Influence of extracellular ATP on mammalian sperm physiology.

In addition to its central role in cellular metabolism, adenosine 5'-triphosphate (ATP) is an important extracellular signalling molecule involved in various physiological processes. In reproduction, extracellular ATP participates in both autocrine and paracrine paths regulating gametogenesis, gamete maturation and fertilisation. This review focusses on how extracellular ATP modulates sperm physiology with emphasis on the mammalian acrosome reaction. The presence of extracellular ATP in the reproductive tract is primarily determined by the ion channels and transporters that influence its movement within the cells comprising the tract. The main targets of extracellular ATP in spermatozoa are its own transporters, particularly species-specific sperm purinergic receptors. We also discuss notable phenotypes from knock-out mouse models and human Mendelian inheritance related to ATP release mechanisms, along with immunological, proteomic, and functional observations regarding sperm purinergic receptors and their involvement in sperm signalling.

Case report: A rare case of cyclodialysis after cataract surgery managed exclusively with medical therapy.

We present the case of a 68-year-old female who presented with a substantial cyclodialysis after phacoemulsification identified by anterior segment optical coherence tomography and ultrasound biomicroscopy. Gonioscopy is challenging because of its shallow anterior chamber. Due to lack of response to topical steroids and refusal by the patient for surgical treatment, topical steroids were tapered, and cycloplegic was initiated. After two weeks, the cyclodialysis cleft was closed on optical coherence tomography persisting residual subchoroidal fluid, topical treatment was mantained. Despite of this, visual acuity and hypotony were restored. Finally at 2 months visit subchoroidal fluid was reabsorbed and patient was discharged. Clinics should consider the possibility of cyclodialysis in cases of unjustified hypotony during postoperative period. In addition, we recommend performing anterior segment imaging which includes ultrasound biomicroscopy and optical coherence tomography, when the evaluation of angular structures is unfeasible. Finally, we propose medical management for severe cyclodialysis as the first option considering anti-inflammatory drugs could perpetrate the separation between scleral spur and ciliary muscle.

[Translated article] Clinical and Histopathologic Features of Subungual Melanoma in 57 Mexican Patients: A 5-Year Cohort Study.

Although subungual melanoma is uncommon, it is associated with worse outcomes than melanomas in other locations and accounts for 1% to 23% of all melanomas, depending on the population. The aim of this study was to describe the clinical and histopathologic features of subungual melanoma in a Mexican population. We identified 303 patients with melanoma, and of these, 19% (57 patients with a median age of 71 years) had subungual melanoma. The main sites affected were the lower limbs (52.6%) and the toe (75.4%). The most common histologic subtype was acral lentiginous melanoma (50.9%). Median Breslow thickness was 3 mm, and stage IA tumors were the most common (in 28.1% of patients). Recurrence and metastasis occurred in 19.3% and 8.8% of patients, respectively. The clinical and histopathologic features identified are similar to those described in the literature. Early diagnosis and treatment are crucial for improving prognosis.

Clinical and Histopathologic Features of Subungual Melanoma in 57 Mexican Patients: A 5-Year Cohort Study. / Características clínicas e histopatológicas de melanoma subungueal en 57 pacientes mexicanos: una cohorte de 5 años.

Although subungual melanoma is uncommon, it is associated with worse outcomes than melanomas in other locations and accounts for 1% to 23% of all melanomas, depending on the population. The aim of this study was to describe the clinical and histopathologic features of subungual melanoma in a Mexican population. We identified 303 patients with melanoma, and of these, 19% (57 patients with a median age of 71 years) had subungual melanoma. The main sites affected were the lower limbs (52.6%) and the toe (75.4%). The most common histologic subtype was acral lentiginous melanoma (50.9%). Median Breslow thickness was 3 mm, and stage IA tumors were the most common (in 28.1% of patients). Recurrence and metastasis occurred in 19.3% and 8.8% of patients, respectively. The clinical and histopathologic features identified are similar to those described in the literature. Early diagnosis and treatment are crucial for improving prognosis.

