Reflections on the Origin of Coded Protein Biosynthesis.

The principle of continuity posits that some central features of primordial biocatalytic mechanisms should still be present in the genetically dependent pathway of protein synthesis, a crucial step in the emergence of life. Key bimolecular reactions of this process are catalyzed by DNA-dependent RNA polymerases, aminoacyl-tRNA synthetases, and ribosomes. Remarkably, none of these biocatalysts contribute chemically active groups to their respective reactions. Instead, structural and functional studies have demonstrated that nucleotidic α-phosphate and ß-d-ribosyl 2' OH and 3' OH groups can help their own catalysis, a process which, consequently, has been called "substrate-assisted". Furthermore, upon binding, the substrates significantly lower the entropy of activation, exclude water from these catalysts' active sites, and are readily positioned for a reaction. This binding mode has been described as an "entropy trap". The combination of this effect with substrate-assisted catalysis results in reactions that are stereochemically and mechanistically simpler than the ones found in most modern enzymes. This observation is consistent with the way in which primordial catalysts could have operated; it may also explain why, thanks to their complementary reactivities, ß-d-ribose and phosphate were naturally selected to be the central components of early coding polymers.

Robotic-assisted versus laparoscopic splenectomy in children: a costeffectiveness study.

Laparoscopic elective splenectomy is considered as a safe surgical treatment of spleen non-traumatic blood disorders. However, robotic assisted splenectomy is becoming a promising alternative, although there are scarce studies in pediatric patients. Our aim is to compare the effectiveness and associated costs of both procedures in children. A single-institution retrospective study was performed among consecutive children undergoing splenectomy between 2004 and 2021, who were divided according to the surgical approach: LAS group (laparoscopic splenectomy) and RAS group (robotic assisted splenectomy). Demographics, clinical features, intraoperative blood loss, surgery time, length of hospital stay (LOS), postoperative complications, need for postoperative blood transfusion, readmission rate and economic data were compared. A total of 84 patients were included (23 LAS group; 61 RAS group), without demographic or clinical differences between them. RAS patients presented lower intraoperative blood loss (42 ± 15 vs. 158 ± 39 ml; p < 0.021) and shorter surgery time (135 ± 39 vs. 182 ± 68 min; p = 0.043), with no differences in median LOS (3 days in both groups). No intraoperative complications or conversion was reported. Five postoperative complications were observed: 4 in LAS patients (17.4%) versus only one in RAS (1.6%; p = 0.021). One reintervention was required in LAS group due to hemoperitoneum 12 h after splenectomy. RAS patients had lower postoperative blood transfusion requirements (1.6% vs. 13.0%; p = 0.025) and lower readmission rate (3.3 vs. 17.4%; p = 0.042). No differences were observed when comparing the median economic costs ($25,645 LAS vs. $28,135 RAS; p = 0.215). Robotic assisted splenectomy may be considered as a safe and feasible option in children compared to the traditional laparoscopic approach. Level of evidence: III.

The Role of Indocyanine Green in Pediatric Gastrointestinal Surgery: Systematic Review.

The use of near-infrared fluorescence imaging with indocyanine green (ICG) is actually considered as a very useful tool in decision-making strategy during challenging surgical procedures with a growing evidence in the literature. Our aim is to perform a systematic review focusing on ICG applications in gastrointestinal surgery. We conducted a systematic review with narrative synthesis in conformity with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines using PubMed, Medline, and EMBASE databases to identify articles describing the gastrointestinal perioperative use of ICG in children. We extracted data on study design, demographics, surgical indications, ICG dose, and perioperative outcomes. Eleven articles, including 94 pediatric patients, from 2013 to 2022 met the inclusion criteria for narrative synthesis in our systematic review, of which 6/11 (54.5%) were case reports, 4/11 (36.4%) were retrospective studies, and 1/11 (0.1%) were case series. Current clinical applications of ICG in gastrointestinal pediatric surgery included: esophagogastric surgery in 4/11 articles (36.4%), intestinal and pancreatic surgery in 3/11 articles (27.2%), and colorectal surgery in 4/11 articles (36.4%). ICG fluorescence in gastrointestinal pediatric surgery is a promising and safe technology that facilitates intraoperative localization of anatomical structures to achieve a more precise dissection and avoid injury to other adjacent tissues. It can be considered as a meaningful tool for assessing intestinal viability, as it provides objective data on tissue perfusion, and can impact the intraoperative decision in reconstructive surgeries requiring anastomosis. Future studies are needed to confirm these initial promising results. The lack of comparative and prospective studies is still the main limitation.

