diverse, intermixed neuronal subpopulations, and our data, as far as the cytoarchitectonics is concerned, did not support the tripartite STN subdivision.

Possibility of Human Gender Recognition Using Raman Spectra of Teeth.

Gender determination of the human remains can be very challenging, especially in the case of incomplete ones. Herein, we report a proof-of-concept experiment where the possibility of gender recognition using Raman spectroscopy of teeth is investigated. Raman spectra were recorded from male and female molars and premolars on two distinct sites, tooth apex and anatomical neck. Recorded spectra were sorted into suitable datasets and initially analyzed with principal component analysis, which showed a distinction between spectra of male and female teeth. Then, reduced datasets with scores of the first 20 principal components were formed and two classification algorithms, support vector machine and artificial neural networks, were applied to form classification models for gender recognition. The obtained results showed that gender recognition with Raman spectra of teeth is possible but strongly depends both on the tooth type and spectrum recording site. The difference in classification accuracy between different tooth types and recording sites are discussed in terms of the molecular structure difference caused by the influence of masticatory loading or gender-dependent life events.

Investigation of WWII/postwar mass burials in Croatia-The implementation of the Croatian model of searching for the imprisoned and missing persons.

The search for victims of World War Two (WWII) and the immediate aftermath period (postwar) in Croatia started together with the search for Homeland War victims in 1991. It continued through years, most often sporadically and in a non-homogenous way. It was just with the adoption of the Law on Research, Arrangement and Maintenance of Military Cemeteries, Cemeteries of Victims of WWII and Postwar Period in 2013 that the search became more structured and gained a formal governmental body responsible for the organization and supervision of the activities related to it. It was then that the well-established model of searching for Homeland War victims, based on many years of field work and research, started to be implemented in the search and analysis of WWII/postwar victims. The model represents a continuously growing and developing project which encompasses a wide variety of steps and procedures, from the investigation of alleged burial locations to the analysis and reburial of recovered mortal remains. From its implementation in 2016, it allowed the successful investigation of 1300 alleged burial locations, 484 field surveys, 42 exhumations and the recovery of remains of more than 1600 individuals. Besides, care for military cemeteries, marking of mass burial sites and arranging of international treaties are conducted in order to guarantee proper handling, relocation and repatriation of all those that perished during WWII and the postwar period.

Accidental death from a fall from height at workplace turned out to be a COVID-19 death.

Coronavirus 2019 disease (COVID-19) has rapidly evolved into a pandemic during the first months of 2020. The general extent of organ involvement, as well as microscopic changes in the organs of the diseased remains insufficiently documented so far. Herein we are reporting the case of a Caucasian male who apparently died in a workplace allegedly by a fall from height and was found to be SARS-CoV-2 positive just prior to the autopsy. The circumstances of his death required a medicolegal investigation. Gross autopsy findings included minor external injuries and focal subarachnoid hemorrhage together with ribs and sternum fractures resulting from resuscitation attempt. In addition, visceral congestion was noticed, especially visible in lungs. Traumatic findings suitable to explain sudden death were absent. However, lungs histopathology analysis revealed diffuse alveolar damage with massive capillary congestion. Pathology in other organs included myocardial fibrosis, left ventricular hypertrophy, severe generalized atherosclerosis, glomerulosclerosis, focal liver necrosis, liver microabscesses and intrahepatal cholestasis. Lab test confirmed the presence of SARS-CoV-2 in nasopharygeal swab taken postmortem. Only afterwards, the investigation brought out that he was suffering complaints consistent with respiratory infection in the days before death. Based on all findings, the death was ruled as natural, caused by COVID-19. Learnings from this case urge medical staff in the autopsy suite to consider the presence of SARS-CoV-2 in sudden deaths during the pandemic, to enable adequate personal protection gear is worn and to ensure the proper cause of death is determined where misleading circumstances exist.

