Challenges and Approaches of Culturing the Unculturable Archaea.

Since Carl Woese's discovery of archaea as a third domain of life, numerous archaeal species have been discovered, yet archaeal diversity is poorly characterized. Culturing archaea is complicated, but several queries about archaeal cell biology, evolution, physiology, and diversity need to be solved by culturing and culture-dependent techniques. Increasing interest in demand for innovative culturing methods has led to various technological and methodological advances. The current review explains frequent hurdles hindering uncultured archaea isolation and discusses features for more archaeal cultivation. This review also discusses successful strategies and available media for archaeal culturing, which might be helpful for future culturing practices.

Biofilms as Battlefield Armor for Bacteria against Antibiotics: Challenges and Combating Strategies.

Bacterial biofilms are formed by communities, which are encased in a matrix of extracellular polymeric substances (EPS). Notably, bacteria in biofilms display a set of 'emergent properties' that vary considerably from free-living bacterial cells. Biofilms help bacteria to survive under multiple stressful conditions such as providing immunity against antibiotics. Apart from the provision of multi-layered defense for enabling poor antibiotic absorption and adaptive persistor cells, biofilms utilize their extracellular components, e.g., extracellular DNA (eDNA), chemical-like catalase, various genes and their regulators to combat antibiotics. The response of biofilms depends on the type of antibiotic that comes into contact with biofilms. For example, excessive production of eDNA exerts resistance against cell wall and DNA targeting antibiotics and the release of antagonist chemicals neutralizes cell membrane inhibitors, whereas the induction of protein and folic acid antibiotics inside cells is lowered by mutating genes and their regulators. Here, we review the current state of knowledge of biofilm-based resistance to various antibiotic classes in bacteria and genes responsible for biofilm development, and the key role of quorum sensing in developing biofilms and antibiotic resistance is also discussed. In this review, we also highlight new and modified techniques such as CRISPR/Cas, nanotechnology and bacteriophage therapy. These technologies might be useful to eliminate pathogens residing in biofilms by combating biofilm-induced antibiotic resistance and making this world free of antibiotic resistance.

Bilateral disease and early age at presentation are associated with shorter survival in patients with congenital heart disease and intraluminal pulmonary vein stenosis.

PURPOSE: Pulmonary vein stenosis (PVS) is a progressive disease that is frequently lethal. We have previously identified neoproliferation of myofibroblasts as the mechanism for progressive intraluminal PVS. PVS occurs in association with other congenital heart diseases (CHD) and in structurally normal hearts. This study sought to describe the spectrum of CHD seen with PVS and explore risk factors associated with mortality. METHODS: All patients diagnosed over a 12-year period with a combination of PVS involving ≥2 vessels and CHD were identified. Cases were categorized according to major anatomic and physiologic categories. Patient and disease characteristics associated with time to death were explored. RESULTS: Eighty-two cases followed longitudinally at our institution were analyzed. Anatomic diagnoses included nonheterotaxy + anomalous pulmonary venous return (29%), heterotaxy + anomalous veins (20%), two ventricles + normal veins (22%), and single ventricle + normal veins (29%). Median age at diagnosis was 5.3 months (0-24 years). Despite multiple treatments, there were 35 (43%) deaths in the group with an estimated survival of 71%, 64%, and 44% at 1, 2, and 5 years, respectively. Bilateral disease at diagnosis (hazard ratio [HR] 3.9 [1.7, 9.2], P= .002), age <5 months at diagnosis (HR 3.4 [1.6, 7.6], P= .002), and involvement of >2 pulmonary veins at diagnosis (HR 3.7 [1.6, 8.8], P= .003) were associated with shorter time to death in univariate analysis. In multivariable analysis, both bilateral disease (HR 2.9 [1.2, 7.1]P= .02) and age <5 months at diagnosis (HR 2.4 [1.1, 5.6]P= .03) were independently associated with time to death. CONCLUSION: Bilateral disease and earlier age at diagnosis are independent predictors of poor survival in patients with CHD and PVS, while patients with unilateral disease presenting at an older age have a better prognosis. These findings are helpful in risk stratification of patients with CHD and multivessel PVS.

A prospective phase II trial of vinblastine and methotrexate in multivessel intraluminal pulmonary vein stenosis in infants and children.

OBJECTIVE: To determine the safety and efficacy of the chemotherapeutic agents vinblastine and methotrexate in the treatment of children with progressive multivessel intraluminal pulmonary vein stenosis (PVS). METHODS: Children received weekly vinblastine and methotrexate for a period of 1 year. Outcomes (for patients receiving ≥1 month of chemotherapy) were classified separately for patients with isolated PVS and PVS with congenital heart disease (CHD). Primary efficacy outcome was "response to treatment" categorized by echocardiographic criteria of response. Survival to 1 year was also evaluated. All adverse events were classified according to Cancer Therapy Evaluation Program, Common Terminology Criteria version 3.0. Events were further classified as related to chemotherapy, cardiac, or other causes. RESULTS: Among 29 patients enrolled, 28 received at least one dose of chemotherapy and were evaluable for toxicity, while 23 were evaluable for response (21 CHD, 2 isolated). Both patients in the isolated group had progressive disease and died. Overall, 33% (7/21) of patients with PVS and CHD had stable disease; 1-year survival of 38%; and four patients continue in remission (93, 96, 124, and 125 months after treatment initiation). While both cardiac-related (19%) and chemotherapy-related (53%) toxicities were common, most were asymptomatic laboratory changes. Grade 3 (13%) and grade 4 (4%) toxicities were reversible, and no treatment-related grade 5 toxicities were observed. CONCLUSION: We report on the first prospective trial of chemotherapy for infants and children targeting the presence of myofibroblastic cells within the lesions of PVS based on myofibroblastic proliferation associated with desmoid tumors of infancy. The toxicity profile resulted in numerous treatment delays and interruptions that, combined with limited information on the natural history of PVS in this patient population, hampered our ability to determine the true efficacy of this approach. These results will be important as a baseline for clinical trials in this patient population.