Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

Annual pulses of copper-enriched sediment in a North American river downstream of a large lake following the catastrophic failure of a mine tailings storage facility.

Failures of mine tailings storage facilities (TSF) can have profound and long-lasting effects on the downstream receiving environment. Virtually all spills to date have been into river systems without large lakes that may buffer downstream impacts. In August 2014, the failure of the Mount Polley copper (Cu)-gold mine TSF in British Columbia, Canada, released ~25 × 106 m3 of water and solids; globally, this is the second largest TSF spill in history. Over 18 × 106 m3 was delivered to Quesnel Lake, which is ~9 km from the TSF and is the third deepest lake in North America, and a crucial habitat for Pacific salmon and trout populations. We determined the sediment-associated Cu concentrations and fluxes in Quesnel River, downstream of the lake, from August 2014 to February 2021 based on the analysis of >400 samples of sediment, mainly collected using a continuous-flow centrifuge. During each winter since the spill, Cu concentrations in the fluvial sediment in the upper reaches of the river (~35 km from the TSF) were elevated relative to regional background concentrations and samples collected before the spill. Maximum Cu concentrations were ~410 mg kg-1 which exceeds Canadian sediment quality guidelines for the protection of aquatic organisms (197 mg kg-1). Monitoring of Quesnel Lake since the spill shows that these annual pulses in the winter are due to resuspension of unconsolidated tailings and sediments at the bottom of Quesnel Lake, during autumnal lake turnover, which become mixed throughout the water column and subsequently flow into Quesnel River. Results show that while large lakes may buffer downstream aquatic systems from contaminated sediment, they may prolong the environmental impact. These findings are crucial in understanding how lake processes may modify the effects of TSF spills on downstream aquatic systems.

Long-term oncological outcomes of endoscopic full-thickness resection after previous incomplete resection of low-risk T1 CRC (LOCAL-study): study protocol of a national prospective cohort study.

BACKGROUND: T1 colorectal cancer (CRC) without histological high-risk factors for lymph node metastasis (LNM) can potentially be cured by endoscopic resection, which is associated with significantly lower morbidity, mortality and costs compared to radical surgery. An important prerequisite for endoscopic resection as definite treatment is the histological confirmation of tumour-free resection margins. Incomplete resection with involved (R1) or indeterminate (Rx) margins is considered a strong risk factor for residual disease and local recurrence. Therefore, international guidelines recommend additional surgery in case of R1/Rx resection, even in absence of high-risk factors for LNM. Endoscopic full-thickness resection (eFTR) is a relatively new technique that allows transmural resection of colorectal lesions. Local scar excision after prior R1/Rx resection of low-risk T1 CRC could offer an attractive minimal invasive strategy to achieve confirmation about radicality of the previous resection or a second attempt for radical resection of residual luminal cancer. However, oncologic safety has not been established and long-term data are lacking. Besides, surveillance varies widely and requires standardization. METHODS/DESIGN: In this nationwide, multicenter, prospective cohort study we aim to assess feasibility and oncological safety of completion eFTR following incomplete resection of low-risk T1 CRC. The primary endpoint is to assess the 2 and 5 year luminal local tumor recurrence rate. Secondary study endpoints are to assess feasibility, percentage of curative eFTR-resections, presence of scar tissue and/or complete scar excision at histopathology, safety of eFTR compared to surgery, 2 and 5 year nodal and/or distant tumor recurrence rate and 5-year disease-specific and overall-survival rate. DISCUSSION: Since the implementation of CRC screening programs, the diagnostic rate of T1 CRC is steadily increasing. A significant proportion is not recognized as cancer before endoscopic resection and is therefore resected through conventional techniques primarily reserved for benign polyps. As such, precise histological assessment is often hampered due to cauterization and fragmentation and frequently leads to treatment dilemmas. This first prospective trial will potentially demonstrate the effectiveness and oncological safety of completion eFTR for patients who have undergone a previous incomplete T1 CRC resection. Hereby, substantial surgical overtreatment may be avoided, leading to treatment optimization and organ preservation. Trial registration Nederlands Trial Register, NL 7879, 16 July 2019 ( https://trialregister.nl/trial/7879 ).

Change of carbon source causes dramatic effects in the phospho-proteome of the archaeon Sulfolobus solfataricus.

