Liver Specimens Research Articles

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Is M30 staining a reliable marker predicting graft rejection and fibrosis in protocolar liver biopsies in post-liver transplant pediatric patients?

Liver transplantation is currently a treatment of choice in patients with end-stage liver disease. Acute cellular rejection (ACR), antibody-mediated rejection (AMR), and chronic rejection (ChR) are major causes of graft injury. Therefore, new markers predicting graft rejection are investigating. Apoptosis has been recently proposed as one of the mechanisms contributing to liver fibrosis in liver grafts. Coarse needle liver biopsy is still a gold standard in monitoring post-transplant pathologies. The aim of this study was to assess the utility of immunohistochemical (IHC) staining for M30 (cytokeratin 18), as a prognostic marker of rejection in pediatric recipients of liver transplant and predicting marker of liver fibrosis and worse follow-up. The study enrolled 55 biopsies from 55 patients aged 2.37 to 18.9 years (median 13.87 years) who underwent protocolar liver biopsies taken 1-17 years after liver transplantation (median 8.36 years). The control group (positive control group) consisted of 26 biopsies from 16 patients in whom acute ACR was diagnosed. IHC staining for M30 (cytokeratin 18) and histochemical Azan staining were performed in all liver specimens. The following changes were re-evaluated in each specimen: features of ACR (the severity was assessed using RAI/Rejection Activity Index/Scale, which ranges from 3-9 points and include 3 histopathological changes suggestive of rejection), AMR or ChR; severity of fibrosis (Ishak Scale); presence of cholestasis and steatosis. Clinical parameters including laboratory tests of liver function (AST, ALT, GGTP, bilirubin) were also evaluated. M30 expression correlated with presence of acute cellular rejection. However, no relationship was found between M30 expression and severity of fibrosis. M30 staining, marker of apoptosis, seems to be a promising marker predicting acute cellular rejection.

Comparison of R1ρ Imaging Between Rapid Acquisition with Relaxation Enhancement (RARE) and Ultrashort TE (UTE) Sequence in the Assessment of Rat Liver Iron Overload at 11.7T.

Since the most prominent effect of iron is increasing R2* and R2 relaxation rates, the iron-overload liver shows little signal with conventional T1ρ sequences like RARE. Whereas UTE MR imaging sequences can detect the signal from short T2/T2* relaxation components in tissues. This study aims to evaluate the difference in R1ρ profiles and compare the correlations between RARE-based and UTE-based sequences with LIC in assessing rat liver iron overload. Iron dextran (Sigma, 100 mg Fe/ml) was injected into thirty-five rats (25-100 mg/kg body weight), while the rats in the control group were injected with saline (n=5). The liver specimen was taken after one week. A portion of the largest hepatic lobe was extracted to quantify the LIC by inductively coupled plasma, and the remaining liver tissue was stored in 4% buffered paraformaldehyde for 24 h before MRI. Spin-lock preparation with RARE readout and 2D UTE readout pulses were developed to quantify R1ρ on a Bruker 11.7T MR system. The mean R1ρ value of the rat liver with UTE-based R1ρ sequence was significantly higher compared to the RARE-based R1ρ sequence (p<0.001). Spearman's correlation analysis (two-tailed) indicated that the R1ρ values were significantly correlated with LIC for both UTE-R1ρ and RARER1ρ sequences (r = 0.727, P < 0.001, and r = 0.712, P < 0.001, respectively). The current study adds to evidence that there is a correlation between iron concentration and R1ρ. Moreover, the UTE-based R1ρ sequence is more sensitive to the liver iron than the RAREbased R1ρ sequence. R1ρ might serve as a complementary imaging biomarker for liver iron overload quantification.

Comparison of Penton and Hexon Genes of Fowl Adenovirus Serotype 11 in Molecular Detection of IBH in Broilers.