High Ligation of the Inferior Mesenteric Artery in Left-Sided Colon and Rectal Cancer Resection: Rates of Success and Outcomes.

BACKGROUND: High ligation of the inferior mesenteric artery, defined as ligation before the takeoff of the left colic artery, is often described as the gold standard in low left-sided colon and rectal cancer surgery. The aim of this study is to quantify the rate of ligation at the described level at a single academic center. Additionally, we examined the relationship between level of ligation and cancer-related outcomes. METHODS: This retrospective cohort study included patients ages 18 and over with low left-sided colon, rectal, and anal cancers undergoing surgical resection. Radiographic evidence of high ligation was defined as ligation of the inferior mesenteric artery before the takeoff of the left colic artery. Patients with and without radiographic evidence of high ligation on CT were compared. Secondary outcomes include lymph node yield and positivity, need for adjuvant therapy, and time from surgery to adjuvant therapy. RESULTS: 169 patients (54% male) were included in the study. 61.5% of operative reports described high ligation of the IMA. There was radiographic evidence of high ligation in 55.6% of total patients and in 70.2% of patients where high ligation was intended. There was no significant difference in surgeon experience, surgical procedure, or surgical approach. There was no difference in lymph node yield, time to adjuvant chemotherapy, or recurrence rates. CONCLUSION: This study demonstrates good technical success rate of high ligation of the inferior mesenteric artery but shows no difference in short-term patient-measured outcomes between high and low ligation (or successful and unsuccessful high ligation).

Use of audiovisual devices in transnasal endoscopy without sedation to improve tolerance. A prospective clinical trial.

INTRODUCTION AND AIM: Transnasal endoscopy (TNE) has proven its diagnostic utility, but it has not been widely accepted given that it is performed without sedation. There are no previous studies on the use of methods to improve its tolerability. Our aim was to evaluate the tolerability of TNE, when simultaneously performed with an audiovisual device as a distractor. METHODS: We evaluated 50 patients, 10 of whom did not agree to participate. The performance of the procedure was explained, using an audiovisual device. Before randomization, we applied anxiety and depression scores. Patients were divided into 2 groups: Group I (using an audiovisual device during the procedure) and Group II (without a device). Anxiety and numeric pain rating scales were used, and vital signs were monitored and recorded before, during, and after the endoscopy. An overall procedure satisfaction score was applied at the end of the study and 24 h later. RESULTS: Mean age was 41.6 years and 35 of the patients were women (87.5%). The most frequent indication for TNE was refractory gastroesophageal reflux disease. There were no severe comorbidities, and none of the patients had a significant anxiety or depression score. One patient in Group II did not tolerate TNE due to nasal pain. There was no statistically significant difference between groups, regarding anxiety, pain, vital signs, and satisfaction scale. CONCLUSION: Our study showed that TNE was well tolerated and had a high acceptance rate in our patients. The use of distracting audiovisual devices did not increase tolerance to the endoscopic procedure.

Morphometric Analysis of Prostate Zonal Anatomy After Transurethral Resection of Prostate and Holmium Laser Enucleation of Prostate Using Magnetic Resonance Imaging: A Pilot Study.

OBJECTIVE: The primary purpose was to compare the completeness of adenomectomy and zonal anatomy of prostate on magnetic resonance imaging prostate after transurethral resection of prostate and Holmium enucleation of prostate. The secondary purpose was to investigate the relationship between preoperative total prostate volume and postoperative transition zone and peripheral zone volume after both procedures. MATERIAL AND METHODS: A retrospective review of all patients who underwent transurethral resection of pros- tate or Holmium enucleation of prostate over 3 years (2017-2020) and had at least 1 postoperative magnetic resonance imaging prostate was performed. Volume estimations of the prostate and individual zones were per- formed, and statistical comparisons were made to evaluate morphometric changes between the 2 procedures. RESULTS: A total of 9 patients (mean age, 71.8 years) underwent transurethral resection of prostate and 12 patients (mean age, 66.9 years) underwent Holmium enucleation of prostate. The median pre-operative prostate volume in the Holmium enucleation of prostate group was higher than the transurethral resection of prostate group (101.5 g vs. 62 g; P =.102). However, there was a significant difference in the resected tissue weight favoring Holmium enucleation of prostate over transurethral resection of prostate (P value=.004). The postoperative transition zone and peripheral zone volume as calculated by magnetic resonance imaging remained relatively constant in both procedures. The peripheral zone volume on postoperative magnetic res- onance imaging was found to be independent of transition zone volume even for very large-sized prostates. CONCLUSION: A well-performed transurethral resection of prostate or Holmium enucleation of prostate cannearly completely eliminate the transition zone volume, irrespective of the size of the prostate as confirmed by magnetic resonance imaging prostate. Additionally, the peripheral zone volume is consistent across the entire spectrum of the prostate size.