Protein phosphorylation is known to occur in Archaea. However, knowledge of phosphorylation in the third domain of life is rather scarce. Homology-based searches of archaeal genome sequences reveals the absence of two-component systems in crenarchaeal genomes but the presence of eukaryotic-like protein kinases and protein phosphatases. Here, the influence of the offered carbon source (glucose versus tryptone) on the phospho-proteome of Sulfolobus solfataricus P2 was studied by precursor acquisition independent from ion count (PAcIFIC). In comparison to previous phospho-proteome studies, a high number of phosphorylation sites (1318) located on 690 phospho-peptides from 540 unique phospho-proteins were detected, thus increasing the number of currently known archaeal phospho-proteins from 80 to 621. Furthermore, a 25.8/20.6/53.6 Ser/Thr/Tyr percentage ratio with an unexpectedly high predominance of tyrosine phosphorylation was detected. Phospho-proteins in most functional classes (21 out of 26 arCOGs) were identified, suggesting an important regulatory role in S. solfataricus. Focusing on the central carbohydrate metabolism in response to the offered carbon source, significant changes were observed. The observed complex phosphorylation pattern hints at an important physiological function of protein phosphorylation in control of the central carbohydrate metabolism, which might particularly operate in channeling carbon flux into the respective metabolic pathways.

Complementation of Sulfolobus solfataricus PBL2025 with an α-mannosidase: effects on surface attachment and biofilm formation.

Compared to Sulfolobus solfataricus P2, the S. solfataricus mutant PBL2025 misses 50 genes (SSO3004-3050), including genes coding for a multitude of enzymes possibly involved in sugar degradation or metabolism. We complemented PBL2025 with two of the missing proteins, the α-mannosidase (SSO3006, Ssα-man) and the ß-galactosidase LacS (SSO3019), and performed comparative fluorescence microscopy and confocal laser scanning microscopy to analyze the recombinant strains. We demonstrated that the Ssα-man complemented strain resembled the S. solfataricus P2 behavior with respect to attachment of cells to glass and growth of cells in static biofilms. During expression of the Ssα-man, but not LacS, glucose and mannose-containing extracellular polymeric substance (EPS) levels changed in the recombinant strain during surface attachment and biofilm formation. These results suggest that the Ssα-man might be involved in the modulation of the EPS composition and/or in the de-mannosylation of the glycan tree, which is attached to extracellular glycosylated proteins in S. solfataricus. On the other hand, LacS expression in PBL2025 reduced the carbohydrate content of the isolated total EPS implying a role in the modulation of the produced EPS during static biofilm formation. These are the first enzymes identified as playing a role in archaeal EPS formation.

Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus.

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

Plasma concentrations of N-terminal brain natriuretic peptide in healthy children, adolescents, and young adults: effect of age and gender.

Children with congenital heart disease need adequate diagnostic classification regarding their cardiovascular status (CVS). N-terminal brain natriuretic peptide (N-BNP) plasma concentration indicates dysfunction of the cardiovascular system and guides decisions concerning treatment and prognosis. Reference values are established for adults, with age-dependent increasing values and higher values in women. To avoid misclassification concerning the CVS, a large group of healthy children and adolescents can be used show the relationship between gender, age, and N-BNP and these can serve as reference values. N-BNP was measured in 434 healthy subjects (240 female and 194 male) with ages ranging from 0 to 32 years without any cardiovascular disease or renal or hepatic impairment. Measurements were performed with an electrochemiluminescence immunoassay from Roche Diagnostics. Mean N-BNP decreased from 12.6 fmol/ml (0-9 years; n = 79) to 9.41 fmol/ml (10-14 years; n = 154) and in adolescents from 6.1 (15-19 years; n = 99) to 4.8 fmol/ml (> 19 years; n = 102) in adults (p < 0.05). Mean N-BNP concerning gender did not differ in any age group younger than 19 years. In contrast, the adult female group had 78% higher N-BNP compared to the male group (p < 0.05). There was a significant peak in N-BNP at the age of 12-14 years. This study shows that reference values for N-BNP differed profoundly in children compared to adults and were up to 260% higher in children without any gender difference. Therefore, these reference values will help to avoid CVS misclassification in children for the biomarker N-BNP.

