Comparison of different in situ hybridization techniques for the detection of various RNA and DNA viruses

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probe

Status, sources and contamination levels of organochlorine pesticide residues in urban and agricultural areas: a preliminary review in central–southern Italian soils

Organochlorine pesticides (OCPs) are synthetic chemicals commonly used in agricultural activities to kill pests and are persistent organic pollutants (POPs). They can be detected in different environmental media, but soil is considered an important reservoir due to its retention capacity. Many different types of OCPs exist, which can have different origins and pathways in the environment. It is therefore important to study their distribution and behaviour in the environment, starting to build a picture of the potential human health risk in different contexts. This study aimed at investigating the regional distribution, possible sources and contamination levels of 24 OCP compounds in urban and rural soils from central and southern Italy. One hundred and forty-eight topsoil samples (0–20 cm top layer) from 78 urban and 70 rural areas in 11 administrative regions were collected and analysed by gas chromatography–electron capture detector (GC–ECD). Total OCP residues in soils ranged from nd (no detected) to 1043 ng/g with a mean of 29.91 ng/g and from nd to 1914 ng/g with a mean of 60.16 ng/g in urban and rural area, respectively. Endosulfan was the prevailing OCP in urban areas, followed by DDTs, Drins, Methoxychlor, HCHs, Chlordane-related compounds and HCB. In rural areas, the order of concentrations was Drins > DDTs > Methoxychlor > Endosulfans > HCHs > Chlordanes > HCB. Diagnostic ratios and robust multivariate analyses revealed that DDT in soils could be related to historical application, whilst (illegal) use of technical DDT or dicofol may still occur in some urban areas. HCH residues could be related to both historical use and recent application, whilst there was evidence that modest (yet significant) application of commercial technical HCH may still be happening in urban areas. Drins and Chlordane compounds appeared to be mostly related to historical application, whilst Endosulfan presented a complex mix of results, indicating mainly historical origin in rural areas as well as potential recent applications on urban areas. Contamination levels were quantified by Soil Quality Index (SoQI), identifying high levels in rural areas of Campania and Apulia, possibly due to the intensive nature of some agricultural practices in those regions (e.g., vineyards and olive plantations). The results from this study (which is in progress in the remaining regions of Italy) will provide an invaluable baseline for OCP distribution in Italy and a powerful argument for follow-up studies in contaminated areas. It is also hoped that similar studies will eventually constitute enough evidence to push towards an institutional response for more adequate regulation as well as a full ratification of the Stockholm Convention

MicroRNAs modulate SARS-CoV-2 infection of primary human hepatocytes by regulating the entry factors ACE2 and TMPRSS2.

BACKGROUND AND AIMS Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection. METHODS We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed. RESULTS We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome. CONCLUSION We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19

Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication

Funding Information: We thank Thorsten Wolff, Daniel Bourquain, Jessica Schulz, and Christian Mache from the Robert-Koch Institute and Martin Beer from the Friedrich Loeffler Institute (FLI) for providing isolates of SARS-CoV-2 variants. We thank Anna Kraft and Gabriele Czerwinski (both FLI) for support in the preparation of samples for pathology, and Catherine Hambly (University of Aberdeen) for help with daily energy expenditure measurements. We would like to thank Cathrin Bierwirth (University Medical Center Göttingen), Isabell Schulz, Anne-Kathrin Donner, and Frank-Thorben Peters for excellent technician assistance and Jasmin Fertey and Alexandra Rockstroh for providing the virus stocks for the mice experiment (Fraunhofer Institute IZI Leipzig). We acknowledge support by the Open Access Publication Funds of the Göttingen University. KMS was a member of the Göttingen Graduate School GGNB during this work. This work was funded by the COVID-19 Forschungsnetzwerk Niedersachsen (COFONI) to MD, by the Federal Ministry of Education and Research Germany ( Bundesministerium für Bildung und Forschung; BMBF ; OrganSARS , 01KI2058 ) to SP and TM, and by a grant of the Max Planck Foundation to DG. Declaration of interests AS, HK, EP, and DV are employees of Immunic AG and own shares and/or stock-options of the parent company of Immunic AG, Immunic Inc. Some of the Immunic AG employees also hold patents for the Immunic compounds described in this manuscript (WO2012/001,148, WO03006425). KMS, AD, and MD are employees of University Medical Center Göttingen, which has signed a License Agreement with Immunic AG covering the combination of DHODH inhibitors and nucleoside analogs to treat viral infections, including COVID-19 (inventors: MD, KMS, and AD). The other authors declare no conflict of interest.Peer reviewedPublisher PD

Vaccine-associated enhanced respiratory pathology in COVID-19 hamsters after T(H)2-biased immunization

Vaccine-associated enhanced respiratory disease (VAERD) is a severe complication for some respiratory infections. To investigate the potential for VAERD induction in coronavirus disease 2019 (COVID-19), we evaluate two vaccine leads utilizing a severe hamster infection model: a T helper type 1 (T(H)1)-biased measles vaccine-derived candidate and a T(H)2-biased alum-adjuvanted, non-stabilized spike protein. The measles virus (MeV)-derived vaccine protects the animals, but the protein lead induces VAERD, which can be alleviated by dexamethasone treatment. Bulk transcriptomic analysis reveals that our protein vaccine prepares enhanced host gene dysregulation in the lung, exclusively up-regulating mRNAs encoding the eosinophil attractant CCL-11, T(H)2-driving interleukin (IL)-19, or T(H)2 cytokines IL-4, IL-5, and IL-13. Single-cell RNA sequencing (scRNA-seq) identifies lung macrophages or lymphoid cells as sources, respectively. Our findings imply that VAERD is caused by the concerted action of hyperstimulated macrophages and T(H)2 cytokine-secreting lymphoid cells and potentially links VAERD to antibody-dependent enhancement (ADE). In summary, we identify the cytokine drivers and cellular contributors that mediate VAERD after T(H)2-biased vaccination