Acellular flowable dermal matrix for ostomy reconstruction: a safe and effective minimally invasive technique.

BACKGROUND: Acellular dermal matrices have long been used for complex abdominal wall closure and, more recently, for ostomy reconstruction. PURPOSE: To describe ostomy reconstruction with acellular flowable dermal matrix (AFDM) in a pediatric patient with a complex abdominal wall defect. CASE REPORT: A 14-year-old female who was diagnosed with unspecific hemorrhagic colitis at age 6 months and who underwent several abdominal surgeries (including total colectomy and terminal ileostomy) reported frequent, severe periostomy skin ulcerations secondary to improper fitting and ostomy bag leakage due to multiple periostomy skin retraction as a result of multiple abdominal scars. Under sedation, 2 small (5 mm) peristomal skin incisions were made, through which dissection and release of dermal scar tissue was performed. Afterwards, AFDM 40 mL was injected subcutaneously until a uniform, flat surface around the ostomy was achieved. The patient was discharged a few hours postoperatively, after verification of proper fit of the ostomy bag with no leakage. At 18-month follow-up, the patient was very satisfied with the result, with fewer ostomy bag changes and improved quality of life. CONCLUSION: This case report indicates that AFDM is a safe and effective minimally invasive technique for ostomy reconstruction, with minimal complications and satisfactory medium-term results.

Simultaneous Robotic-Assisted Splenectomy and Cholecystectomy in Children: Is It Safe and Effective?

Background: Hematologic conditions such as hereditary spherocytosis, sickle cell disease, and idiopathic thrombocytopenic purpura are frequently linked to cholelithiasis. In instances where symptoms are present, simultaneous cholecystectomy and splenectomy are commonly recommended. Our aim was to assess the outcomes of robotic-assisted procedures conducted for simultaneous surgical issues involving the spleen and gallbladder in pediatric patients. Materials and Methods: We have made a simultaneous retrospective study of children with hereditary hematological diseases who underwent combined robotic-assisted splenectomy and cholecystectomy at our institution from January 2010 to December 2021. Demographics, clinical features, intraoperative data, length of hospital stay, postoperative complications, and follow-up outcomes were analyzed. Results: A total of 11 patients (6 male; 5 female) were included, with a mean age of 13.9 ± 4.4 years (range 8-17). Hereditary spherocytosis was the most common disease (7 cases), followed by sick cell disease (4 cases), with associated symptomatic gallbladder litiasis in all of them. Both operations were carried out using the da Vinci® Surgical Si System in a single docking robotic platform (four robotic arms). Median total surgery time was 145 minutes (Q1-Q3: 115-162). Minimal intraoperative bleeding was recorded (mean 45 ± 15 mL), with no intraoperative complications or conversion. Median length of hospital stay was 3 days (Q1-Q3: 2-4). There were no cases of surgical wound infections or postoperative bleeding documented. Conclusion: Simultaneous robotic-assisted splenectomy and cholecystectomy can be considered safe and feasible interventions in children with hematological diseases that affect both the spleen and the gallbladder. However, further research is needed to enhance the existing evidence and establish a standardized approach.

Robotic-assisted vs. Laparoscopic Heller's Myotomy for Achalasia in Children.

