A non-radioactive electrophoretic mobility shift assay for the detection of heat shock element (HSE)-binding activity in Neurospora crassa

A non-radioactive electrophoretic mobility shift assay based on the digoxigenin detection system had been developed and applied to the analysis of the interaction between the heat shock transcription factor and the heat shock element of N. crassa

Abnormal microglia and enhanced inflammation-related gene transcription in mice with conditional deletion of Ctcf in Camk2a-Cre-expressing neurons

CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations inCTCFcause intellectual disability and autistic features. Knocking outCtcfin mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked outCtcfpostnatally in glutamatergic forebrain neurons under the control ofCamk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+(CtcfCKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age.CtcfCKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA fromCtcfCKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically fromCtcfCKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia inCtcfCKO hippocampus. Finally, we found that microglia inCtcfCKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm thatCtcfKO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction.SIGNIFICANCE STATEMENTCCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations inCTCFcause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lackingCtcfinCamk2a-expressing neurons (CtcfCKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate thatCtcfCKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron–microglia interactions are factors in the pathology of CTCF deficiency.</jats:p

Genome Sequences of Two Copper-Resistant Escherichia coli Strains Isolated from Copper-Fed Pigs.

The draft genome sequences of two copper-resistant Escherichia coli strains were determined. These had been isolated from copper-fed pigs and contained additional putative operons conferring copper and other metal and metalloid resistances

Could recombinant insulin compounds contribute to adenocarcinoma progression by stimulating local angiogenesis?

Negative effects on the progression of adenocarcinomas by hyperinsulinaemia and the insulin analogue glargine (A21Gly,B31Arg,B32Arg human insulin) have recently been suggested. Most actions of this insulin analogue have hitherto been explained by direct stimulation of growth potential of neoplastic cells and by its IGF-1 related properties. However, insulin-stimulated angiogenesis could be an additional factor involved in tumour progression and clinical outcomes associated with cancer. Five types of human adenocarcinoma (breast, colon, pancreas, lung and kidney) were evaluated for the presence of insulin receptors (IRs) on angiogenic structures. In an in vitro angiogenesis assay, various commercially available insulin compounds were evaluated for their potential to increase capillary-like tube formation of human microvascular endothelial cells (hMVEC). Insulin compounds used were: human insulin, insulin lispro (B28Lys,B29Pro human insulin), insulin glargine and insulin detemir (B29Lys[e-tetradecanoyl],desB30 human insulin). Insulin receptors were found to be strongly expressed on the endothelium of microvessels in all evaluated adenocarcinomas, in addition to variable expression on tumour cells. Low or no detectable expression of IRs was seen on microvessels in extratumoral stroma. Incubation with commercially available insulin compounds increased capillary-like tube formation of hMVEC in vitro. Our results suggest that all tested insulin compounds may stimulate tumour growth by enhancing local angiogenesis. Future studies need to confirm the association between insulin therapy in type 2 diabetes and tumour progressio

The effect of Covid-19 on pediatric surgical case volume.