Trasplante pulmonar ex vivo: primera experiencia en Chile y Latinoamérica / Lung transplant with ex vivo lung perfusion, Chilean and Latin American first experience

Background: The number of patients waiting for a lung transplant worldwide greatly exceeds the number of available donors. Ex vivo lung perfusion is a useful tool that allows marginal donor lungs to be evaluated and reconditioned for a successful lung transplantation. Aim: To describe the first Chilean and Latin American experience in ex vivo lung perfusion for marginal donor lungs before transplantation. Material and Methods: Descriptive analysis of all ex vivo lung perfusion conducted for marginal donor lungs at a private clinic, from April 2019 to October 2020. High risk donor lungs and rejected lungs from other transplantation centers were included. The "Toronto Protocol" was used for ex vivo lung perfusion. Donor lung characteristics and recipient outcomes were studied. Results: During the study period, five ex vivo lung perfusions were performed. All lungs were reconditioned and transplanted. No complications were associated. There were no primary graft dysfunctions and only one chronic allograft dysfunction. There was no mortality during the first year. The median arterial oxygen partial pressure/fractional inspired oxygen ratio increased from 266 mm Hg in the donor lung to 419 after 3 hours of ex vivo lung perfusion (p = 0.043). Conclusions: ex vivo lung perfusion is a safe and useful tool that allows marginal donor lungs to be reconditioned and successfully transplanted.

Anastomosing hemangioma of the ovary mimics metastatic ovarian cancer.

Anastomosing hemangioma (AH) is an uncommon benign vascular tumor reported to occur in the kidney and, in rare instances, the ovary. While most cases of AH in the ovary are incidental findings, we report a case of ovarian AH presenting with abdominal ascites and elevated CA-125 suggestive of metastatic ovarian cancer. Postoperative histopathologic examination demonstrated a tumor consisting of numerous vascular spaces lined by benign-appearing endothelial cells with exuberant hilus cell hyperplasia. These characteristics led to the diagnosis of anastomosing hemangioma of the ovary. A summary of the characteristics of AH, along with a review of all previously reported cases and possible theories for its presentation, are discussed.

Resultados de un programa de recuperación mejorada (PRM) en cirugía colorrectal: efectos en la estadía hospitalaria / Shortening of hospital length of stay after the implementation of an Enhanced Recovery Program for elective colorectal surgery

Resumen Introducción Los Programas de Recuperación Mejorada (PRM) en cirugía colorrectal disminuyen las complicaciones y acortan la estadía hospitalaria. La implementación de un PRM representa un desafío en nuestro sistema de salud público, dado la alta demanda y recursos limitados. Objetivo Evaluar el efecto en la estadía hospitalaria (EH) posterior a la implementación de un PRM en cirugía colorrectal electiva en un hospital público. Materiales y Método Estudio retrospectivo, que incluyó pacientes entre 15 y 85 años operados electivamente de resecciones colorrectales en un hospital público entre 1 de enero de 2015 y 31 de diciembre de 2016. Se compararon dos grupos: 1) total de pacientes operados electivamente 1 año posterior a la implementación del PRM (post-PRM) y 2) total de pacientes operados electivamente un año previo a la implementación de PRM (pre-PRM), en términos de EH, reingresos, complicaciones y mortalidad a 30 días. Se realizó análisis de tiempo a evento para comparar la EH entre ambos grupos. Resultados Un total de 144 pacientes fueron incluidos: 92 en el grupo post-PRM y 52 en el grupo pre-PRM. No hubo diferencias clínicas entre ambos grupos. La mediana de EH en el grupo post-PRM fue 2 días menor que en el grupo control (5 vs 7 días, test de log-rank, p = 0,03). No hubo diferencia en complicaciones, reingresos ni mortalidad. Conclusión La implementación de un PRM en cirugía colorrectal electiva en un hospital público se asocia a una disminución de la EH sin aumento de morbimortalidad.

