A Chimeric ORF Fusion Phenotypic Reporter for Cryptococcus neoformans.

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans.

Aortic remodelling based on false lumen communications in patients undergoing acute type I dissection repair with AMDS hybrid prosthesis: a substudy of the DARTS trial.

OBJECTIVES: The Dissected Aorta Repair Through Stent (DARTS) Implantation trial demonstrated positive proximal aortic remodelling following aortic dissection repair with the AMDS hybrid prosthesis. In this study, we look to identify predictors of aortic remodelling following aortic dissection repair with AMDS including whether communications between branch vessels and the false lumen (FL) predict aortic growth. METHODS: The DARTS implantation trial included patients who underwent acute DeBakey type I aortic dissection (ATAD I) repair with the AMDS from March 2017 to January 2019. Anatomic measurements were collected from original computerized tomography scans. Measurements were taken at zones 2, 3, 6 and 9. Patients were grouped based on the number of FL communications with the supra-aortic branch vessels or visceral branch vessels. RESULTS: Forty-seven patients were included in the original DARTS implantation trial. Patients with FL communications with the supra-aortic branch vessels tended to have significant growth at zone 3 (P = 0.02-0.0018), while greater numbers of visceral FL communications tended to predict aortic growth at zones 3 (P = 0.003), 6 (P = 0.017-0.0087) and 9 (P = 0.0016-0.0003). CONCLUSIONS: Aortic remodelling following ATAD I repair using the AMDS may be predicted by local FL communications with branch vessels. Patients undergoing ATAD I repair were more likely to experience significant aortic growth in zone 3 with more head vessel communications and in zones 3, 6 and 9 with more visceral FL communications. Predictors of aortic remodelling may help to guide initial surgical management for aortic dissection patients.

Implantation of ascyrus medical dissection stent, our first-hand experience.

BACKGROUND: Acute type A aortic dissection is associated with high perioperative morbidity and mortality. Ascyrus Medical Dissection Stent (Cryolife, Kennesaw, USA) is a novel uncovered hybrid stent graft developed to be used as an adjunct to standard surgical approach to promote true lumen expansion and enhance aortic remodeling. METHODS: From March 2021 to March 2022, four consecutive patients presented with acute Debakey type I aortic dissection and underwent emergent surgical repair with Tirone David procedure and implantation of Ascyrus Medical Dissection Stent. We reviewed patient's files retrospectively and described the perioperative outcomes. RESULTS: All four device implantations were successful. Overall 30-day mortality was 0%. Malperfusion that was present in two patients pre-operatively improved after Ascyrus Medical Dissection Stent implantation. No aortic reinterventions were needed. No aortic injury related to the device was noted. Favourable changes in aortic true lumen and false lumen dimensions were found in most of our patients but the stent was compressed at the isthmus in one patient. CONCLUSION: Ascyrus Medical Dissection Stent is a reliable and secure device. However, its benefits remain unclear when it comes to a positive remodeling and seems less likelihood comparable to a frozen elephant trunk. The main reason seems to be an insufficient radial force of the stent.

Comparing the Dosing Period in Package Inserts of Antimicrobial Agents Between Japan and the United States.

Introduction: The duration of antimicrobial therapy is a critical evaluation index of antimicrobial stewardship (AS). The inclusion of the dosing period on package inserts provides a strong reason for clinical intervention by pharmacists in cases where physicians prescribe inappropriate dosing periods. This study investigated differences in the description of dosing periods in antimicrobial package inserts between Japan and the U.S. Methods: We conducted a survey comparing differences in the dosing period of oral and injectable antimicrobials approved and marketed in Japan and the U.S. as of May 1, 2021. The Fisher exact test was used to compare the presence or absence of a description of the dosing period on the package insert between these two countries. Results: We evaluated 69 antimicrobial agents, of which 34 were oral; and 35 were injectable agents. In Japan, 20 (29.0%) of the antimicrobials had package inserts stating the dosing periods, compared with 58 (84.1%) in the U.S. (p < 0.001). Conclusions: It was found that the information on the duration of administration was missing from the package insert in Japan compared to the U.S. Lack of information on the duration of administration may lead to long-term administration by the treating physician and also make it difficult for the pharmacist to inquire about the administration. It is expected that the inclusion of scientifically-based dosing periods in all package inserts will promote AS among physicians and pharmacists who are not specialists in infectious disease therapy.