Fatal motorcycle crashes in wide urban area of Zagreb, Croatia-A 10-year review.

Objective: The objective of our study was to determine the prevalence of alcohol and drug intoxication among fatally injured motorcyclists in a wide urban area of Zagreb, Croatia. Methods: We conducted a single-center observational retrospective study over a 10-year period (2007-2016) in 3 counties covering an area including 1.2 million residents. We reviewed the records on fatally injured motorcyclists, collecting information relating to sex, age, cause of death, time of death in relation to the time of the crash, and the circumstances of the crash (time of day, day of the week, season). Blood alcohol concentration (BAC) and toxicology analysis results were collected and analyzed. Results: We identified 163 deaths (95.7% males, 4.3% females). Overall, 64.2% of the victims were 20 to 39 years old. The majority (50.9%) of those fatally injured were responsible for causing the traffic crash; the rest were determined not to be responsible or the responsibility could not be determined. The most frequent causes of death were multiple injuries (55.8%) and isolated head trauma (23.3%). The rider's BAC was above the legal limit for driving (>0.50 g/kg) in 53.8% of cases, with a mean BAC of 1.91 g/kg. There was no difference in riding a motorcycle with a BAC above the legal limit between groups defined as younger (≤39 years of age) and older (≥40 years of age). The number of people with an illegal BAC was significantly higher during weekends than during the work week. The BAC of riders who were responsible for the crash was significantly higher than that of those who were not responsible or whose responsibility could not be determined. Use of illegal drugs or nontherapeutic use of legal drugs was not common and was detected in 10.4% of fatally injured riders. Conclusions: Alcohol intoxication has a major role in motorcycle crash-related mortality. A significant difference in BAC between fatally injured riders responsible for the accident and those who were not responsible implies that measures directed toward prevention of drinking and driving behavior could lower the number of fatal motorcycle crashes. Weekend measures, especially during spring and summer, could have particularly significant effects.

Acetabular osteometric standards for sex estimation in contemporary Croatian population.

AIM: To determine the sexual dimorphism of acetabular measurements in contemporary Croatian population and to provide a discriminant function equation for sex estimation. METHODS: The sample consisted of 200 adult pelvic bones (100 male and 100 female) from positively identified victims of Croatian War of Independence. In total, 96 left (48 male and 48 female) and 104 right (52 male and 52 female) acetabula were measured. One author measured two acetabular parameters using a sliding caliper: acetabular diameter (AD) and transverse acetabular diameter (TAD). Another author re-measured the acetabula of 40 randomly selected individuals to determine the inter-observer error. RESULTS: Both measured variables showed significant sexual dimorphism. Men had significantly higher values for AD and TAD than women. Receiver operating characteristic curve analysis showed that the cut-off point for prediction of male sex when using acetabular diameter was higher than 54 mm. For transverse acetabular diameter it was higher than 52 mm. The discriminant function was generated by using both acetabular variables, with 88% of accuracy in sex estimation. Inter-observer error was not significant. CONCLUSION: The acetabular measurements can be used for sex estimation in contemporary Croatian population with high accuracy.

Changes in neurofilament 200 and tyrosine hydroxylase expression in the cardiac innervation of diabetic rats during aging.