Bioenergetics and solute uptake under extreme conditions.

The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.

Beneficial effects of alpha-lipoic acid and ascorbic acid on endothelium-dependent, nitric oxide-mediated vasodilation in diabetic patients: relation to parameters of oxidative stress.

The impairment of nitric oxide (NO)-mediated vasodilation in diabetes has been attributed to increased vascular oxidative stress. Lipoic acid has been shown to have substantial antioxidative properties. The aim of this study was to assess the effect of lipoic acid on NO-mediated vasodilation in diabetic patients in comparison with the well-recognized effect of ascorbic acid. Using venous occlusion plethysmography, we examined the effects of lipoic acid (0.2 mM) and ascorbic acid (1 and 10 mM) on forearm blood flow responses to acetylcholine, sodium nitroprusside and concomitant infusion of the NO-inhibitor, N(G)-monomethyl-L-arginine, in 39 diabetic patients and 11 control subjects. Plasma levels of antioxidants and parameters of lipid peroxidation were measured and correlated to endothelial function tests. Lipoic acid improved NO-mediated vasodilation in diabetic patients, but not in controls. NO-mediated vasodilation was improved by ascorbic acid at 10 mM, but not 1 mM. Improvements of endothelial function by ascorbic acid and lipoic acid were closely related. The beneficial effects of lipoic acid were positively related to plasma levels of malondialdehyde and inversely related to levels of ubiquinol-10. These findings support the concept that oxidative stress contributes to endothelial dysfunction and suggest a therapeutic potential of lipoic acid particularly in patients with imbalance between increased oxidative stress and depleted antioxidant defense.

Detection of neonatal carnitine palmitoyltransferase II deficiency by expanded newborn screening with tandem mass spectrometry.

The introduction of tandem mass spectrometry to newborn screening has substantially expanded our ability to diagnose metabolic diseases in the newborn period. We report the first case of neonatal carnitine palmitoyltransferase deficiency II detected by expanded newborn screening with tandem mass spectrometry. The neonate presented with dysmorphic facial features, structural malformations, renal failure, seizures, and cardiac arrythmias and died on the third day of life. This experience illustrates the importance of expanded newborn screening to avoid missing a metabolic diagnosis in early infantile death.

MR findings in athletes with pubalgia.

OBJECTIVE: To describe the MR findings in athletes with pubalgia. DESIGN AND PATIENTS: Pelvic MR images of 32 athletes (30 men, 2 women) with pubalgia were studied. T1-weighted and T2-weighted (SE and FSE) and STIR images in the axial and coronal planes were obtained on a 1.5-T system. Images were reviewed for general pelvic pathology. Special attention was given to the pubic symphysis, groin and pelvic musculature, and to the abdominal wall musculature. RESULTS: Thirty surgically confirmed cases comprise the study group. Abnormalities in the following were found: pubic symphysis (21/30), abdominal wall (27/30), groin musculature, including rectus abdominis (21/30), pectineus (6/30), and adductor muscle group (18/30). CONCLUSIONS: Pubalgia is a complex process which is frequently multifactorial. The MRI findings can alter the surgical approach.

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters.

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.

Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with Type II diabetes mellitus.

AIMS/HYPOTHESIS: Tetrahydrobiopterin is an essential cofactor of nitric oxide synthase, and its deficiency decreases nitric oxide bioactivity. Our aim was to find whether supplementation of tetrahydrobiopterin could improve endothelial dysfunction in diabetic patients. METHODS: Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine (0.75-3.0 microg x 100 ml(-1) x min(-1)) and to the endothelium-independent vasodilator sodium nitroprusside (0.1-1.0 microg x 100 ml(-1) x min(-1)) before and during concomitant intra-arterial infusion of tetrahydrobiopterin (500 microg/min) were measured by venous occlusion plethysmography in 12 control subjects and 23 patients with Type II (non-insulin-dependent) diabetes mellitus. RESULTS: In control subjects, tetrahydrobiopterin had no effect on the dose-response curves to acetylcholine and sodium nitroprusside. In contrast, in diabetic patients, the attenuated endothelium-dependent vasodilation to acetylcholine was considerably improved by concomitant treatment with tetrahydrobiopterin, whereas the endothelium-independent vasodilation was not affected. This beneficial effect of tetrahydrobiopterin in diabetic patients could be completely blocked by N(G)-monomethyl-L-arginine. CONCLUSION/INTERPRETATION: These findings suggest the possibility that endothelial dysfunction in Type II diabetes might be related to decreased availability of tetrahydrobiopterin.