Fowl Adenoviruses (FAdVs) are widely distributed pathogens across the globe. The FAdVs from serotypes FAdV 2, 3, 8a, 8b, 9, and 11 are responsible forinclusion bodyhepatitis (IBH). Recently, increased mortality and IBH-suspected lesions were observed in 8-10-day-old broiler chickens in West Azerbaijan Province, Iran. In this regard, the present study aimed to compare penton and hexon genes of ADDV11 in the molecular detection of IBH in broiler chickens. In total, 100 liver specimens were collected from 10 suspected farms, and their DNAs were extracted. Two polymerase chain reactions (PCRs) were applied; one targeting the L1 region of the hexon gene and another aiming at the penton gene. Based on the findings, 60% of samples showed positive results in both PCRs and phylogenetic analysis clustered the studied viruses into serotype 11 (species D) FAdV. The detected FAdVs also shared a multitude of homologies with previously published serotype 11 viruses from Iran and those identified in Pakistan, Saudi Arabia, India, China, and Canada. This research not only provides an update on circulating FAdVs in Iran, but also introduces the penton gene as an alternative target for IBH diagnosis. Considering that IBH is a primary disease in Iran with both horizontal and vertical routes of transmission, urgent preventive measures are needed.

A new highly apomorphic species of Bujurquina (Teleostei: Cichlidae) from a reverse flowing river in the Peruvian Amazon, with a key to the species in the genus

Bujurquina is the most widely distributed and species-rich genus of cichlids in the western Amazon of South America. In this study we describe a new species from Peru from a hypothesized reverse flowing river system. Prior to the origin of the modern Amazon River at 4.5 Ma, this river system had its headwaters on the Iquitos arch, one of several main structural arches (swells) in the Amazon. Prior to the origin of the modern Amazon these arches formed topographic barriers of drainage basins in lowland Amazonia. For our analyses we use morphological and molecular data, analyzed through multivariate statistics and molecular phylogenies, respectivelly. For all valid species in the genus (except B. cordemadi and B. pardus) we additionally for the first time provide photographs of live specimens. Based on DNA phylogeny and coloration patterns we demonstrate that Bujurquina is divided into two main clades and based on this we provide a dichotomous key for all the species.

Two new species of Tibouchina (Melastomataceae) from the Brazilian Cerrado

We describe here two new species of Tibouchina from the Brazilian Cerrado. Tibouchina falcifolia is endemic to the state of Goiás and it can be readily recognized by the lanceolate to linear, conduplicate, pendulous leaves, and very conspicuous hypanthium scales. It is morphologically related to Tibouchina aegopogon var. angustifolia, especially by the narrow leaves, but differs by the older branches decorticant (vs. older branches non-decorticating), slightly discolorous leaves (vs. strongly discolorous leaves), with the adaxial surface covered by trichomes (vs. covered by scales). Tibouchina longisquamata is a species endemic to the state of Tocantins and it can be readily recognized by the oval, flat, patent leaves and hypanthium with long scales. It shares with Tibouchina llanorum the oval, slightly discolorous leaves, with a conspicuous petiole, and hypanthium covered with long scales. Tibouchina longisquamata differs from T. llanorum by its subcylindrical branches (vs. 4-6 angulose) and leaves with the adaxial surface covered by trichomes (vs. covered by scales). According to our assessments, based on IUCN criteria, T. falcifolia must be considered as endangered (EN) because its occurrence is restricted, and its populations are small; T. longisquamata is indicated in Deficient Data (DD), with only two known collections. We present here complete descriptions of the new species, taxonomic comments, illustrations made from dehydrated material, photos of live specimens (for T. falcifolia), plates with microscopic details, a distribution map, and an updated list of species of the genus in the Brazilian Cerrado, with its occurrences by state.

Russell’s Viper Bite and Its Immediate Outcome In Bangladesh

Background: Russell’s viper (Daboia russelii) is found in Asia, throughout Indian subcontinent, much of South East Asia, Southern China and Taiwan. Russell’s viper was seemed to be rare in Bangladesh. Anecdotes suggest that Russell’s viper (Daboia russelii) was an important cause of mortality in the 1920s, but no case of envenoming by this species has since been reported in Bangladesh. Russell’s viper bite has been reported and it is the one of the common snake bite at Rajshahi region which has great morbidity and mortality as well. Methods: This is a prospective observational study was conducted in the Medicine department of Rajshahi Medical College Hospital. We treated total 171 patients from 2013 to December 2022. Russell’s viper envenomation confirmed either by brought dead or live snake specimen, photograph, bedside 20MWBCT and other symptoms. Results: In the management of RV bite patient in the Rajshahi Medical College Hospital, we found that most of the patients are delayed in getting hospitalization after bite due to visit to traditional healer. In our study 96% were male, and 4% were female, mostly farmer and 90% bite site in the lower limb during work in the paddy field. The clinical presentation was pain and local swelling (100%), blood oozing from local site (77%), bruising (44%), haematuria (25%), hypotension (30%), oliguria and few with DIC. In lab findings, coagulopathy (24%), raised CPK (60%), 67% Patients had AKI (raised creatinine and RBC in urine), among them 50% needed dialysis. Few patients also develop multiorgan failure. All patients received polyvalent Anti-venomsupplied by GOB (Incepta Bangladesh Ltd.) which works against cobra, krait, Russell’s viper bite. Despite maximum available support at RMCH, around 30% patient died. Conclusion: It’s high mortality indicates that this polyvalent antivenom might not be working properly. We may need to increase the initial dose or we need to provide monovalent antivenom specific to RV in Bangladesh and early treatment at Upazila Health Complex to reduce the rate of death due to Russell’s viper bite. Bangladesh J Medicine 2023; Vol. 34, No. 2(1) Supplement: 204

