Potential reservoirs of antimicrobial resistance in livestock waste and treated wastewater that can be disseminated to agricultural land.

Livestock manure, dairy lagoon effluent, and treated wastewater are known reservoirs of antibiotic resistance genes (ARGs), antibiotic-resistant bacteria (ARB), and virulence factor genes (VFGs), and their application to agricultural farmland could be a serious public health threat. However, their dissemination to agricultural lands and impact on important geochemical pathways such as the nitrogen (N) cycle have not been jointly explored. In this study, shotgun metagenomic sequencing and analyses were performed to examine the diversity and composition of microbial communities, ARGs, VFGs, and N cycling genes in different livestock manure/lagoon and treated wastewater collected from concentrated animal feeding operations (CAFOs) and a municipal wastewater treatment plant along the west coast of the United States. Multivariate analysis showed that diversity indices of bacterial taxa from the different microbiomes were not significantly different based on InvSimpson (P = 0.05), but differences in ARG mechanisms were observed between swine manure and other microbiome sources. Comparative resistome profiling showed that ARGs in microbiome samples belonged to four core resistance classes: aminoglycosides (40-55 %), tetracyclines (30-45 %), beta-lactam-resistance (20-35 %), macrolides (18-30 %), and >50 % of the VFGs that the 24 microbiomes harbored were phyletically affiliated with two bacteria, Bacteroidetes fragilis and Enterobacter aerogenes. Network analysis based on Spearman correlation showed co-occurrence patterns between several genes such as transporter-gene and regulator, efflux pump and involved-in-polymyxin- resistance, aminoglycoside, beta-lactam, and macrolide with VFGs and bacterial taxa such as Firmicutes, Candidatus Themoplasmatota, Actinobacteria, and Bacteroidetes. Metabolic reconstruction of metagenome-assembled genome (MAGs) analysis showed that the most prevalent drug resistance mechanisms were associated with carbapenem resistance, multidrug resistance (MDR), and efflux pump. Bacteroidales was the main taxa involved in dissimilatory nitrate reduction (DNRA) in dairy lagoon effluent. This study demonstrates that the dissemination of waste from these sources can increase the spread of ARGs, ARB, and VFGs into agricultural lands, negatively impacting both soil and human health.

Breast surgeons updating on the thresholds of COVID-19 era: results of a multicenter collaborative study evaluating the role of online videos and multimedia sources on breast surgeons education and training.

OBJECTIVE: Current trends show a rise of attention given to breast cancer patients' quality of life and the surgical reconstructive result. Along with this trend, surgical training quality and efficacy are gaining importance and innovative training methods such as online videos shared on social media portals, are becoming main updating tools. In hazardous times like COVID-19 pandemic nowadays, online communication becomes of vital importance and adaptation and innovation are fundamental to keep research and education alive. The authors aimed to investigate the role of video and multimedia sources on the daily activity and surgical training of a representative group of surgeons specifically dedicated to oncologic, oncoplastic and reconstructive breast surgeries. MATERIALS AND METHODS: A survey was produced and administered to 20 major Italian Breast Centers. Collected data were analyzed with Fisher's Exact Test. RESULTS: From October 2019 to March 2020, a total of 320 surveys were collected. Among the responders, there were 188 trainees (intern medical doctors and residents) and 110 faculty, 72% of them belonged to a plastic surgery environment, while 28% to general surgery environment. Almost all respondents have ever watched videos concerning breast surgery. CONCLUSIONS: The results of the study show how breast surgeons rely on videos and web platforms, mostly YouTube, when searching for training info about surgical procedures. Social media offer great opportunities for sharing knowledge and diffusion of new ideas but greater attention to their reliability is mandatory.

The use of dermal regeneration template (Pelnac®) in a complex upper limb trauma: the first Italian case report.