Development of Marker Recycling Systems for Sequential Genetic Manipulation in Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7.

Marine-derived fungi are emerging as prolific workhorses of structurally novel natural products (NPs) with diverse bioactivities. However, the limitation of available selection markers hampers the exploration of cryptic NPs. Recyclable markers are therefore valuable assets in genetic engineering programs for awaking silent SM clusters. Here, both pyrG and amdS-based recyclable marker cassettes were established and successfully applied in marine-derived fungi Aspergillus sp. SCSIO SX7S7 and Spiromastix sp. SCSIO F190, respectively. Using pyrG recyclable marker, a markerless 7S7-∆depH strain with a simplified HPLC background was built by inactivating a polyketide synthase (PKS) gene depH and looping out the pyrG recyclable marker after depH deletion. Meanwhile, an amdS recyclable marker system was also developed to help strains that are difficult to use pyrG marker. By employing the amdS marker, a backbone gene spm11 responsible for one major product of Spiromastix sp. SCSIO F190 was inactivated, and the amdS marker was excised after using, generating a relatively clean F190-∆spm11 strain for further activation of novel NPs. The collection of two different recycle markers will guarantee flexible application in marine-derived fungi with different genetic backgrounds, enabling the exploitation of novel structures in various fungi species with different genome mining strategies.

Combining aortic arch dissection stent implantation and root surgery for aortic dissection type A.

BACKGROUND: Acute aortic dissection type A (AADA) is associated with high perioperative morbidity and mortality. A novel non-covered hybrid prosthesis (AMDS, CryoLife, Kennesaw, USA) can be easily implanted to stabilize the true lumen. However, the role of AMDS for patients requiring additional aortic root surgery has not been described. METHODS: Between 2010 and 2020 a total of n = 370 patients underwent surgery for AADA in our department. Of those, n = 120 underwent treatment for aortic root in addition to proximal arch replacement without resection of the aorta beyond the innominate artery (Control, n = 111) and were compared to patients who received additional AMDS implantation (AMDS, n = 9). RESULTS: Aortic valve repair was performed in 48.6% (Control) and in 55.6% of AMDS patients. Cardiopulmonary bypass (Control: 248 ± 76 min, AMDS: 313 ± 53 min, P < 0.01) time as well as circulatory arrest time of the lower body (Control: 30 ± 15 min, AMDS: 52 ± 12 min, P < 0.01) was prolonged in the AMDS group. Nevertheless, postoperative in-hospital morbidity such as dialysis (Control: 22.4%, AMDS: 11.1%, P = 0.68) and stroke (Control: 17.0%, AMDS: 22.2%, P = 0.65) were comparable. In-hospital death (Control: 21.8%, AMDS: 11.1%, P = 0.68) and the compound end-point MACCE (Control: 38.7%, AMDS: 44.4%, P = 0.74) did also not differ. CONCLUSIONS: Addressing the arch by implantation of AMDS prolongs cardiopulmonary bypass and circulatory arrest time, however without relevant impairments of short-term outcome. Combining root surgery with replacement of the proximal aortic arch and AMDS implantation seems feasible and safe as it did not impair the early postoperative outcome.

Thoraflex Hybrid vs. AMDS: To replace the arch or to stent it in type A aortic dissection?