Unique Morphologic Findings in the Liver After Stereotactic Radiation for Cholangiocarcinoma.

Newer radiotherapy techniques, such as stereotactic body radiation, have been increasingly used as part of the treatment of cholangiocarcinomas, particularly as a bridge to liver transplantation. Although conformal, these high-dose therapies result in tissue injury in the peritumoral liver tissue. This retrospective study characterized the morphologic changes in the liver after stereotactic body radiation in a series of liver explant specimens with perihilar cholangiocarcinoma. The morphologic changes in the irradiated zone were compared against the nonirradiated background liver parenchyma to control for chemotherapy-related changes. Of the 21 cases studied, 16 patients (76.2%) had underlying primary sclerosing cholangitis, and 13 patients (61.9%) had advanced liver fibrosis. The average duration between completion of radiotherapy and liver transplantation was 33.4 weeks (range: 6.29 to 67.7). Twelve patients (57.1%) had no residual tumor in the liver. The most frequent histologic changes in the peritumoral irradiated liver tissue were sinusoidal congestion (100%), sinusoidal edematous stroma (100%), and hepatocellular atrophy (100%), followed by partial/complete occlusion of central veins (76.2%), sinusoidal cellular infiltrates (76.2%), and hepatocyte dropout (66.7%). The findings in the radiated areas were more extensive than in the background liver ( P <0.01). Sinusoidal edematous stroma was striking and dominated the histologic findings in some cases. Over time, there was less sinusoidal congestion but more hepatocyte dropout (r s =-0.54, P =0.012 and r s =0.64, P =0.002, respectively). Uncommon findings, such as foam cell arteriopathy in the liver hilum, were also observed. In summary, postradiation liver specimens have distinctive morphologic findings.

Steatosis and Steatohepatitis in Autopsy Specimens of Liver

Background: Liver is the site of many diseases, many of which become symptomatic while some are diagnosed only on autopsy. Involvement of liver is secondary to cardiac, metabolic, social problem like alcoholism, drug toxicity, herbal remedies and environmental exposures. Objectives: This study aims at finding the frequency of steatosis and steatohepatitis in autopsy specimens of liver. Methods: This was a descriptive cross-sectional study carried out at the Department of Pathology, Dhaka Medical College, Dhaka over a period of 2011-2012. Liver samples of postmortem cases from the Department of Forensic Medicine, Dhaka Medical College and autopsy samples of liver received at the Department of Pathology, Dhaka Medical College, sent from different parts of Bangladesh via postal service for histopathological examination were included in this study. Result: Satisfactory liver tissue samples for histological evaluation were available in 200 autopsy specimens (155 male and 45 female) with a mean age of 39.50±14.76 years. Out of 200 cases, the most common lesion was steatosis in 35(17.50%) cases. Steatosis mild, moderate and severe were found in 20(10%) cases, 9(4.50%) cases and 6(3.0%) cases respectively. Steatohepatitis was found in 16(8.0%) cases. Steatohepatitis with fibrous portal expansion, steatohepatitis with bridging fibrosis and steatohepatitis with cirrhosis were detected in 7(3.5%), 6(3%) and 3(1.5%) cases respectively. Conclusion: A high prevalence of steatosis and steatohepatitis was detected in postmortem liver samples of Bangladesh. Since both diseases can have serious clinical consequences, they should be considered as an important threat to the health of the general population of our country.