INTRODUCTION: Robotic-assisted Heller-Dor procedure has been proposed as an alternative minimally invasive approach to traditional laparoscopy for the treatment of achalasia in children. Our aim is to compare the effectiveness, safety and associated costs between both procedures. METHODS: A retrospective single center study was conducted among consecutive children operated for achalasia (Heller-Dor operation) between 2005 and 2021, who were divided into two groups according to the surgical approach: laparoscopic (LAP-group) or robotic (ROB-group). Demographics, clinical features, surgery time, length of hospital stay (LOS), postoperative complications, long-term outcomes and economic data were compared between both groups. RESULTS: A total of 24 patients were included (12 in LAP-group; 12 in ROB-group), with no demographic or clinical differences between them. ROB-group patients presented lower intraoperative blood loss (23 ± 15 vs. 95 ± 15 ml; p < 0.001), shorter surgery time (178 ± 25 vs. 239 ± 55 min; p = 0.009) and shorter LOS, with a median of 2 days (Q1-Q3: 2-3) when compared to LAP-group (4 days [Q1-Q3:3-5]; p = 0.008). Three post-operative complications were reported, all in LAP-group (2 esophageal perforations and 1 esophageal tightness). After a median follow-up of 11 years, ROB-group patients presented fewer recurrences (0 vs 5; p = 0.039), less reintervention rate (0 % vs 41.7 %; p < 0.039) and lower associated economic costs (28,660$ vs. 60,360$; p < 0.001). CONCLUSION: This is the first comparative study of robotic and laparoscopic treatment of achalasia in children. Initial outcomes of robotic-assisted Heller-Dor procedure suggests some intraoperative (less blood loss and surgical time) and postoperative advantages (fewer complications and reinterventions). However, long-term studies with larger numbers of patients are needed. TYPE OF STUDY: Retrospective comparative study. LEVEL OF EVIDENCE: III.

Neutrophil-to-lymphocyte ratio as a predictor of postoperative complications and readmissions after appendectomy in children.

To analyze the role of neutrophil-to-lymphocyte ratio (NLR) in predicting the development of postoperative complications and readmission after appendectomy in children. A retrospective single-centered case-control study was conducted on children who underwent appendectomy between 2017 and 2020. Demographics, time since symptoms onset, laboratory tests at admission, postoperative complications, and readmissions in the first 30 days after surgery were recorded. Sensitivity and specificity analysis of the parameters evaluated were determined by the area under the curve (AUC) represented on the receiver operating characteristic (ROC) curves. A total of 1,214 patients (765 males; 449 females) were included, with a median age at diagnosis of 10.4 years. The median time from symptom onset was 24 h. During the first 30 days after surgery, 149 postoperative complications were reported (12.3%). NLR at admission presented the highest AUC (0.753), with a cut-off point of 10.5 for maximum sensitivity (68.7%) and specificity (86.1%). Readmissions were reported in 45 cases (3.7%). NLR at admission presented an AUC of 0.794 significantly higher than neutrophils (0.696), leukocytes (0.654), and time since symptom onset (0.622), making these differences statistically significant (p < 0.001). The cut-off point of NLR > 12.4 was estimated, with a maximum sensitivity and specificity of 71.0% and 82.3% for predicting readmission. NLR is an independent predictor of postoperative complications and readmission in children with acute appendicitis. While its application in routine clinical practice has yet to be established, the NLR may provide clinicians with a tool for identifying high-risk surgical patients.

Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse Alphaproteobacteria that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo. However, they were significantly more stable than wild-type RirA when exposed to O2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.

Mucosal appendicitis: How can it be differentiated from nonappendicitis?