BACKGROUND: Acute type A aortic dissection (ATAAD) is a life-threatening medical emergency that requires urgent surgical intervention. The mainstay surgical approach to treating ATAAD with aortic arch involvement is total arch replacement (TAR). The frozen elephant trunk (FET) procedure involves TAR with hybrid endovascular stenting of the DTA in a single step using a hybrid prosthesis (HP). The prime example of a FET HP is Thoraflex Hybrid Prosthesis (THP). Another treatment option is the novel Ascyrus Medical Dissection Stent (AMDS) that is deployed as a non-covered stent along with the aortic arch as an adjunct to prior hemi-arch replacement. AIMS: This comparative review highlights the clinical applications and outcomes of THP and AMDS in the treatment of ATAAD and discusses the main differences between both approaches. METHODS: A comprehensive literature search was conducted using multiple electronic databases including PubMed, Google Scholar, Ovid, Scopus and Embase. RESULTS: TAR with FET can be considered the superior approach to managing ATAAD with arch involvement relative to AMDS with hemi-arch replacement due to more optimal clinical outcomes. Upon comprehensively searching the literature, early mortality was substantially lower with FET ranging from 0-11% compared to 12.5-18.7% using AMDS, with more favourable long-term survival. The incidence of kidney injury and new stroke post-FET ranged from 3-20% and 5-16%, and 11-37.5% and 0-18.8% following AMDS implantation. However, evidence supporting the use of AMDS is extremely limited. Meanwhile, TAR with FET is a well-established and well-described procedure for ATAAD repair. CONCLUSION: Despite the novel nature of AMDS, its clinical safety and effectiveness are yet to be proven. In conclusion, THP remains the best evidenced-based approach to treat ATAAD in this era.

Early Results of a Novel Hybrid Prosthesis for Treatment of Acute Aortic Dissection Type A With Distal Anastomosis Line Beyond Aortic Arch Zone Zero.

Introduction: Acute aortic dissection type A (AADA) is associated with high perioperative morbidity and mortality. A novel non-covered hybrid prosthesis (Ascyrus Medical Dissection Stent (AMDS) Hybrid Prosthesis, Cryolife/Jotec, Hechingen, Germany) can be easily implanted to stabilize the true lumen (TL), improve remodeling, and preserve organ perfusion. Although developed for implantation in aortic zone 0, occasionally, partial replacement of the aortic arch and further distal implantation of AMDS may appear favorable. Implantation of AMDS with anastomosis line beyond zone 0 has not been described yet. Materials and Methods: Between 08/2019 and 12/2020, a total of n = 97 patients were treated due to AADA at a single University hospital. Of those, n = 28 received an AMDS hybrid prosthesis, of whom in eight patients, due to intraoperative finding the distal anastomosis line was placed distal to the brachiocephalic trunk. Three patients had AMDS implantation in zone I and four were treated by implantation of the prostheses in zone II, and one patient had the implantation performed in zone III. Clinical outcome and the development of a proportional area of TL and false lumen (FL) at defined levels of the thoracic aorta were analyzed. Results: None of the surviving patients (87.5%) showed signs of clinical malperfusion (i.e., stroke, spinal cord injury, and need for dialysis). A postoperative CT scan showed an open TL in all patients. The proportion of TL with respect to total aortic diameter (TL+FL) was postoperatively significantly higher in zone III (p = 0.016) and at the level of T11 (p = 0.009). The mean area of TL+FL was comparable between pre- and postoperative CT-scan (p = n.s.). One patient with preoperative resuscitation died of multiple organ failure on extracorporeal life support on postoperative day 3. Conclusion: Implantation of AMDS can be safely performed in patients who need partial replacement of the aortic arch beyond zone 0. The advantages of the AMDS can be combined with those of the total arch repair (remodeling of the arch and prevention of TL collapse) without the possible disadvantages (risk of spinal cord injury).

In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild-type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains.

Aspergillus oryzae acetamidase catalyzes degradation of ethyl carbamate.

Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60 kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18 h indicated that AmdS was stable in 0%-40% ethanol solutions. The optimum temperature of AmdS was 40 °C. This enzyme showed urethanase activity at pH 6.4-9.6 and exhibited its highest activity at pH 9.6. The Km value of AmdS for EC was 8.2 mM, similar to the Km value (7.6 mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC.

Repair of acute type A dissection with distal malperfusion using a novel hybrid arch device.

Acute type A aortic dissection remains a high-risk surgical condition, and mortality among those presenting with malperfusion is up to 3-fold higher. Despite the added technical challenge of distal aortic arch interventions in the acute setting, it may be necessary to resolve distal malperfusion in patients with this disorder.  The ideal arch intervention to address acute type A aortic dissection complicated by malperfusion should address the following objectives: (1) to relieve distal malperfusion by expanding the distal true lumen and depressurizing the false lumen; (2) to avoid compromising arch branches without requiring additional arch branch interventions; (3) to minimize the risk of spinal cord ischemia; and (4) to minimize the operative duration and circulatory arrest time. The use of an uncovered aortic arch stent that is delivered in an antegrade manner during circulatory arrest, concomitantly with hemiarch replacement, therefore represents an attractive solution in the management of acute type A aortic dissection complicated by malperfusion. This strategy does not require complex arch reconstruction and may thus be a feasible option among cardiac and vascular surgeons in lower volume aortic centers.  Here we present a step-by-step approach to acute type A aortic dissection repair with hemiarch repair and delivery of an uncovered arch stent for a patient presenting with malperfusion.