NIMA-related kinase 6 as an effective target inhibits the hepatocarcinogenesis and progression of hepatocellular carcinoma

BackgroundNIMA-related kinase 6 (NEK 6) is over-expressed in some tumor cell lines and tissues. However, its expression in hepatocellular carcinoma (HCC) and its correlation with clinical features remain unclear. MethodsTotal RNA from HCC liver tissues, other liver specimens, and hepatic cell lines was extracted and QPCR was adopted to detect NEK6 expression. The correlation between NEK6 expression and the clinical characteristics of HCC was analyzed. Scratch assay, Transwell assay, and tumor-formation assay were used to evaluate the effects of NEK6 on the HCC progression in vitro and in vivo. ResultsThe expression of NEK6 was up-regulated in HCC tissues and HCC cell lines: Li-7 and HepG2. The overexpression of NEK6 was correlated with hepatitis B virus infection and tumor diameter (P = 0.045). When down-regulated the expression of NEK6, both the migration and invasion capabilities of Li-7 and HepG2 cells and the growth of xenograft tumors were suppressed. (P < 0.05). ConclusionsNEK6 expression was up-regulated in HCC and correlated with the progression, suggesting it might be a valuable biomarker and a potential therapeutic target for HCC.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

A checklist of the Scarabaeoidea (Coleoptera) of Chile with exemplar live-photographed.

An updated list of the Scarabaeoidea (Coleoptera) species registered in Chile is given, which includes current names, synonyms, and photographs of live specimens of 88 species. The photographs cover 35% of the species present in the country, with all families represented except for Glaresidae and Ochodaeidae. For each species, the geographical distribution is presented from north to south by administrative regions of the country. For species shared with neighboring countries, the distribution by provinces or departments is indicated, while for those that have a worldwide distribution, the countries, or regions where they have been reported are cited. This list includes species whose presence in continental and insular Chile has been verified and confirmed by studying specimens from collections, excluding all records considered erroneous. The systematic ordering is carried out at the family, subfamily, and tribe level following the current classification of the group; genera and species are presented in alphabetical order. Paranimbus peruanus (Erichson) (Aphodiinae) is recorded for the first time in Chile based on specimens collected in the locality of Visviri (Arica and Parinacota Region). The known distribution is extended for the following species: Allidiostoma landbecki (Philippi), A. simplicifrons (Fairmaire), Archophileurus chaconus Kolbe, Brachysternus germaini (Ohaus), Chiasognathus beneshi Lacroix, Cyclocephala modesta Burmeister, Frickius costulatus Germain, Martinezostes posticus (Germar), Martinezostes ruizi Gutiérrez, Megathopa villosa Eschscholtz, Oogenius chilensis Ohaus, O. kuscheli Gutiérrez, Oryctomorphus maculicollis Guérin-Méneville, Paranimbus zoai Dellacasa, Paranomala undulata peruviana (Guérin-Méneville), Phytholaema fenestra Smith & Mondaca, Tesserodoniella meridionalis Vaz-de-Mello & Halffter, Macrodactylus farinosus Philippi, and Ulata argentina Saylor. Considering the great morphological variability and differences in the color pattern of the integument, Chiasognathus grantii holometallicus Rataj, 2012 and Chiasognathus megalodon Rataj, 2013 are considered new synonyms of Chiasognathus grantii Stephens, 1831. The study of the syntype of Copris punctatissima Curtis, 1844 confirms that this species belongs to the genus Homocopris Burmeister, 1846 and that is not conspecific with H. torulosus (Eschscholtz, 1822), therefore, the combination Homocopris punctatissimus (Curtis, 1844) new combination is established. The study of the syntype of Copris valdiviana Philippi, 1859 confirms that this species is a synonymy of Homocopris torulosus and not of H. puntatissimus. The morphological study of specimens of the genus Germarostes Paulian, 1982 and the comparison with Chilean specimens of Germarostes posticus (Germar, 1843), confirms that this last species really belongs to the genus Martinezostes Paulian, 1982, therefore, the combination Martinezostes posticus (Curtis, 1843) new combination is established. Based on the inconsistencies that exist with the current definition of the subfamily Melolonthinae, and on the results of a previous phylogenetic analysis, Lichniini is elevated to the category of subfamily, placing Lichniinae new status very close to Aclopinae. There are 79 genera, 251 species and 10 subspecies of Scarabaeoidea in Chile, belonging to the families Geotrupidae (three genera, 10 species), Glaresidae (one genus, one species), Hybosoridae (one genus, six species), Trogidae (one genus, 11 species), Lucanidae (nine genera, 31 species, nine subspecies), Ochodaeidae (one genus, one species), and Scarabaeidae (63 genera, 191 species, one subspecies). In addition, Onthophagus pilauco Tello, Verdú, Rossini, & Zunino, 2021, the first fossil scarab from Chile, is included. Approximately 58% of the listed species are endemic to the country, 39% are native to South America and 3% correspond to introduced species with a wide global distribution.