OBJECTIVES: Mucosal appendicitis is defined by neutrophilic infiltration limited to the mucosa, with no transmural invasion; it is currently a controversial entity. The aim of our study was to determine whether mucosal appendicitis represents an early stage of acute appendicitis (AA) or should be considered a negative appendectomy. METHODS: A retrospective study was performed of children with suspected AA who underwent surgical treatment between 2017 and 2020. The participants were divided into 2 groups according to histologic appendiceal findings: mucosal appendicitis (MA) and negative appendicitis (NA). Demographic, clinical, ultrasound, and laboratory features were compared between the groups. RESULTS: A total of 1269 patients with suspected appendicitis in whom appendectomy was performed were included, with a median age of 10.5 years. Mucosal appendiceal inflammation was histologically confirmed in 30 cases (MA group), while no inflammation or other pathologic findings were observed in 25 cases (NA group), with no differences in demographic, clinical, or ultrasound features between the groups. Those in the MA group presented with significantly higher leukocyte and neutrophil counts and higher neutrophil to lymphocyte ratios (NLRs) than those in the NA group (P < .001). The NLR was the parameter with the highest area under the curve (0.736) for the diagnosis of MA. A cutoff of 3.20 was established, with a maximum sensitivity and specificity of 62.5% and 78.9%, respectively. CONCLUSIONS: Mucosal appendicitis presents with laboratory and histologic inflammatory features that can be distinguished from nonappendicitis and should therefore be considered a pathologic entity within the spectrum of AA. Preoperative leukocyte and neutrophil counts and NLRs may help reduce the number of negative appendectomies.

Robotic-assisted versus laparoscopic redo antireflux surgery in children: A cost-effectiveness study.

INTRODUCTION: Robotic-assisted redo fundoplication has some advantages compared to the laparoscopic approach in adults, although to date there are no studies in children. METHODS: A retrospective case-control study was performed among consecutive children who underwent redo antireflux surgery between 2004 and 2020, divided into two groups: LAF group (laparoscopic redo-fundoplication) and RAF group (robotic-assisted redo-fundoplication). Demographics, clinical, intraoperative, postoperative and economic data were compared. RESULTS: A total of 24 patients were included (10 LAF group; 14 RAF group) without demographic or clinical differences. The RAF group presented lower intraoperative blood loss (52 ± 19 vs. 145 ± 69 mL; p < 0.021), shorter surgery time (135 ± 39 vs. 179 ± 68 min; p = 0.009) and shorter length of hospital stay (median 3 days [2-4] vs. 5 days [3-7]; p = 0.002). The RAF group presented a higher rate of symptom improvement (85.7% vs. 60%; p = 0.192) and lower overall associated economic costs (25 800$ vs. 45 500$; p = 0.012). CONCLUSION: Robotic-assisted redo antireflux surgery may offer several benefits over the laparoscopic approach. Prospective studies are still needed.

Neutrophil-to-lymphocyte ratio as a predictor of surgical site infection in acute appendicitis.

Background: Surgical site infection (SSI) is one of the most common complications after appendectomy, which carries high associated morbidity. Therefore, it is essential to determine SSI predictive factors in order to prevent its occurrence. The aim of this study is to explore the role of neutrophil-to-lymphocyte ratio (NLR) as a predictor of SSI after appendectomy in children. Methods: A single-center, retrospective cohort study was performed in children who underwent appendectomy between 2017-2020. Demographics, time since symptoms onset, laboratory tests at admission, ultrasound appendiceal diameter, rate of complicated appendicitis, surgical aproach, surgery time and SSI rate were analyzed. Follow-up was performed during hospitalization and at outpatient clinic at 2 weeks and 30 days postoperatively to assess the surgical wound aspect. Diagnostic cut-off values of these markers for SSI prediction were based on the significance in the univariate analysis. Variables with a P value <0.05 in the univariate analysis were then entered into the multivariate analysis. Results: A total of 1,136 patients (710 males; 426 females) were included. SSI was reported in 53 patients (4.7%) during the 30-day follow-up after appendectomy (SSI group), with no demographic differences with the control group. Time since symptoms onset was significantly higher in SSI group (24 vs. 18 hours; P=0.034), as well as ultrasound appendiceal diameter (10.5 vs. 8.5 mm; P=0.010). Complicated appendicitis was observed in about 60% of both groups, without differences in surgical approach between them. Surgery time was statistically higher in the SSI group (62.4 vs. 47.9 min; P<0.001). SSI group presented higher counts of leukocytes, neutrophils and NLR than control group (P<0.001). NLR was the parameter with the highest area under the curve (AUC) (AUC =0.808; P<0.001), with a cut-off point of 9.8 with maximum sensitivity (77.8%) and specificity (72.7%). NLR was an independent predictive factor for SSI in the multivariate analysis [odds ratio (OR) 1.82 (1.13-2.73); P<0.01]. Conclusions: NLR value at admission was the most promising predictive factor for the development of SSI in children undergoing appendectomy. It is an easy, simple, inexpensive, and rapid method to detect patients at high risk for SSI. However, further prospective studies are still needed to confirm these results.

