Cross-genus Boot-up of Synthetic Bacteriophage in Staphylococcus aureus Using a New and Efficient DNA Transformation Method

Staphylococcus aureus is an opportunistic pathogen causing a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but methods are needed for efficient transformation of large DNA molecules into S. aureus to boot-up (i.e., rescue) infectious phages. We present a new, efficient and reproducible DNA transformation method, NEST (Non-Electroporation Staphylococcus Transformation), for S. aureus to boot-up of purified phage genomic DNA (at least 150 kb in length tested) and whole yeast-assembled synthetic phage genomes. This method is a powerful new tool for transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. Importance The continued emergence of antibiotic resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy, but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages, but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional non-model Siphophage from a constructed synthetic genome. This method will facilitate not only the engineering of Staphylococcus and Enterococcus phages for therapeutic applications but also the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

Open Cholecystostomy Under Local Anesthesia for Acute Cholecystitis in the Elderly and High-Risk Surgical Patients

Background The morbidity and mortality rates associated with cholecystectomy for acute cholecystitis are higher in the critically ill elderly population. As an alternative to cholecystectomy, we report the results of treatment of acute cholecystitis in the elderly after open cholecystolithotomy with cholecystostomy tube placement under local anesthesia. Methods A case series was performed on 5 patients from August 2007 to April 2010 who presented with acute cholecystitis and underwent an open cholecystolithotomy and tube placement. Thirty-day mortality, intra- and immediate-postoperative complications, clinical improvement after drainage, additional biliary procedures needed, and outcome after cholecystostomy tube removal were recorded. Results Open cholecystolithotomy and tube placement were performed successfully in all patients and permitted resolution of the acute attack in all after a mean period of 3.75 days. Thirty-day mortality was 0%. Patients did not experience any intraoperative complications. We observed 100% rate of successful short-term outcomes in our patients including resolution pain, and objectively, normalization of leukocytosis, and defervescence. None of the patients required emergency cholecystectomy. All patients had their cholecystostomy tubes removed at a mean postoperative day 27. There were no cases of biliary leakage or tube dislodgement. There were no recurrences of acute cholecystitis within the mean follow-up of 20.75 months. Discussion Emergency open cholecystolithotomy and cholecystostomy tube placement represent an effective, safe, and definitive alternative treatment strategy for acute gallstone cholecystitis in selected elderly patients with a mortality rate of 0% in the authors’ experience.

Fluorescence Lifetime Imaging Microscopy of Porphyrins in Helicobacter pylori Biofilms

Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. Helicobacter pylori (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the ‘fit-free’ phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.

Stem Cells in Clinical Trials for Pelvic Floor Disorders: a Systematic Literature Review

Pelvic floor disorders (PFDs) include a series of conditions that can be poorly tolerated, negatively affecting the quality of life. Current treatment options show unsatisfactory results and new ones are therefore needed. Stem cell (SC) therapy might be an alternative treatment strategy. This systematic review aims to define the state of art of SC therapy for PFDs in clinical trials, by systematically reviewing the available evidence. A systematic search strategy was conducted up to November 7, 2020, in PubMed, Scopus, Cochrane Library, and ISI Web of Science. Preclinical studies on animal models were not considered. Studies were included when the patients were affected by any PFDs and cells were isolated, cultured, and characterized as SC. The study protocol was registered in PROSPERO (CRD42020216551). A total of 11 prospective clinical studies were included in the final assessment, specifically 7 single-arm studies dealing with SC therapy for stress urinary incontinence and 4 with anal incontinence. Among the latter, there were two prospective, single-arm studies and two randomized controlled trials. No papers concerning the use of SC for prolapse repair were retrieved. Due to the great heterogeneity, data pooling was not possible. Stem cell injection resulted in a safe procedure, with few mild adverse side effects, mostly related to harvesting sites. However, a clear beneficial impact of SC treatment for the treatment of pelvic floor disorders could not be demonstrated. Further larger targeted studies with control arms are needed before any conclusions can be made.

Current Healthcare Utilization and Alternative Strategies for Rotator Cuff Tendinopathy (239)

Objectives: Rotator cuff disease is one of the most common reasons for people to seek upper extremity specialty care. Rotator cuff tendinopathy is typically a part of the normal aging process and is often well adapted. Healthcare utilization for the condition varies widely and can range from formal physical therapy to surgery. Evidence of the effectiveness of some interventions for rotator cuff tendinopathy is lacking and for others, such as steroid injection or subacromial decompression, suggests no benefit. There is an opportunity to streamline care for this common condition and, in doing so, conserve finite healthcare resources. The purpose of this study was to characterize the current healthcare utilization for rotator cuff tendinopathy using a large claims database in an effort to identify opportunities to streamline care and develop value-based treatment algorithms. Methods: Using de-identified records in a large claims data base that includes over 350 private-sector payers, we identified patients with a new visit claim between April and June 2016 in one state with an ICD-10 diagnosis code for rotator cuff tendinopathy. The most common atraumatic diagnoses and the frequency of interventions, such as surgery, physical or occupational therapy, advanced imaging, and injections were tracked. We recorded all return visits (including post-operative visits), tests, treatments, and costs for 3 months prior and 6 months after diagnosis. We then calculated the potential cost savings using an alternative treatment strategy for rotator cuff tendinopathy based on value-based care principals. Results: The percentage of enrollees undergoing specific interventions was MRI 19%; physical therapy visit 29%; injections 52%; return visits 44%; and surgery 17%. Alternative treatment strategies, such as a reliance on home exercises rather than formal physical therapy, avoidance of injections and limiting MRIs to patients considering surgery accounted for a potential mean payment that was 8% of the actual mean payments of claims paid out in the database. Conclusions: Conclusion: Our analysis identified notable variation in care of people with rotator cuff tendinopathy. Alternative treatment strategies identified an opportunity for improving value given growing evidence that limiting some of the more commonly used interventions is unlikely to diminish health and might even improve it by increasing self-efficacy.

BALLOON VALVULOPLASTY FOR EARLY BIOPROSTHETIC AORTIC VALVE STENOSIS IN SEPTUAGENARIAN - A RAY OF HOPE

Calcic aortic stenosis (AS) remains a major cause of mortality and morbidity in the aging population . Surgical AV Replacement (SAVR) and Transfemoral Aortic Valve Implantation (TAVI) are available treatment options. With improvements in long term patient survival after AVR and increases in overall longevity, more patients are now seen with Prosthetic Aortic Valve failure. The management of patients with stenotic aortic bioprostheses is usually surgical. However, a proportion of such patients are unt for such procedures. The technique of aortic balloon valvuloplasty as an alternative treatment strategy for such patients is explored. We report a case of seventy-three-year-old male with prosthetic aortic valve stenosis treated with balloon valvuloplasty with promising intermediate term outcome and describe the growing valve in valve procedure. Hence this case is reported to enhance our knowledge and potentiate literature regarding the management strategy of prosthetic aortic valve stenosis in old age.

Mechanistic Insights in Biofilm Eradication Potential of Tyrosol Functionalized Chitosan Gold Nanoparticles against Candida spp.

In the present study, tyrosol functionalized chitosan gold nanoparticles (Chi-TY-AuNPs) were prepared as an alternative treatment strategy to combat fungal infections. Various biophysical techniques were used to characterize the synthesized Chi-TY-AuNPs. The antifungal and antibiofilm activities of Chi-TY-AuNPs were evaluated against C. albicans and C. glabrata and efforts have been made to elucidate the possible mechanism of action. Chi-TY-AuNPs showed a high fungicidal effect against both sessile and planktonic cells of Candida spp. Additionally, Chi-TY-AuNPs completely eradicated (100%) the mature biofilms of both the Candida spp. FESEM analysis highlighted the morphological alterations in Chi-TY-AuNPs treated Candida biofilm cells. Effect of Chi-TY-AuNP's on the ECM components showed significant reduction in protein content in C. glabrata biofilm and substantial decrease in extracellular DNA (eDNA) content of both the Candida spp. ROS generation analysis using DCFDA-PI staining showed high ROS levels in both the Candida spp., whereas pronounced ROS production was observed in Chi-TY-AuNPs treated C. glabrata biofilm. Biochemical analysis revealed decreased ergosterol content in Chi-TY-AuNP's treated C. glabrata cells, while inconsequential changes were observed in C. albicans. Furthermore, the transcriptional expression of selected genes (ergosterol biosynthesis, efflux, sterol importer, and glucan biogenesis) was reduced in C. glabrata in response to Chi-TY-AuNP's except ERG11 and CDR1. Conclusively the result showed the biofilm inhibition and biofilm eradication efficacy of Chi-TY-AuNPs in both the Candida spp. Findings of the present study manifest Chi-TY-AuNP's as a potential therapeutic solution to Candida biofilm-related chronic infections and overcome biofilm antifungal resistance.

Optimum and Sustainable Cooling Technology Selection for Different Climatic Conditions

Global warming has led to rising electricity demands due to soaring cooling load, resulting in different technologies being implemented with renewable energy options. Renewable energy has been used to partially or fully operate these cooing systems through different technology routes in both conventional and hybrid modes. The feasibility of a particular cooling process is influenced by several technological, economic, environmental and other related factors. Selection of the appropriate route also requires consideration of external factors such as local weather, cooling load requirements and the potential of possible renewable energy. Multi-criteria decision analysis is a useful tool to systematically arrive at the right option from several possible options. This tool is used to assess the feasibility of eight technology routes for three different climatic conditions. Other than the direct cooling processes, two routes of renewable energy utilization, namely, the solar photovoltaic system and solar thermal system, are considered. The normalized decision matrix is established and weighted decision matrix is estimated, and the best solution and the worst solution values are obtained by using equations. This study is performed for three climatic zones under the Koppen classification, namely, the tropical maritime arid condition with average midday temperature from 40 to 45 °C, with two different relative humidity ranges, namely, dry area and maritime area. Additionally, the temperate continental climatic zone is analyzed for comparison. The results of this study will help decision makers to judiciously implement air conditioning systems in the above climatic zones. The distance of each waste treatment strategy from the overall best alternative treatment strategy and the overall worst alternative treatment strategy is obtained. Finally, the cooling strategies are ranked for the best option for the cooling mechanism to be adopted for the three climatic conditions.

Enteric Microbiota-Mediated Serotonergic Signaling in Pathogenesis of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a chronic functional disorder that affects the gastrointestinal tract. Details regarding the pathogenesis of IBS remain largely unknown, though the dysfunction of the brain-gut-microbiome (BGM) axis is a major etiological factor, in which neurotransmitters serve as a key communication tool between enteric microbiota and the brain. One of the most important neurotransmitters in the pathology of IBS is serotonin (5-HT), as it influences gastrointestinal motility, pain sensation, mucosal inflammation, immune responses, and brain activity, all of which shape IBS features. Genome-wide association studies discovered susceptible genes for IBS in serotonergic signaling pathways. In clinical practice, treatment strategies targeting 5-HT were effective for a certain portion of IBS cases. The synthesis of 5-HT in intestinal enterochromaffin cells and host serotonergic signaling is regulated by enteric resident microbiota. Dysbiosis can trigger IBS development, potentially through aberrant 5-HT signaling in the BGM axis; thus, the manipulation of the gut microbiota may be an alternative treatment strategy. However, precise information regarding the mechanisms underlying the microbiota-mediated intestinal serotonergic pathway related to the pathogenesis of IBS remains unclear. The present review summarizes current knowledge and recent progress in understanding microbiome–serotonin interaction in IBS cases.