DART-HRMS allows the detection of toxic alkaloids in animal autopsy specimens and guides the selection of confirmatory methods in accidental plant poisoning

BackgroundIn cases of suspected animal poisonings or intoxications, there is the need for high-throughput, rapid and accurate analytical tools capable of giving rapid answers and, thus, speeding up the early stages of investigations. Conventional analyses are very precise, but do not meet the need for rapid answers capable of orienting the decisions and the choice of appropriate countermeasures. In this context, the use of ambient mass spectrometry (AMS) screening methods in toxicology laboratories could satisfy the requests of forensic toxicology veterinarians in a timely manner. ResultsAs a proof of principle, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was applied to a veterinary forensic case in which 12 of a group of 27 sheep and goats died with an acute neurological onset. Because of evidence in the rumen contents, the veterinarians hypothesized an accidental intoxication after ingestion of vegetable materials.The DART-HRMS results showed abundant signals of the alkaloids calycanthine, folicanthidine and calycanthidine, both in the rumen content and at the liver level. The DART-HRMS phytochemical fingerprinting of detached Chimonanthus praecox seeds was also compared with those acquired from the autopsy specimens. Liver, rumen content and seed extracts were then subjected to LC-HRMS/MS analysis to gather additional insights and confirm the putative assignment of calycanthine anticipated by DART-HRMS.HPLC-HRMS/MS confirmed the presence of calycanthine in both rumen contents and liver specimens and allowed its quantification, ranging from 21.3 to 46.9 mg kg−1 in the latter. This is the first report detailing the quantification of calycanthine in liver after a deadly intoxication event. Significance and noveltyOur study illustrates the potential of DART-HRMS to offer a rapid and complementary alternative to guide the selection of confirmatory chromatography-MSn strategies in the analysis of autopsy specimens from animals with suspected alkaloid intoxication. This method offers the consequent saving of time and resources over those needed for other methods.

Abstract 674: Transgelin Deficiency Is Associated With Elevated Low-density Lipoprotein Cholesterol And Atherogenesis In Mice.

Genetic hyperlipidemias are a major risk factor for atherosclerotic cardiovascular disease due to life-long exposure to elevated plasma lipids. However, the causality of hyperlipidemia is not known in a large proportion of patients, suggesting that unknown genes remain to be discovered. Applying a CRISPR/Cas9 screen, we recently identified transgelin (TAGLN), an actin binding protein, as a new gene involved in LDL metabolism. TAGLN supports LDL endocytosis presumably by interacting with actin filaments during LDL endocytosis; however, if TAGLN impacts on plasma lipid levels and atherogenesis in vivo is unknown. Aim: To investigate TAGLN’s impact on lipid profile and atherosclerosis development in a Tagln -knockout (KO) mouse model. Methods: Tagln -KO (n=18) and wild-type (WT) (n=18) mice were fed a high-fat diet (42% Cal from fat) for 12 weeks. We collected plasma for lipoprotein analysis, liver specimens for histopathology and lipid extraction/quantification, and aortas for quantifying atherosclerotic plaque area. Results: Tagln -KO mice showed higher total cholesterol than WT mice (20% increase, mean±SD: 299±51 vs. 251±59 mg/dL, p=0.014), without changes in triglyceride levels (p=0.36). Lipoprotein subfraction analysis by FPLC revealed an increase in the lipids associated with the LDL fraction of Tagln -KO mice. Although no differences were observed in hepatic histology or triglyceride content, Tagln -KO animals showed an increase in the hepatic content of total cholesterol (mean±SD: 376±65 vs. 317±70 μg/100 mg, p=0.012). Finally, Tagln -KO mice developed about twice more atherosclerotic lesion area than WT mice (Median(IQR): 0.997(0.347-1.451) vs 0.428(0.336-0.859), p=0.015). Conclusions: In line with a reduction in LDL uptake in hepatocytes, shown by our previous in vitro data, TAGLN deficiency was associated with hypercholesterolemia and high LDL cholesterol in mice, likely due to decreased hepatic LDL clearance. Despite the modest hypercholesterolemia, Tagln -KO mice showed disproportionally increased atherosclerosis, suggesting that TAGLN may regulate other atherogenic mechanisms, beyond LDL cholesterol.