Changes in sensory and sympathetic innervation during diabetes mellitus (DM) can be a predictor of arrhythmias, silent myocardial ischemia, and chronic heart failure, but knowledge about these changes is still unsatisfactory. We analyzed whether prolonged DM induces changes in density of sensory and sympathetic nerve terminals of rat's heart and whether it contributes to cardiomyopathy during aging. DM was induced by i/p injecting 55 mg/kg streptozotocin to male Sprague-Dawley rats, while a control group received a citrate buffer. DM in the rats was validated by measuring blood glucose level. Animals were sacrificed after 2 weeks, 2 months, 6 months, and 12 months. Five areas of cardiac sections were analyzed. Antibodies raised against tyrosine hydroxylase (TH) and neurofilament 200 kDa (NF 200) were used to detect sympathetic and sensory fibers. TH immunoreactive fiber density increased in DM groups 2 weeks after induction, reaching a peek after 2 months, while in the later stages of DM (6 and 12 months), there was no significant difference compared to control. NF 200 immunoreactive fiber density increased 2 weeks after induction compared to control. There was no consistent pattern of change during the given period in both the DM or control groups. In the DM group, we found thickening of the left ventricle wall (P<.05) as the sign of cardiomyopathy. Our findings suggest that hyperglycemia as a hallmark of DM in early stages can lead to proliferation of sympathetic and sensory nerve terminals. This finding can contribute to a better understanding of the occurrence of arrhythmias and silent myocardial ischemia in DM.

Case of Fatal Starvation: Can Stable Isotope Analysis Serve to Support Morphological Diagnosis and Approximate the Length of Starvation?

The diagnosis of death as a result of starvation is established on anthropological measurements, visual appearance of the deceased on external and internal examination, microscopic analysis, laboratory testing, and exclusion of other causes of death. Herein, we present our findings on a case of 95-year-old man who died of starvation. After the diagnosis of starvation was established by traditional forensic medicine methods, we have conducted retrospective segmental analysis of stable carbon (δ13 C) and nitrogen (δ15 N) isotope ratios in hair sample. This method reveals periods of starvation through decrease in δ13 C and increase in δ15 N along the strand of hair. Our analysis revealed the decrease of 0.6 ‰ in δ13 C during the last 10-12 weeks prior to death, similar as reported in other investigations. Also, a decrease of 0.7 ‰ in δ15 N during the last 8-10 weeks prior to death was determined that was different than observed in previous studies.

Suicidal chemistry: combined intoxication with carbon monoxide and formic acid.

Herein, we present a rare case of suicidal intoxication with carbon monoxide produced via reaction of formic and sulphuric acid with additional toxic effect of formic acid. The deceased was a 22-year-old men found dead in the bathroom locked from the inside. A bucket filled with liquid was found next to him, together with an almost empty canister labeled "formic acid" and another empty unlabeled canister. The postmortem examination revealed corrosive burns of the face, neck and chest, cherry-pink livor mortis, corrosive injury to the oropharyngeal area and trachea, subpleural petechiae, 100 mL of blood in stomach and superficial erosions of stomach mucosa. Toxicology analysis revealed 30% of carboxyhemoglobin in the femoral blood and the presence of the formic acid in various samples. Quantitative analysis of formic acid was performed by measuring methyl ester derivative of formic acid by using headspace gas chromatography with flame ionization detection. The highest concentration of formic acid was measured in the lungs (0.55 g/kg), gastric content (0.39 g/L), and blood (0.28 g/L). In addition, it was established that content of the unlabeled canister had a pH value of 0.79 and contained sulphuric ions. Morphological and toxicology findings suggested that the main route of exposure to formic acid was inhalation of vapors with a possible ingestion of only small amount of liquid acid. The cause of death was determined to be combined intoxication with carbon monoxide and formic acid.

Death by band-aid: fatal misuse of transdermal fentanyl patch.

We present a case of fatal intoxication by the application of a transdermal fentanyl patch upon a superficial bleeding abrasion of a 2-year-old girl. The grandmother discovered the body of the child in bed at approximately 7 a.m. External examination revealed a properly developed, nourished, and hydrated child, with some vomit in the nostrils and inside the mouth. There was no evidence of trauma besides small contusions and abrasions on the knees, with a patch placed over the largest abrasion. Closer inspection revealed that this was transdermal fentanyl patch. Internal examination and microscopic analysis revealed regurgitation of stomach content, cerebral and pulmonary edema, and liver congestion. Toxicology analysis revealed trace levels of fentanyl in the blood just above the limit of detection (2 ng/mL), while concentrations in the urine, liver, and kidney were approximately 102, 28, and 10 ng/mL, respectively. Investigation discovered that the child injured her knee while playing the evening before. The grandmother applied the patch to cover the injury, unaware that she had used a fentanyl transdermal patch instead of simple band-aid. Although fatal intoxications are uncommon among young children in high-income countries, it is of major interest to raise awareness of such events especially since a great majority of these are preventable. The presented case points at the need for more thorough education of users and more strict rules in prescribing and handling of this potent medicine. As well, we find this case to be a useful contribution to the evaluation of postmortem fentanyl concentrations in fatal intoxication in a small child.

