Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines

Transgenic mouse lines are invaluable tools for neuroscience but, as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, of either sex, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, although rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study

SHIV-162P3 Infection of Rhesus Macaques Given Maraviroc Gel Vaginally Does Not Involve Resistant Viruses

Maraviroc (MVC) gels are effective at protecting rhesus macaques from vaginal SHIV transmission, but breakthrough infections can occur. To determine the effects of a vaginal MVC gel on infecting SHIV populations in a macaque model, we analyzed plasma samples from three rhesus macaques that received a MVC vaginal gel (day 0) but became infected after high-dose SHIV-162P3 vaginal challenge. Two infected macaques that received a placebo gel served as controls. The infecting SHIV-162P3 stock had an overall mean genetic distance of 0.294±0.027%; limited entropy changes were noted across the envelope (gp160). No envelope mutations were observed consistently in viruses isolated from infected macaques at days 14–21, the time of first detectable viremia, nor selected at later time points, days 42–70. No statistically significant differences in MVC susceptibilities were observed between the SHIV inoculum (50% inhibitory concentration [IC50] 1.87 nM) and virus isolated from the three MVC-treated macaques (MVC IC50 1.18 nM, 1.69 nM, and 1.53 nM, respectively). Highlighter plot analyses suggested that infection was established in each MVC-treated animal by one founder virus genotype. The expected Poisson distribution of pairwise Hamming Distance frequency counts was observed and a phylogenetic analysis did not identify infections with distinct lineages from the challenge stock. These data suggest that breakthrough infections most likely result from incomplete viral inhibition and not the selection of MVC-resistant variants

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations

Microbial sulfate reduction and metal attenuation in pH 4 acid mine water

Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2–3 ‰ heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic