The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.

BackgroundAustralia’s health disparity, combined with evolving technologies, has evoked increasing interest and funding in health services that could address inequities. One such emerging service is tele-medicine.ObjectiveThe purpose of this report is to discuss and evaluate the current literature regarding patient and practitioner satisfaction with tele-medicine, and more specifically tele-dermatology.MethodsWe searched for literature relevant to tele-dermatology use among Australia’s indigenous population. We synthesized the literature in our report and identified elements of tele-dermatology not yet researched.ResultsMost significantly, all available research is currently based on descriptive studies and there is no validated tool to assess the efficacy of tele-dermatology.LimitationsNo published research currently exists on the use of tele-dermatology among Australia’s indigenous population.ConclusionA review of the literature shows that tele-dermatology is considered a valuable service, particularly to patients living in rural areas who might not otherwise have access to specialist care.

A novel mechanism is proposed for ring formation in the biosynthetic pathway to thiotetronate antibiotics thiolactomycin and Tü 3010.

Background

Microbial natural products (NPs) especially of the Streptomyces genus have been regarded as an unparalleled resource for pharmaceutical drugs discovery. Moreover, recent progress in sequencing technologies and computational resources further reinforces to identify numerous NP biosynthetic gene clusters (BGCs) from the genomes of Streptomyces. However, the majority of these BGCs are silent or poorly expressed in native strains and remain to be activated and investigated, which relies heavily on efficient genome editing approaches. Accordingly, numerous strategies are developed, especially, the most recently developed, namely, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system reveals remarkable higher accuracy and efficiency for genome editing in various model organisms including the Streptomyces. In this mini review, we highlight the application of CRISPR/Cas9-based approaches in Streptomyces, focus on the editing of BGCs either in vivo or in vitro, as well as target cloning of large-sized BGCs and heterologous expression in a genetically manipulatable host, for discovery, characterization, reengineering, and production of potential pharmaceutical drugs.

Objectives The aim of sclerotherapy is to induce fibrosclerosis of superficial veins. We postulated that inadvertent entry of sclerosants into deep veins can result in sclerotic occlusion, deep vein sclerosis, a non-thrombotic process distinct from spontaneous deep vein thrombosis. The aim of this study was to assess the role of d-dimer in differentiating between deep vein sclerosis and deep vein thrombosis. Methods Proximal trunks of great and small saphenous veins were treated with endovenous laser ablation. Venous tributaries and perforators were treated with foam ultrasound guided sclerotherapy. Ultrasound studies of lower limb deep veins were performed before and one week after the procedures, to detect deep vein occlusions (DVOs). d-dimer levels were measured for DVOs and long-term ultrasound studies monitored the recanalisation rates. Results In a six-year period, 9143 procedures were performed in 1325 patients for bilateral varicose veins. This included 1124 endovenous laser ablation and 8019 foam ultrasound guided sclerotherapy procedures. A total of 259 DVOs (2.83%) were identified on ultrasound which included 251 deep vein sclerosis (2.74%), seven deep vein thrombosis (0.07%) and one endovenous heat-induced thrombosis (EHIT, 0.08%). d-dimer values <0.5 µg/mL excluded deep vein thrombosis s, 0.5–1.0 µg/mL were more likely to be associated with deep vein sclerosis and >1.0 µg/mL were a more likely to be associated with deep vein thrombosis. Lower sclerosant concentrations and higher foam volumes were associated with increased risk of DVO ( p <  .0001). No significant relationship was found between DVO and gender or thrombophilia. Deep vein thrombosis and EHIT cases but not deep vein sclerosis patients were anticoagulated. None had thromboembolic complications. Patients were followed up for a median of 299 days (37–1994 days). Recanalisation rates were 71.1% for deep vein sclerosis (92.3% competent) and 71.4% for deep vein thrombosis (60.0% competent). Conclusions Deep vein sclerosis is a relatively benign clinical entity distinct from deep vein thrombosis and does not require anticoagulation. Majority of affected veins on long-term follow-up regain patency and competence. d-dimer can be used to assist in differentiating deep vein sclerosis from deep vein thrombosis.

Background Osteopenia or osteoporosis is one of the many comorbidities in patients with Epidermolysis Bullosa (EB). Current literature on the prevalence of osteoporosis in EB is scarce. Objective This review will analyse the current literature in the field of patients with compromised bone health in EB and any articles on the prevalence of such diseases in EB groups. Methods A systematic search for articles related to bone health and epidermolysis bullosa (EB) (1946‐2017) was performed on seven databases: MEDLINE, EMBASE and EBM, PubMed, ProQuest, Scopus and Web of Science. Search terms: epidermolysis bullosa, osteop*, bone mass, bone mineral*, fracture, dual X‐ray absorptiometry, vitamin D, calcium, nutrition, exercise and physical activity. Abstracts from all search results were screened, and reference scanning of the search results was performed. Eighty‐three articles met the selection criteria and were considered for review. Letters to the editor and abstract‐only articles were excluded. Articles were favoured based on citation count, impact factor of their journal and study sample size. The search included all languages. Results The searches yielded a total of 1309 articles including 717 duplicates. The remaining 592 articles were screened by title and abstracts. Eighty‐three full‐text articles were analysed. Twenty‐one articles directly relating to bone health in EB were included. Three descriptive studies and one case‐control study were found, indicating a need for research of larger scale. Conclusion Further investigations into osteoporosis in EB, especially the milder forms of EB, are valuable in providing evidence to support guideline developments for EB bone health management.

BackgroundThe BIOCHIP is a novel multiplex indirect immunofluorescence technique used in the serological diagnosis of bullous pemphigoid and pemphigus. The BIOCHIP method combines the screening of autoantibodies and target antigen‐specific substrates in a single miniature incubation field.ObjectiveTo evaluate the diagnostic accuracy of the new immunofluorescence BIOCHIP multiplex tool in pemphigus and bullous pemphigoid.MethodsFor the validation of the BIOCHIP, sera from patients with BP (n = 38), PF (n = 8) and pemphigus vulgaris (PV) (n = 23) were used. In addition, sera from disease control patients (n = 63) and healthy volunteers (n = 39) were used. The multiplex BIOCHIP and direct immunofluorescence (DIF) were performed for all BP, PF and PV patients. Additional indirect immunofluorescence (IIF) was performed on patients with BP, and ELISA was performed on patients with pemphigus.ResultsThe BIOCHIP mosaic showed a sensitivity of 86.8% and specificity of 85% for BP180 or BP230 being positive in BP. It demonstrated a sensitivity of 75% and specificity of 97.7% for Dsg1 in PF. The BIOCHIP was found to have a sensitivity of 60.9% and specificity of 73.6% for Dsg3 in PV.ConclusionThe BIOCHIP mosaic‐based immunofluorescence test is potentially a simple, time and effort saving test that can aid in the diagnosis and screening of BP, PV and PF. However, there is potential for interpretation bias and a learning curve that needs to be taken into consideration.