Background Enhanced Recovery Programs (ERP) in colorectal surgery has proved to decrease complications and shorten hospital stay. The implementation of an ERP could be a difficult task in a high-demanding public health system with limited resources. Aim Evaluate the effect on length of stay (LOS) after the implantation of an ERP for colorectal surgery in a resource-limited public hospital. Materials and Method This retrospective study included patients aged 15-85 years who underwent elective colorectal surgery at a resource-limited public hospital in Chile between January 1, 2015 and December 31, 2016. We compared two groups: (1) total number of patients operated electively after 1-year of ERP implementation (post-ERP group) and (2) total number of patients electively operated 1-year previous ERP (pre-ERP group) in terms of LOS, readmissions, complications, and 30-day mortality. A time to event analysis was performed to evaluate differences in LOS. Results A total of 144 patients were included in this study: 92 in the post-ERP group and 52 in the pre-ERP group. There were no clinical differences between groups. The median LOS in the post-ERP group was 2 days lower than that in the control group (5 vs 7 days, log-rank test p = 0.03). There were no differences in complications, readmissions, and 30-day mortality. Conclusions The implementation of an ERP for colorectal surgery in a resource-limited hospital was associated with decreased LOS without increasing morbidity.

Recomendación Clínica: "Recomendaciones para el manejo de pacientes con COVID-19 con indicación terapéutica de ventilación mecánica que eventualmente son conectados a máquinas de anestesia" / Recommendations for patient management with COVID-19 with therapeutic indication mechanical ventilation that eventually are connected to anesthesia machines

La pandemia de COVID-19 producida por SARS-CoV-2 actualmente en curso anticipa una gran demanda por ventiladores mecánicos (VM), ya que un porcentaje relevante de los contagiados cae rápidamente en insuficiencia respiratoria y requiere de cuidados intensivos. Anticipándose a ese exceso de demanda y considerando que es muy probable que el número actual de ventiladores mecánicos en las unidades de cuidados intensivos (UCI) sean insuficientes, se ha solicitado a la SACH un informe técnico en relación al uso de las máquinas de anestesia como VM.

Validation of dominant and secondary sequence utilization in PI-RADS v2 for classifying prostatic lesions.

INTRODUCTION: To assess the secondary sequence rule in The Prostate Imaging Reporting Data System (PI-RADS) version 2 by comparing the detection of Grade group 1+ (GG1+) and 2+ (GG2+) cancers in PI-RADS 3, an upgraded PI-RADS 4, and true (non-upgraded) PI-RADS 4 targets. MATERIALS AND METHODS: We analyzed a total of 589 lesions scored as PI-RADS 3 or 4 obtained from 434 men who underwent mpMRI-US fusion biopsy from September 2015 to November 2017 for evaluation of GG1+ and GG2+ prostate cancer. PI-RADS 4 lesions were differentiated into those that were 'upgraded' to PI-RADS 4 based on the secondary sequence and those that were 'true' PI-RADS 4 based on the dominant sequence. RESULTS: The odds of detecting a GG2+ cancer was significantly higher for an upgraded 4 (peripheral zone (PZ): OR 5.06, 95%CI 2.04-12.54, p < 0.001, transitional zone (TZ): OR 3.08, 95%CI 1.04-9.08, p = 0.042) and true 4 (PZ: OR 5.82, 95%CI 3.10-10.94, p < 0.0001, TZ: OR 2.43, 95%CI 1.14-5.18, p = 0.022) lesions compared to PI-RADS 3 lesions. Additionally, we found no difference in the odds of detecting a GG2+ prostate cancer between a true PI-RADS 4 (OR 1.15, 95%CI 0.49-2.71 p = 0.746) and upgraded 4 (referent) in the PZ. Similar non-significance was noted between true 4 (OR 0.79, 95%CI 0.26-2.38 p = 0.674) and upgraded 4 lesions in the TZ for detection of GG2+ cancers. CONCLUSIONS: Upgraded PI-RADS 4 and true 4 targets have a higher odds of detecting GG1+ and GG2+ compared to PI-RADS 3 in the PZ and TZ. Our findings validate the revised scoring system for PI-RADS.