Introduction The COVID-19 pandemic has had an unprecedented ef- fect on hospital systems. Policy changes lead to de- creased hospital visits as well as surgical case volume. The literature on pediatric surgical case volume during the pandemic is sparse. Throughout the country, hospitals sought various policies to preserve personal protective equipment and other hospital resources, and to minimize avoidable peri- and postoperative sequelae due to COVID-19 infection. Our hospital first placed a hold on all elective surgeries. Later, all elective cases required a preoperative negative COVID test prior to proceed- ing. We sought to review the sequelae of our hospital’s policy in response to COVID-19. We identified trends in surgical case volume and cancellations due to a positive COVID-19 test. We also reviewed postoperative out- comes of cases with a positive test. Material and Methods This study was approved by the institutional IRB. Data was retrospectively collected on all surgical cases at our children’s hospital between March 2019 and March 2021. We marked the start of the COVID-19 pandemic as March 2020, when elective cases were suspended. A required preoperative negative COVID-19 test was im- plemented in May 2020. We identified pre-operative COVID-19 test results,the posted urgency of each case and 30-day outcomes from medical records. Results From March 2019 to March 2021, we identified 25,496 completed surgeries and 3,503 cancellations. 12,024 ca- ses proceeded during the first year of the pandemic, which appeared lower, compared to pre-pandemic case numbers. Of those, 2,785 (23%) cases were considered urgent or emergent. The average number of completed monthly cases fell from a pre-pandemic number of 1,123 to a pandemic number of 925. When comparing to a pre- pandemic month, average monthly case volume declined by 19%, with the largest decline noted to be 66%. There was a monthly average of 189 total cancellations between March 2020 and March 2021. 34 (18%) of those were for a positive preoperative COVID test. A total of 139 sur- geries commenced despite concomitant COVID-19 in- fection. 25 (18 %) had identifiable respiratory symptomsdocumented preoperatively. 13 (9 %) were deemed to have a respiratory complication afterward. Of those, three patients (2%) had a prolonged, and one (1%) had an unexpected reintubation. The remaining nine (6%) pa- tients had a prolonged oxygen requirement. Conclusion The COVID pandemic left operating rooms struggling to determine how to safely provide care to patients. This study demonstrated how the policies of one hospital af- fected the operating room case volume and how conco- mitant COVID infection affected outcomes in those that proceeded with surgery

Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essen- tial component of the cellular homeostatic machinery in eukary- otes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co- chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilita- tor of Hsp90-mediated folding of kinase and non-kinase clients— including Tsc2—thereby preventing their ubiquitination and proteasomal degradation

Metal resistance and its association with antibiotic resistance

Antibiotic resistance is recognised as a major global threat to public health by the World Health Organization. Currently, several hundred thousand deaths yearly can be attributed to infections with antibiotic-resistant bacteria. The major driver for the development of antibiotic resistance is considered to be the use, misuse and overuse of antibiotics in humans and animals. Nonantibiotic compounds, such as antibacterial biocides and metals, may also contribute to the promotion of antibiotic resistance through co-selection. This may occur when resistance genes to both antibiotics and metals/biocides are co-located together in the same cell (co-resistance), or a single resistance mechanism (e.g. an efflux pump) confers resistance to both antibiotics and biocides/metals (cross-resistance), leading to co-selection of bacterial strains, or mobile genetic elements that they carry. Here, we review antimicrobial metal resistance in the context of the antibiotic resistance problem, discuss co-selection, and highlight critical knowledge gaps in our understanding

Inoculation of Weaned Pigs by Feed, Water, and Airborne Transmission of Salmonella enterica Serotype 4,[5],12:i:-

Salmonella enterica serotype 4,[5],12:i:- (STM) has become an increasing problem for food safety and has been often detected in pork products. For this study, weanling pigs were exposed to STM-contaminated feed, water, or air to determine possible STM transmission routes. An uninoculated control group of pigs was included. The STM was monitored daily in feces and rectal and nasal swabs. The STM colonization was most prevalent in tissues from tonsil, lower intestine, and mesenteric lymph nodes. No differences in lesion severity were observed between inoculated and control pigs. Contaminated feed, water, and aerosolized particles caused infection in weaned pigs; however, no STM colonization was observed in skeletal muscle destined for human consumption. Based on the results from this study, STM contamination in pork products most likely results from cross-contamination of meat by digesta or lymph node tissue during processing

Inoculation of Weaned Pigs by Feed, Water, and Airborne Transmission of Salmonella enterica Serotype 4,[5],12:i:-

Salmonella enterica serotype 4,[5],12:i:- (STM) has become an increasing problem for food safety and has been often detected in pork products. For this study, weanling pigs were exposed to STM-contaminated feed, water, or air to determine possible STM transmission routes. An uninoculated control group of pigs was included. The STM was monitored daily in feces and rectal and nasal swabs. The STM colonization was most prevalent in tissues from tonsil, lower intestine, and mesenteric lymph nodes. No differences in lesion severity were observed between inoculated and control pigs. Contaminated feed, water, and aerosolized particles caused infection in weaned pigs; however, no STM colonization was observed in skeletal muscle destined for human consumption. Based on the results from this study, STM contamination in pork products most likely results from cross-contamination of meat by digesta or lymph node tissue during processing