Neocortical calretinin neurons in primates: increase in proportion and microcircuitry structure.

In this article we first point at the expansion of associative cortical areas in primates, as well as at the intrinsic changes in the structure of the cortical column. There is a huge increase in proportion of glutamatergic cortical projecting neurons located in the upper cortical layers (II/III). Inside this group, a novel class of associative neurons becomes recognized for its growing necessity in both inter-areal and intra-areal columnar integration. Equally important to the changes in glutamatergic population, we found that literature data suggest a 50% increase in the proportion of neocortical GABAergic neurons between primates and rodents. This seems to be a result of increase in proportion of calretinin interneurons in layers II/III, population which in associative areas represents 15% of all neurons forming those layers. Evaluating data about functional properties of their connectivity we hypothesize that such an increase in proportion of calretinin interneurons might lead to supra-linear growth in memory capacity of the associative neocortical network. An open question is whether there are some new calretinin interneuron subtypes, which might substantially change micro-circuitry structure of the primate cerebral cortex.

Shot through the heart-firepower and potential lethality of air weapons.

Although certain air weapons attain muzzle velocities comparable to some firearms, their successful use for committing suicide is rarely seen. Herein, we report a case of a young female died shortly after urgent admission to the hospital with penetrating gunshot wound to the chest. The autopsy revealed perforation of the pericardium and two perforative defects to the right heart ventricle. Injury was inflicted by a 0.177 caliber pellet, fired from an air rifle found near her body. Investigation ruled the death as suicide. The used air rifle fired the pellet with the energy density of 1.9 J/mm(2) , substantially above the threshold for inflicting injury. The pellet penetrated through two layers of cotton fabric and several layers of tissue to inflict fatal injury to the heart. In addition, we provide an insight into the wounding potential of air weapons, which illustrates the need for greater caution in handling those devices.

Changes in cardiac innervation during maturation in long-term diabetes.

Diabetic autonomic neuropathy being a common complication of diabetes mellitus (DM) is related to an increased risk of cardiovascular mortality. However, mechanisms underlying changes of innervation density in affected hearts remain insufficiently understood. Hence, the aim of this study was to describe quantitative changes of intra-myocardial nerve terminals in hearts of diabetic rats of various ages. Male Sprague-Dawley rats were injected with 55mg/kg streptozotocin (STZ) (DM group) or with citrate buffer (control). After 2weeks, 2months, 6months and 12months, sections of their hearts were analyzed in five areas-left ventricle, interventricular septum, right ventricle, anterior and posterior wall. Nerve fibers were visualized immunohistochemically, using antibody against a general neuronal marker, protein gene product 9.5 (PGP 9.5). Significant increase in total nerve fibers from all heart areas was observed 2weeks and 2months after diabetes induction, followed by a decrease at 6months and again increase at 12months was observed in both control and diabetic rats. Significant difference between control and diabetic rats was visible after 2weeks and 2months, with diabetic rats exhibiting significantly more nerve fibers. There were no consistent differences in quantity of nerve fibers in different areas of the heart within a particular age-related group of animals. In conclusion, cardiac innervation undergoes dynamic changes both in control and in diabetic rats, with a time-dependent significant increase in neuronal fiber density in diabetic animals. This novel information may contribute to our understanding of pathophysiological changes associated with diabetic cardiac neuropathy.