Reflections on the Origin and Early Evolution of the Genetic Code.

Examination of the genetic code (GeCo) reveals that amino acids coded by (A/U) codons display a large functional spectrum and bind RNA whereas, except for Arg, those coded by (G/C) codons do not. From a stereochemical viewpoint, the clear preference for (A/U)-rich codons to be located at the GeCo half blocks suggests they were specifically determined. Conversely, the overall lower affinity of cognate amino acids for their (G/C)-rich anticodons points to their late arrival to the GeCo. It is proposed that i) initially the code was composed of the eight (A/U) codons; ii) these codons were duplicated when G/C nucleotides were added to their wobble positions, and three new codons with G/C in their first position were incorporated; and iii) a combination of A/U and G/C nucleotides progressively generated the remaining codons.

How does the number of training years in pediatric surgery affect appendectomy outcomes?

INTRODUCTION: Appendectomy has traditionally been considered as a training operation for junior pediatric surgeons during their training period. However, with the increase of laparoscopic appendectomy, there has been a growing concern about the performance of this procedure by junior trainees. Our aim is to analyze intra-/postoperative appendectomy outcomes according to the number of training years during Pediatric Surgical residency training program. METHODS: A retrospective study was performed in patients who underwent appendectomy between 2018 and 2021 in our institution, who were divided into 5 groups according to the number of training years of the junior surgeon who performed the intervention (Y1-Y5). Demographics, complicated appendicitis rate, operation time, and postoperative complications were compared. A stratified analysis according to the technique performed (open/laparoscopic) was performed. RESULTS: A total of 1274 appendectomized patients were analyzed, of which 1257 (98.7%) were operated on by junior trainees (81 in Y1; 407 in Y2; 337 in Y3; 261 in Y4; and 171 in Y5) without demographic differences between groups. As the year of training increased, an elevation in complicated appendicitis rate was observed, although without statistically significant differences. However, laparoscopic/open appendectomies ratio increased with increasing year of training (p < 0.001). Operative time decreased significantly with increasing year of training (p < 0.001), both in open and laparoscopic appendectomies. There were no significant differences in postoperative complications, nor in the stratified analysis according to surgical technique. CONCLUSION: Appendectomy performed by junior pediatric surgery trainees can be considered a safe procedure from the first year of training, regardless of the technique used.

Neutrophil-to-Lymphocyte Ratio as a Predictor of the Need for Surgical Treatment in Children's Intussusception.