Foraminifera associated with cold seeps in marine sediments

Cold seep foraminifera have attracted considerable attention as they provide valuable insights into the study of cold seeps. This study provides a comprehensive overview of the manifestations of foraminifera in cold seep environments and methane seepage activities. Certain taxa of benthic foraminifera, such as Uvigerina, Bolivina, Bulimina, Chilostomella, Globobulimina, Nonionella, Melonis, Epistominella, Cibicidoides, and Globocassidulina, are known to inhabit geochemical conditions induced by methane-rich environments and may feed on associated methanotrophic microbial communities. Secondary mineralization on foraminifera shells is a widespread manifestation in seep sediments, and alters the microstructure, elementary composition, and isotopic signatures of foraminifera. On one hand, the precipitation of secondary authigenic Mg-rich, Mn-rich, Sr-rich, and Ba-rich calcite coatings have been observed on microfossils. On the other hand, micron-sized crystal pyrite and gypsum aggregates can also grow on the foraminifera walls. The negative δ13C and positive δ18O anomalies in both planktonic and benthic foraminifera from seep-associated sediments can serve as proxies for tracing past seepage activities, either in their live form or being adulterated by methane-derived authigenic carbonate after deposition. Seeping activities are recognized with a significant impact on benthic foraminifera, and the presence of cold seep-related species and significant isotopic anomalies in shells can be used to reconstruct past methane seepage events. Intensive methane seepage tends to suppress benthic foraminifera populations, while moderate intensity seepage may lead to a thriving benthic foraminiferal community, with hypoxia-enduring taxa such as Uvigerina, Melonis, and Bulimina being predominant. In contrast, oxygen-loving epibenthic taxa such as Cibicidoides often occur in areas of low methane flux. Compared to planktonic foraminifera, the single species of benthic foraminifera can provide a more comprehensive record of seepage evolution. Live benthic specimens are preferred for in-situ seep studies, while the superimposition of secondary minerals on the original shells should not be ignored when observing dead individuals. The significance of the evolution of methane seepage, changes in environmental parameters of the living habitat, and species sensitivity in cold seeps are emphasized in explaining the variation in foraminiferal assemblages and fluctuations in stable isotopes.

The genus Pupa Röding, 1798 (Mollusca, Gastropoda, Acteonidae) in New Caledonia with notes on Recent species.

The study of newly collected, live specimens of Pupa (Acteonidae) from New Caledonia and French Polynesia, revealed the presence of six distinct species in this region, including an undescribed species. All these species are re-described and/or named using molecular and morphological evidence as well as a review of the literature and examination of photographs of the type material of described species. Moreover, a review of the literature and type material suggests the existence of six additional species from other geographic regions across the Indo-Pacific. Illustrations of all these species are provided as well as remarks on possible synonymies. This study is the first species-level review of this group using modern taxonomic techniques.

“Spice”-related deaths in and around Munich, Germany: A retrospective look at the role of synthetic cannabinoid receptor agonists in our post-mortem cases over a seven-year period (2014–2020)

Synthetic cannabinoid receptor agonists (SCRAs, “Spice”) are a diverse group of recreational drugs, with their structural and pharmacological variability still evolving. Forensic toxicologists often rely on previous reports to assess their role in intoxication cases. This work provides detailed information on the “Spice”-related fatalities around Munich, Germany, from 2014 to 2020. All cases underwent an autopsy. Pharmaceutical and illicit drugs were detected and quantified in post-mortem peripheral blood or liver by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Based on circumstantial evidence, only those cases for which a prior consumption was suspected underwent additional analyses for SCRAs and other new psychoactive substances in post-mortem blood, liver or antemortem specimens. Drug concentrations, pathological findings at autopsy and case histories were considered to assess and rank the SCRAs’ involvement in each death. Concentration ranges for the individual substances in blood were defined and their distribution patterns over the investigated period were determined and correlated with their legal status and local police seizures. We identified 41 different SCRAs among 98 fatalities. 91.8% were male, at a median age of 36 years. SCRAs played a causative role in 51%, contributory role in 26%, and an insignificant role in 23% of cases. In correlation with local police seizures and legal status, 5F-ADB was the most prevalent in our cases, followed by 5F-MDMB-PICA and AB-CHMINACA. Cumyl-CBMICA and 5F-MDMB-P7AICA were among the least frequently detected SCRAs. “Spice”-related fatalities and SCRAs’ causative role have significantly decreased among our cases since the German New Psychoactive Substances Act.