Intraoperative effects and post operative recovery quality after racemic ketamine or S (+) ketamine administered to male dogs undergoing elective neutering surgery

Anesthesia with S (+) ketamine results in better recoveries in humans, cats and horses than with racemic ketamine at dose rates of only 50-66 % of the racemic ketamine. This prospective, blinded, randomized trial compared anesthesia induction with intravenous S (+) ketamine (2mg/Kg, n= 20) and racemic ketamine (4 mg/Kg, n=20) in 40 dogs undergoing castration. Sedation was performed with intramuscular medetomidine and butorphanol in both groups. For analgesia lidocaine was applied locally. Anaesthesia was monitored as under clinical circumstances. Sixty minutes after S (+)- or racemic ketamine atipamezole was administered and recovery quality assessed with standardized scores 0, 10, 20, 30, and 60 minutes thereafter. During the intraoperative phase, three dogs in the S (+) ketamine group needed an extra bolus of intravenous S (+) ketamine to maintain adequate anaesthesia depth. Another two dogs, one in each group, showed spasms immediately post-operatively which needed treatment and were excluded from comparison. Respiration was better maintained with S (+) ketamine. Recovery was not different between the groups. Half dose of S (+) ketamine provided similar anaesthesia quality, cardiopulmonary function and recovery as racemic ketamine. Only dogs with S (+)-ketamine needed redosing to finish the surgery. This might suggest that duration of action of S (+) ketamine is slightly less than that of racemic ketamine, as has been suggested in other species. S (+) Ketamin führt in der Anästhesie von Menschen, Katzen und Pferden bei Dosierungen von nur 50-66% des razemischen Ketamins, zu einer besseren Aufwachphase. Diese prospektive, blinde, randomisierte Studie vergleicht die Narkosewirkung von IV S (+) Ketamin (2mg/kg, n = 20) gegenüber das IV razemische Ketamin (4 mg/kg, n = 20) bei 40 Hünden, bei denen eine elektive Kastration durchgeführt wurde. Die Sedierung wurde in beiden Gruppen mit IM Medetomidin- und Butorphanolgabe durchgeführt. Zusätzlich wurde lokal Lidocain verabreicht. Während der Anästhesie- Ueberwachung wurde sechzig Minuten nach S (+) Ketamin- oder razemischer Ketamingabe, Atipamezol verabreicht und danach die Aufwachphase zu den Zeiten 0, 10, 20, 30 und 60 Minuten beobachtet. Während der intraoperativen Phase brauchten drei Hünde aus der S (+) Ketamin- Gruppe, einen zusätzlichen IV Bolus S (+) Ketamin, um eine adäquate Anästhesie- Tiefe zu gewährleisten. Zwei weitere Hunde, je aus einer Ketamin-Gruppe, zeigten nach Operationsende Krämpfe, die eine Nachbehandlung benötigten und wurden aus der Studie ausgeschlossen. Die Respiration war besser mit S (+) Ketamin. Zwischen den zwei Gruppen zeigte sich in der Aufwachphase aber kein Unterschied. Die halbe Dosis von S (+) Ketamin hat eine ähnliche Anästhesie Qualität, Auswirkung auf Herz-Lungen-Funktion und Aufwachphase als das razemische Ketamin. Nur Hunde anästhesiert mit S (+) Ketamin benötigten eine Nachdosierung um die Operation zu beenden. Dies könnte darauf hindeuten, dass die Dauer der Wirkung von S (+) Ketamin etwas geringer als die von razemischem Ketamin, wie bei anderen Spezies vorgeschlagen wurde

Hemodynamics in the Extracorporeal Aortic Cannula: Review of Factors Affecting Choice of the Appropriate Size

To minimize hemolysis rate and damage to blood due to characteristics of aortic cannulae, critical blood velocity, blood cannula interactions and shear stress ranges for extracorporeal circulation (ECC) must be determined. At blood flow rates and viscosity levels typically experienced during ECC mean blood velocity, critical blood velocity, shear stress, and kinetic energy are determined in several aortic cannulae, 3-8 mm. Twenty-five percent glycerol is the priming solution. In ten Fr and twenty-four Fr aortic cannulae at 25°C, the velocity above which flow becomes turbulent occurs at flow rates of 975 ccjmin and 2750 ccjmin respectively. Turbulence within these cannulae occurs at flow rates above 650 ccjmin and 2000 ccjmin at 37°C. The recommended flow rate for the cannula sizes tested, determined by the average flow rate at each limit studied, resulted in maximum recommended flows of 883, 1283,2134,2899,3840, and 4954 ccjmin for cannulae sizes 3-8 mm respectively. Blood velocity and shear stress at typical ECC flow rates caused significant turbulence and hemolysis, which must be minimized by the choice of cannulae of the correct size