Dermal regeneration template (DRT) has been well widely implicated in the reconstruction of full-thickness injury. We present our experience and our clinical application of Pelnac® to achieve wound closure with complex acute, upper limb, full-thickness defect post-trauma. A 22-year-old boy presented a soft tissues loss of the back of the hand and forearm with tendon's involvement and exposure. The wound was treated with Pelnac®; the silicone layer was removed at postoperative day 30 and dermal regeneration template was reapplied at the level of the residual tendon exposure; a split-thickness skin graft (0.2 to 0.3 mm) was inserted. Clinically, the reconstructed areas demonstrated good granulation tissue at 14 days with a good take of the skin graft. There were no major acute graft loss, rejection or associated infections cells through downregulating TLR4 expression.

Phenotypic correction of diabetic mice by adenovirus-mediated glucokinase expression.

Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector-mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 +/- 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 +/- 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 +/- 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 +/- 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 +/- 291 pg/ml; vehicle, 1,861 +/- 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.

Treatment of type 2 diabetes by adenoviral-mediated overexpression of the glucokinase regulatory protein.

The enzyme glucokinase (GK) plays a central role in glucose homeostasis. Hepatic GK activity is acutely controlled by the action of the GK regulatory protein (GKRP). In vitro evidence suggests that GKRP reversibly binds to GK and inhibits its activity; however, less is known about the in vivo function of GKRP. To further explore the physiological role of GKRP in vivo, we used an E1/E2a/E3-deficient adenoviral vector containing the cDNA encoding human GKRP (Av3hGKRP). High fat diet-induced diabetic mice were administered Av3hGKRP or a control vector lacking a transgene (Av3Null). Surprisingly, the Av3hGKRP-treated mice showed a significant improvement in glucose tolerance and had lower fasting blood glucose levels than Av3Null-treated mice. A coincident decrease in insulin levels indicated that the Av3hGKRP-treated mice had sharply improved insulin sensitivity. These mice also exhibited lower leptin levels, reduced body weight, and decreased liver GK activity. In vitro experiments indicated that GKRP was able to increase both GK protein and enzymatic activity levels, suggesting that another role for GKRP is to stabilize and/or protect GK. These data are the first to indicate the ability of GKRP to treat type 2 diabetes and therefore have significant implications for future therapies of this disease.

Pancreatic beta-cell K(ATP) channel activity and membrane-binding studies with nateglinide: A comparison with sulfonylureas and repaglinide.

Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes. The aim of this study was to determine whether nateglinide's interaction at the K(ATP) channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models. Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1). The relative order for displacement of [(3)H]glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide. The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide. Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for [(3)H]glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1. Finally, the inability to measure [(3)H]nateglinide binding suggests that nateglinide dissociates rapidly from SUR1. Direct interaction of nateglinide with K(ATP) channels in rat pancreatic beta-cells was investigated with the patch-clamp method. The relative potency for inhibition of the K(ATP) channel was repaglinide > glibenclamide > nateglinide. Kinetics of the inhibitory effect on K(ATP) current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide. The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide. These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K(ATP) channel.

Detection of transforming growth factor alpha in normal, malignant, and hyperproliferative human keratinocytes.

Transforming growth factor alpha (TGF-alpha) is a 50-amino acid peptide, previously demonstrated only in transformed cell lines and human tumors, which is structurally homologous to epidermal growth factor (EGF). TGF-alpha expression in keratinocytes from normal individuals, patients with psoriasis, and patients with malignant skin diseases was investigated using an mAb raised against synthetic human TGF-alpha. mAb A1.5 reacted with TGF-alpha, but not EGF, in a sensitive ELISA. Keratinocytes in eight nodular basal cell carcinomas, one morpheic basal cell carcinoma, and one squamous cell carcinoma demonstrated intense membranous immunoperoxidase staining with mAb A1.5. Of even greater interest was the observation that the overlying normal epidermis, as well as the epidermis from five normal skin specimens, were stained by the mAb. Keratinocytes in plaques from 18 psoriasis patients were more intensely stained than those from normal skin. Cultured normal keratinocytes demonstrated membranous staining with mAb A1.5. Absorption of mAb A1.5 with synthetic human TGF-alpha completely removed the reactivity of mAb A1.5 with both basal cell tumors and normal epidermis. The demonstration of TGF-alpha in normal keratinocytes suggests that it plays a role in normal keratinocyte growth, wound healing, and in the pathogenesis of acanthosis.