Metabolism and pharmacokinetics studies of allyl methyl disulfide in rats.

1. Allyl methyl disulfide (AMDS) is one of the main compounds in garlic, whereas its metabolism has not been studied yet. 2. In this work, we first identified the metabolites of AMDS in rat erythrocytes and rats using GC-MS. The transformation mechanism study among different metabolites was then conducted. The apparent kinetics of AMDS in rat erythrocytes and pharmacokinetics of AMDS by oral administration in rats were also studied. 3. The metabolic pathway study showed that AMDS was mainly metabolized in rats to allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2) through mechanisms of reduction, methylation and oxidation. The transformation mechanism study indicated that AMDS was firstly reduced to allyl mercaptan (AM) in rat erythrocytes, and then methylated to allyl methyl sulfide (AMS) by S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and finally oxidized to AMSO and AMSO2 by liver microsomes. The half-life of AMDS in rat erythrocytes was 6.285 ± 0.014 min while the half-lives of its active metabolites AMSO and AMSO2 in vivo were 18.17 and 17.50 h, respectively. Also, the large AUCs of the two active metabolites were observed, indicating potential applications of AMDS for certain pharmacological effects.

Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering.

We have investigated the use of the gene coding for acetamidase (amdS) as a recyclable dominant marker for the methylotrophic yeast Komagataella phaffii in order to broaden its genetic toolbox. First, the endogenous constitutive AMD2 gene (a putative acetamidase) was deleted generating strain LA1. A cassette (amdSloxP) was constructed bearing a codon-optimized version of the Aspergillus nidulans amdS gene flanked by loxP sites for marker excision with Cre recombinase. This cassette was successfully tested as a dominant selection marker for transformation of the LA1 strain after selection on plates containing acetamide as a sole nitrogen source. Finally, amdSloxP was used to sequentially disrupt the K. phaffii ADE2 and URA5 genes. After each disruption event, a Cre-mediated marker recycling step was performed by plating cells on medium containing fluoroacetamide. In conclusion, amdS proved to be a suitable tool for K. phaffii transformation and marker recycling thus providing a new antibiotic-free system for genetic manipulation of this yeast.

Alternate Mediterranean diet score is positively associated with skeletal muscle mass index in middle-aged adults.

Researches have suggested Mediterranean diet might lower the risk of chronic diseases, but data on skeletal muscle mass (SMM) are limited. This community-based cross-sectional study examined the association between the alternate Mediterranean diet score (aMDS) and SMM in 2230 females and 1059 males aged 40-75 years in Guangzhou, China. General information and habitual dietary information were assessed in face-to-face interviews conducted during 2008-2010 and 3 years later. The aMDS was calculated by summing the dichotomous points for the items of higher intakes of whole grain, vegetables, fruits, legumes, nuts, fish and ratio of MUFA:SFA, lower red meat and moderate ethanol consumption. The SMM of the whole body, limbs, arms and legs were measured using dual-energy X-ray absorptiometry during 2011-2013. After adjusting for potential covariates, higher aMDS was positively associated with skeletal muscle mass index (SMI, SMM/height2, kg/m2) at all of the studied sites in males (all P trend<0·05). The multiple covariate-adjusted SMI means were 2·70 % (whole body), 2·65 % (limbs), 2·50 % (arms) and 2·70 % (legs) higher in the high (v. low) category aMDS in males (all P<0·05). In females, the corresponding values were 1·35 % (P trend=0·03), 1·05, 0·52 and 1·20 %, (P trend>0·05). Age-stratified analyses showed that the favourable associations tended to be more pronounced in the younger subjects aged less than the medians of 59·2 and 62·2 years in females and males (P interaction>0·10). In conclusion, the aMDS shows protective associations with SMM in Chinese adults, particularly in male and younger subjects.