Imaging of Unusual Renal Tumors.

PURPOSE OF REVIEW: Renal masses are a wide entity and a common finding in clinical practice. Detection of these masses has increased in the last years, yet mortality rates have slightly decreased. RECENT FINDINGS: According to the World Health Organization classification, there are 8 types, 51 subtypes, and a lot more subsequent subclassifications of renal tumors. Histopathological analysis should always be assessed for final diagnosis of theses tumors. However, imaging can be an important diagnostic guidance. The most common diagnoses of renal tumor are clear cell carcinoma, papillary renal cell carcinoma, angiomyolipoma, and transitional cell carcinoma. Nonetheless, a considerable variety of particular tumors can arise from the kidney, challenging the expertise of radiologists and urologists on this subject. The awareness of these unusual entities is vital for professionals working at a complex medical facility with greater volume of patients. We hereby present uncommon renal tumors and its pathological and radiological features.

Complex design of surgical instruments as barrier for cleaning effectiveness, favouring biofilm formation.

BACKGROUND: Inadequately reprocessed reusable surgical instruments (RSIs) may harbour infectious agents which may then be transferred to a suitable site for replication. AIM: To determine the cumulative effect of 20 cycles of contamination, cleaning (manual or manual followed by automated) and steam sterilization on high-complex-design RSIs used for orthopaedic surgery. METHODS: New flexible medullary reamers and depth gauges were contaminated by soaking in tryptone soya broth, containing 5% sheep blood and 109 cfu/mL of Staphylococcus aureus (ATCC 25923), for 5 min. To mimic a worse-case scenario, RSIs were dried 7 h and subjected to either (a) rinsing in distilled water, (b) manual cleaning or (c) manual plus automated cleaning (reference standard), and steam sterilization. The contamination, cleaning, and sterilization cycle was repeated 20 times. Adenosine triphosphate (ATP) was measured after cleaning procedures; microbial load and residual protein were measured following the 10th and 20th reprocessing, in triplicate. Scanning electron microscopy (SEM) was used to confirm soil and biofilm presence on the RSIs after the 20th reprocessing. FINDINGS: Manual and manual plus automated cleaning significantly reduced the amount of ATP and protein residues for all RSIs. Viable bacteria were not detected following sterilization. However, SEM detected soil after automated cleaning, and soil, including biofilms, after manual cleaning. CONCLUSION: Soil and/or biofilms were evident on complex-design RSIs following 20 cycles of contamination and reprocessing, even using the reference standard method of cleaning. Although the depth gauges could be disassembled, biological residues and biofilm accumulated in its lumen. The current design of these RSIs prevents removal of all biological soil and this may have an adverse effect on patient outcome.

Orbital presentation of Rosai-Dorfman disease. / Presentación orbitaria de enfermedad de Rosai-Dorfman.

OBJECTIVE: To report a case of orbital mass and proptosis, diagnosed as Rosai-Dorfman disease (RDD). METHODS: Clinical case report based on the review of clinical charts, radiological images, and histopathology. RESULTS: A 42-year-old male with orbital mass and proptosis of the right eye. A surgical biopsy was performed, and the diagnosis of RDD was established using microscopy and immunohistochemistry. Definitive management included open tumour cytoreduction, with good response. DISCUSSION: The RDD is a rare, benign, proliferative condition of unknown origin. It rarely affects the cranial cavities, with the orbital presentation being very unusual. Diagnostic confirmation is essential for the best surgical management.