OBJECTIVE: Neutrophil-to-lymphocyte ratio (NLR) is an emerging inflammatory marker in abdominal pathologies. Ileocolic intussusception (ICI) involves a progressive intestinal inflammation, and the effectiveness of nonsurgical treatment (enema) might be related to the inflammation degree, although no previous studies have investigated this relationship. Our aim is to identify predictors of the need for surgical treatment in ICI. MATERIALS AND METHODS: A single-center, retrospective, case-control study was performed in children with ICI, who were treated with initial nonsurgical management between 2005 and 2019. Patients were divided in two groups: A (effective enema) and B (need for surgery). Admission demographic and clinical and laboratory data were analyzed. Specificity and sensitivity of the different parameters as predictors of the need for surgical treatment were determined by receiver operating characteristic (ROC) curves. RESULTS: A total of 511 patients were included (410: group A; 101: group B), without statistically significant demographic differences. Group B presented significantly higher frequency of vomiting, bloody stools, and longer median time since symptoms onset (24 vs. 8 hours; p < 0.001). Group B presented higher median laboratory inflammatory markers than group A: NLR (6.8 vs. 1.8; p < 0.001), neutrophils (10,148 vs. 7,468; p < 0.001), and C-reactive protein (CRP; 28.2 vs. 4.7; p < 0.001). In ROC curve analysis, NLR had an area under the curve of 0.925, higher than neutrophil count (0.776; p = 0.001), CRP (0.670; p = 0.001), and time since symptoms onset (0.673; p = 0.001). It was estimated a cut-off point of NLR greater than 4.52 (sensitivity: 73.2%; specificity: 94.5%). CONCLUSION: High NLR values imply a high degree of bowel inflammation and might anticipate the need for surgical treatment in ICI in children. LEVEL OF EVIDENCE: III.

Structural determinants of DNA recognition by the NO sensor NsrR and related Rrf2-type [FeS]-transcription factors.

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes. Important structural differences reside in the variation of the DNA minor and major groove widths. In addition, different DNA curvatures and different interactions with the protein sensors are observed. We also report studies of NsrR binding to four hmpA1 variants, which indicate that flexibility in the central region is not a key binding determinant. Our study explores the promotor binding specificities of three closely related transcriptional regulators.

Surgical Treatment of Bilateral Tubo-Ovarian Abscess in a Pre-Coitarchal Female.

Tubo-ovarian abscess (TOA) is a complication of pelvic inflammatory disease that is most commonly seen in sexually active females and seldom in pre-coitarchal adolescents. Initial treatment is generally parenteral antibiotics but often requires more invasive surgical procedures. We present the case of a 12-year-old, non-sexually active adolescent, with bilateral TOA who ultimately underwent a sterilizing bilateral salpingectomy and appendectomy for treatment.

Quinolinate Synthase: An Example of the Roles of the Second and Outer Coordination Spheres in Enzyme Catalysis.

The activation energy barrier of biochemical reactions is normally lowered by an enzyme catalyst, which directly helps the weakening of the bond(s) to be broken. In many metalloenzymes, this is a first coordination sphere effect. Besides having a direct catalytic action, enzymes can fix their reactive groups and substrates so that they are optimally positioned and also modify the water activity in the system. They can either activate substrates prior to their reaction or bind preactivated substrates, thereby drastically reducing local entropic effects. The latter type is well represented by some bisubstrate reactions, where they have been defined as "entropic traps". These can be described as "second coordination sphere" processes, but enzymes can also control the reactivity beyond this point through local conformational changes belonging to an "outer coordinate sphere" that can be modulated by substrate binding. We have chosen the [4Fe-4S] cluster-dependent enzyme quinolinate synthase to illustrate each one of these processes. In addition, this very old metalloenzyme shows low in vitro substrate binding specificity, atypical reactivity that produces dead-end products, and a unique modulation of its active site volume.

The Complex Roles of Adenosine Triphosphate in Bioenergetics.

ATP is generally defined as the "energy currency" of the cell. Its phosphoanhydride P-O bonds are often considered to be "high energy" linkages that release free energy when broken, and its hydrolysis is described as "strongly exergonic". However, breaking bonds cannot release energy and ATP hydrolysis in motor and active transport proteins is not "strongly exergonic". So, the relevance of ATP resides elsewhere. As important as the nucleotide are the proteins that undergo functionally relevant conformational changes upon both ATP binding and release of ADP and inorganic phosphate. ATP phosphorylates proteins for signaling, active transport, and substrates in condensation reactions. The ensuing dephosphorylation has different consequences in each case. In signaling and active transport the phosphate group is hydrolyzed whereas in condensation reactions the phosphoryl fragment acts as a dehydrating agent. As it will be discussed in this article, ATP does much more than simply contribute free energy to biological processes.

Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe4S4]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.