Adaptations of the archaeal cell membrane to heat stress.

In extreme environments varying from hot to cold, acidic to alkaline, and highly saline, mainly Archaea are found. Thermophilic and extremely acidophilic Archaea have a membrane that contains membrane spanning tetraether lipids. These tetra-ether membranes have a limited permeability for protons even at the high temperatures of growth and this property makes it possible for thermophilic archaea to maintain a viable proton motive force under the extreme conditions. -Ether lipids cannot be degraded easily and are highly stable which is also a requirement for life under extreme conditions. Psychrophilic and mesophilic Bacteria, and all Archaea adjust the lipid composition of their membranes so that the proton permeability of their membranes remains within a narrow range. This phenomenon is termed 'homeoproton permeability adaptation'. Thermophilic Bacteria are the only prokaryotes that are unable to control the proton permeability of their membranes. These organisms have to rely on the less permeable sodium ions in energy transducing processes in their membrane.

Vulvitis circumscripta plasmacellularis mimicking child abuse.

Vulvitis circumscripta plasmacellularis (VCP) is a rare, benign vulvar disorder that is typically described in adult women. Our case occurred in an 8-year-old girl. The primary diagnostic concern was sexual abuse. VCP may also mimic lichen sclerosus, extramammary Paget's disease, pemphigus vulgaris, fixed drug eruption, squamous cell carcinoma, candidiasis, allergic contact dermatitis, and herpes simplex infection. Evaluation should include a biopsy because the histopathologic features of VCP are distinctive.

Hepatic arterial 5-fluorouracil in patients with liver metastases of colorectal cancer: single-centre experience in 145 patients.

BACKGROUND: Hepatic arterial chemotherapy for liver metastases of colorectal cancer is still under discussion. Mainly because of the technical complications of this mode of treatment and the lack of a survival benefit in randomized studies. We performed an analysis of hepatic arterial 5-fluorouracil (5-FU) chemotherapy in 145 consecutive patients treated at a single institution. PATIENTS AND METHODS: One hundred forty-five patients with inoperable liver metastases from colorectal cancer were included. 5-FU, 1000 mg/m2/day continuous infusion for five days every three weeks, was delivered in the hepatic artery by percutaneous catheter or arterial access device. RESULTS: The response rate was 34% for all patients, 40% in patients with extrahepatic disease, and 15% in patients with i.v. 5-FU-based pretreatment. TTP and OS for all patients were 7.5 and 14.3 months, respectively. In patients with extrahepatic disease or i.v. 5-FU-based pretreatment, OS was significantly shorter compared to patients without extrahepatic disease or 5-FU-based pretreatment (9.7 vs. 19.3 months and 10.1 vs. 17.4 months, respectively), forty-seven percent of patients stopped treatment because of a complication. Complications most often seen in patients with arterial ports were hepatic artery thrombosis (48%) and dislocation of the catheter (22%). CONCLUSIONS: The results of our analysis are in line with previous phase III studies. Extrahepatic disease and i.v. 5-FU-based pretreatment were prognostic for reduced OS. The complication rate of hepatic arterial delivery was worrisome. although, no negative impact on survival could be established. There is a strong need for improvement of hepatic arterial delivery methods before further evaluation of hepatic arterial

Genetic screening of newborns.

Screening of newborn infants for genetic disease began over 35 years ago as a public health measure to prevent mental retardation in phenylketonuria (PKU). It was so successful that tests for several other genetic disorders were added. We review the current status of this screening, including discussions of the genetic disorders often covered and the results of newborn screening for them. We emphasize recent advances. These include expansion of coverage for genetic disorders with the new methodology of tandem mass spectrometry (MS-MS) and the introduction of molecular (DNA) testing to increase the specificity of testing for several disorders, thereby reducing false-positive rates. These and other advances have also produced issues of criteria for screening, missed cases, and appropriate use of stored newborn specimens.