Comparison of conventional cytomorphology, flow cytometry immunophenotyping, and automated cell counting of CSF for detection of CNS involvement in acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVE: Cytospin conventional cytomorphology (CCC) is the standard method for detecting lymphoblasts in cerebrospinal fluid (CSF) of patients with acute lymphoblastic leukemia (ALL) and for guiding treatment decisions. We evaluated flow cytometry immunophenotyping (FCI) performance for improving detection of central nervous system (CNS) involvement in ALL. METHODS: This prospective study included analysis of consecutive CSF samples of patients of all ages with ALL at 3 clinical stages: new diagnosis, relapse suspicion, and after relapse treatment. Manual, cytospin, automated, and FCI methods were compared and their performance statistically assessed. Using FCI as the reference method, optimal CSF cutoff cell count that better correlated with presence of lymphoblasts was established by receiver operating characteristic (ROC) curve analysis. RESULTS: Seventy-seven CSF samples were investigated, 35 (45.4%) from newly diagnosed cases, 30 (39%) suspicion of relapse, and 12 (15.6%) after treatment for relapse. Median manual WBC count in patients with CNS involvement detected by FCI was 3.75 cells/µL (0.0-1280), and this was also the count that best correlated with CNS infiltration (sensitivity, 50.0%; specificity, 82.2%). Compared with FCI, CCC sensitivity and specificity were 28.6% and 100%. Automated CSF WBC count in patients with CNS involvement detected by FCI was 5 (0.0-1578). For automated count, optimal WBC cutoff was 4.5 cells/µL (sensitivity, 62.5%; specificity, 70.5%). CONCLUSION: Flow cytometry immunophenotyping complements conventional cytospin analysis for detection of lymphoblasts in the CSF of ALL patients at any clinical stage.

Evolución de síntomas a corto plazo y grado de satisfacción del paciente tras hemorroidectomía convencional / Evolution of short-term symptoms and degree of patient satisfaction after conventional hemorrhoidectomy

Resumen Introducción La hemorroidectomía es el tratamiento de elección para las hemorroides sintomáticas, reservado para pacientes con hemorroides grado III o IV o sin respuesta a tratamiento conservador. Existen diversas técnicas quirúrgicas, cuyo grado de resolución de síntomas y satisfacción por parte del paciente posee resultados variados. Objetivos Evaluar al corto plazo la sintomatología y grado de satisfacción de los pacientes operados de hemorroidectomía convencional en el Complejo Asistencial Dr. Sótero del Río. Materiales y Métodos Se realizó una encuesta telefónica retrospectiva a pacientes operados de hemorroidectomía convencional durante junio de 2015 a enero de 2016. Se preguntó por síntomas en el preoperatorio, al mes y a los 6 o más meses posoperados. Se evaluó sangrado, prurito, dolor, sensación de prolapso e incontinencia. El análisis estadístico consideró los resultados según los 5 síntomas evaluados y luego dicotomizados según relevancia clínica. Se consideró un valor p significativo < 0,01. Resultados Cuarenta y tres pacientes respondieron la encuesta, con una mediana de edad de 55 años (17-80). La mediana de evaluación fue de 8 meses (6-12). Considerando los síntomas: sangrado, prurito, dolor y prolapso, 35 pacientes (79%) presentaban 3 o más síntomas preoperatorios considerados significativos. La persistencia al seguimiento varía entre un 9 a un 28%. En el análisis estadístico con síntomas dicotomizados, existe una disminución estadísticamente significativa al mes y a los 6 o más meses posoperados. Discusión La hemorroidectomía convencional es bien evaluada por los pacientes en relación a una resolución significativa de síntomas de la enfermedad hemorroidal.

