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The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled "Dosimetry for conducting in vitro evaluations" and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research., (© 2022 JT International SA and The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Non-clinical in vitro studies were conducted to investigate the characteristics of extracts from tobacco free nicotine pouches alongside a reference snus product and/or 1R6F reference cigarette. In vitro investigations were conducted in the Neutral Red Uptake (NRU) cytotoxicity assay, Bacterial Reverse Mutation (Ames) assay, and in vitro Mammalian Cell Micronucleus ( iv MN) assay. These products were also investigated for their oral irritation potential in the EpiGingival™ 3D tissue model. Results from the Ames, in vitro Micronucleus and NRU assays indicated that the tested products were non-mutagenic, non-genotoxic and non-cytotoxic in contrast to results obtained for the 1R6F reference cigarette. Results from Complete Artificial Saliva (CAS) extracts from these products also failed to be classified as irritants (as measured using the MTT assay), in the EpiGingival™ 3D tissue model., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JT International SA funded the project, and all authors were employees of Japan Tobacco Group at the time of the study. Nordic spirit tobacco-free nicotine pouches are manufactured and supplied by JT International., (© 2022 The Authors.)

Phosphoinositide-3-kinase, class III (PIK3C3) is a member of the phosphoinosite-3-kinases family, involved in cell signaling, membrane trafficking, and neurodevelopment. Previous studies have indeed shown an association between PIK3C3 gene variants and both bipolar disorder (BD) and schizophrenia (SZ). Brain-derived neurotrophic factor (BDNF) is a neurodevelopmental factor, which can regulate the PI3K signaling pathway. Associations have been reported between BDNF gene polymorphisms and affective and psychotic disorders. The aim of the present study was to replicate an association between PIK3C3 and BDNF gene variants in SZ and BD and a putative epistasis between the two genes. Patients meeting the DSM-IV criteria of BD and SZ were included in this study (98 BD and 79 SZ) as well as 158 healthy controls. Blood DNA was extracted and genotyping was performed either by the polymerase chain reaction (PCR) technique followed by enzymatic digestion or by the high-resolution melt (HRM) method. Genotype and haplotype association was assessed with the UNPHASED statistical program.The results showed one nominal association with BD (P < 0.02) and two risk haplotypes in both SZ (P < 0.001) and BP (P < 0.0005), which survived multiple testing correction. A modest interaction between a BDNF variant and PI3KC3 polymorphism was observed (P < 0.04).These preliminary results confirm the genetic association of PI3K gene variants with both SZ and BD, and support the hypothesis that SZ and BD share a genetic background.

Several wine isolates of Saccharomyces were analysed for six molecular markers, five nuclear and one mitochondrial, and new natural interspecific hybrids were identified. The molecular characterization of these Saccharomyces hybrids was performed based on the restriction analysis of five nuclear genes (CAT8, CYR1, GSY1, MET6 and OPY1, located in different chromosomes), the ribosomal region encompassing the 5.8S rRNA gene and the two internal transcribed spacers, and sequence analysis of the mitochondrial gene COX2. This method allowed us to identify and characterize new hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii, between S. cerevisiae and Saccharomyces bayanus, as well as a triple hybrid S. bayanusxS. cerevisiaexS. kudriavzevii. This is the first time that S. cerevisiaexS. kudriavzevii hybrids have been described which have been involved in wine fermentation.

This research note addresses the development of an automated molecular typing system for yeast. Specifically, our objectives were to generate specific probes for genotyping yeast with an automated fingerprinting system. We have adapted the RiboPrinter microbial characterization system for use with Saccharomyces cerevisiae yeast using alternative probes based on specific multi-copy gene families. Manual construction and labeling of probes proved successful in initial experiments. Results indicate that this method could be applied to food or clinical environments if the appropriate probes are developed.

Classical microbiological methods for determining antimicrobial compounds in feeds are nonspecific. Thus, there is a need to identify biological activity, and bioautography is used for this purpose. A routine method for detecting the following antimicrobial substances in feeds is described: avilamycin, avoparcin, Zn-bacitracin, erythromycin, flavomycin, furazolidone, lasalocid, monensin, narasin, penicillin, salinomycin, spiramycin, tetracyclines, tylosin, and virginiamycin. Carbadox can be detected by UV light examination of the plates prior to bioautography. Semiquantitative estimations of antibiotic content are compared with quantitative determinations of the above mentioned substances in feeds, except erythromycin, penicillin, and tetracyclines. Detection limits range from 0.1 mg/kg (chlortetracycline) to 20 mg/kg (lasalocid). The method involves agar diffusion of buffered samples, a neutral extraction of polyether antibiotics followed by thin-layer chromatography (TLC), and an acid extraction for other antibiotics followed by TLC. Five test bacteria were used for the main detection by agar diffusion: Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 6538P, Corynebacterium xerosis NCTC 9755, Bacillus cereus ATCC 11778, and B. subtilis ATCC 6633. Identification after TLC was achieved by bioautography with the most sensitive microorganism(s). This method allows one laboratory technician to analyze up to 30 feed samples within 2.5 working days, provided that feeds of the same category are analyzed in the same run, and that labels of additives are available. Qualitative and semiquantitative information are valuable when performing a quantitative antibiotic determination and it provides proof that the activity determined is due to the tested substance. This last feature is essential from the perspective of quality assurance of results.

The cellular Ha-ras oncogene, activated by missense mutations, has been implicated in intrinsic resistance to ionizing radiation. This study shows that the overexpression of the unmutated gene (proto-oncogene) may also be involved in how the cells respond to radiation. The experimental system consisted of mouse NIH 3T3-derived cell lines which carry multiple copies of a transcriptionally activated human c-Ha-ras proto-oncogene. Both tumorigenic (RS485) and revertant nontumorigenic subclones (PR4 and 4C3) which have high levels of ras expression exhibited a marked increase in radioresistance as measured by D0 compared to control NIH 3T3 cells. Other nontransformed cells with elevated levels of ras (phenotypically revertant line 4C8-A10) also had a significantly increased resistance to radiation, further indicating an association between ras and radioresistance. The increased radioresistance of the RS485 and phenotypic revertants could not be explained by a differential expression of the myc or metallothionein I genes or by variations in cell cycle. The correlation between increased ras proto-oncogene expression and radioresistance suggests that the ras encoded p21, a plasma membrane protein, may participate in the cellular responses to ionizing radiation.

Two so far undetected tRNA genes were found close to delta (delta) sequences at the sup4 locus on chromosome X in the genome of Saccharomyces cerevisiae. The two genes were identified from their abundant transcription products in frog oocytes. Hybridisation experiments allowed the mapping of the transcripts in cloned DNA and DNA sequence analysis revealed the presence of one AGGtRNA and one GACtRNA gene. tRNA genes with sequences similar or identical to GACtRNA exist in 14-16 copies per haploid yeast genome, whereas only one copy was detected for AGGtRNA. In vivo labelling of total yeast tRNA with P followed by hybridisation revealed that the unique AGGtRNA gene is transcribed in S. cerevisiae. delta sequences are present 120 bp upstream from the first coding nucleotide in the case of AGGtRNA, 80 bp in the case of GACtRNA and 405 bp in the case of the known UACtRNA (sup4) gene. delta sequences, as part of Ty elements or alone, were also found by other investigators at similar distances upstream of the mRNA start in mutant alleles of protein-coding yeast genes. Although protein-coding genes are transcribed by RNA polymerase II and tRNA genes by RNA polymerase III, the 5' non-coding region of both types of genes could conceivably have a peculiar DNA or chromatin structure used as preferred landing sites by transposable elements.