Vibratome technique revealed initial carcinogenic changes that induce GST-P+ single hepatocytes and minifoci in rat liver

The drastic initial carcinogenic changes that induce single hepatocytes and minifoci positive for GST-P (a specific biomarker of foci and nodules) identified previously in rat livers (K. Satoh, Life Sci. 2018) require elucidation. Notably, after animals were administered benzyl isothiocyanate (BITC, anti-cancer phytochemical, 0.5% by wt) in their basal diet, immunocytochemical staining of vibratome-prepared liver specimens for GST-P revealed that the canalicular networks and bile ducts of the animal livers were heavily and finely stained for GST-P even though the biomarker is a cytosolic enzyme. In addition, the mean diameter of the canaliculi was greatly enlarged. The results thus indicate that GST-P was rapidly synthesized in all hepatocytes but rapidly excreted into bile. Similar results were obtained with animals administered dietary AAF carcinogen (0.04%). The biliary excretion of GST-P was detectable not only in all hepatocytes but also within minifoci, foci and nodules. A new initiation model was therefore proposed assuming that GST-P+ single hepatocytes are formed after injury to canaliculi by carcinogens to decrease the excretion of GST-P from hepatocytes. The key findings from this study and the biomarker analysis using a vibratome technique might help elucidate the ‘unknowable’ mechanism of cancer initiation in rat chemical carcinogenesis.

Point for Enrichment, Point for Welfare-Testing Use of a Laser Pointer with Arapaima gigas.

The arapaima (Arapaima gigas) is one of the largest freshwater fish species, known to exceed 3 m in total length. It is listed as Data Deficient by the IUCN. A. gigas is native to the Amazon River basin where they are an important food source. Arapaimas are also farmed for meat and for live specimens in various South American and Asian countries. Despite decades of keeping the species in public aquariums, little is known of its behaviour and cognitive abilities. This pilot study provides baseline data on using a green laser pointer as environmental enrichment for this species under human care. The data collection included 18 observations before the use of the laser pointer (baseline) and 18 observations during the use of the laser pointer (test). Ten behaviours were monitored, investigating physical contact, activity pattern and habitat use by the fish. During the test, the fish significantly increased their presence in the tank, their level of activity and their use of the habitat. This pilot study provides valuable baseline data for further investigations demonstrating the value of a laser pointer as environmental enrichment for A. gigas under human care.

Rapid Detection and Identification of Fasciola spp. and Dicrocoelium spp. Isolated from the Ruminant Livestock of Northwest Iran Using High-Resolution Melting Analysis (HRM).

The liver flukes of the Fasciola species and Dicrocoelium spp. are recognised as parasites of domestic and wild herbivores. Both species of F. hepatica and F. gigantica as well as D. dendriticum are distributed in Iran. The present study aimed to identify Fasciola spp. and Dicrocoelium spp. using mitochondrial Cox1 (cytochrome c oxidase I) gene by HRM method. Totally, thirty infected liver specimens were collected from the sheep (n:23) and cattle (n:7) at the abattoirs of Qazvin Province, northwest Iran in 2022. DNA extraction and PCR amplification of Cox1 gene were conducted by HRM technique. DnaSP v.5.0 was used for compression of diversity indices of ribosomal 28S rDNA and mitochondrial Cox1 markers of Dicrocoelium spp. The taxonomic status of Dicrocoelium spp. was performed by sequencing and phylogenetic analysis. Overall, 26 and 4 isolates were identified as F. hepatica and F. gigantica, respectively. D. dendriticum was the sole infecting species of Dicrocoelium revealed by HRM analysis. Genomic analysis showed a moderate (28S rDNA genes: 0.600±0.215) to high (Cox1: 0.733±0.155) haplotype diversity for D. dendriticum. The parasite-dependent mitochondrial gene (Cox1) could identify a higher genetic diversity of D. dendriticum compared to nuclear 28S rDNA gene. HRM technique in the present study found to be a reliable technique for identification and genetic diversity of liver flukes but more comprehensive and in-depth studies in different parts of the country are needed.