Background Hemorrhoidectomy is the treatment of choice for symptomatic hemorrhoids, reserved for patients with grade III or IV hemorrhoids or no response to conservative treatment. There are several surgical techniques, whose degree of resolution of symptoms and patient satisfaction has varied results. Objectives To evaluate in the short term the symptomatology and degree of satisfaction of patients undergoing conventional hemorrhoidectomy in Complejo Asistencial Dr. Sótero del Río. Material and Methods A retrospective telephone survey was applied to patients undergoing conventional hemorrhoidectomy from June 2015 to January 2016. The survey includes questions about symptoms in the pre-operative, at the month and at 6 or more months post-operated. Bleeding, pruritus, pain, prolapsed sensation and incontinence were evaluated. The statistical analysis considered the results according to the 5 symptoms evaluated in the survey and then dichotomized according to clinical relevance. A significant p value < 0.01 was considered. Results 43 patients answered the survey, with a median age of 55 years (17-80). The median evaluation was 8 months (6-12). Considering the symptoms: bleeding, pruritus, pain and prolapse, 35 patients (79%) had 3 or more preoperative symptoms considered significant. Persistent follow-up ranges from 9 to 28%. In the statistical analysis with dichotomized symptoms, there is a statistically significant decrease at one month and at 6 or more months post-surgery. Conclusion Conventional hemorrhoidectomy is well evaluated by patients in relation to a significant resolution of hemorrhoidal disease symptoms.

Theoretical Study of the Phosphoryl Transfer Reaction from ATP to Dha Catalyzed by DhaK from Escherichia coli.

Protein kinases, representing one of the largest protein families involved in almost all aspects of cell life, have become one of the most important targets for the development of new drugs to be used in, for instance, cancer treatments. In this article an exhaustive theoretical study of the phosphoryl transfer reaction from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) catalyzed by DhaK from Escherichia coli (E. coli) is reported. Two different mechanisms, previously proposed for the phosphoryl transfer from ATP to the hydroxyl side chain of specific serine, threonine, or tyrosine residues, have been explored based on the generation of free energy surfaces (FES) computed with hybrid QM/MM potentials. The results suggest that the substrate-assisted phosphoryl and proton-transfer mechanism is kinetically more favorable than the mechanism where an aspartate would be activating the Dha. Although the details of the mechanisms appear to be dramatically dependent on the level of theory employed in the calculations (PM3/MM, B3LYP:PM3/MM, or B3LYP/MM), the transition states (TSs) for the phosphoryl transfer step appear to be described as a concerted step with different degrees of synchronicity in the breaking and forming bonds process in both explored mechanisms. Residues of the active site belonging to different subunits of the protein, such as Gly78B, Thr79A, Ser80A, Arg178B, and one Mg2+ cation, would be stabilizing the transferred phosphate in the TS. Asp109A would have a structural role by posing the Dha and other residues of the active site in the proper orientation. The information derived from our calculations not only reveals the role of the enzyme and the particular residues of its active site, but it can assist in the rational design of new more specific inhibitors.

Insights into the Giardia intestinalis enolase and human plasminogen interaction.

Giardia intestinalis is an intestinal parasite that causes diarrhea in humans and animals worldwide. The enolase of G. intestinalis (GiENO) participates in its glycolysis pathway and is abundantly expressed in the parasite cytosol; however, its localization on the surface of trophozoites and cysts has been demonstrated. Enolases from bacteria and parasites can have different functions and are considered moonlighting proteins, for example, as a cell surface plasminogen receptor. In relation to GiENO, no studies have been performed about its possible participation as a plasminogen receptor. In this work, we employed molecular docking and multiscale molecular dynamics (MD) simulations to explore the possible interactions of human plasminogen (HsPLG) with the open and closed GiENO conformations. Our proposed GiENO plasminogen binding site (PLGBs) was identified at Lys266 based on the sequence comparison with bacterial enolase known to act as a plasminogen receptor. Our docking results performed with multiple MD snapshots of the closed GiENO conformation showed that Lys266 preferentially binds to the K5 domain of HsPLG. On the other hand, open GiENO conformations from all-atom and coarse-grained simulations indicated a high preference of the HsPLG K4 domain for lysine residues 186 and 188. Furthermore, we identified a potential N-glycosylation site of GiENO which suggests a possible explanation for the parasite cell surface localization or host mucin oligosaccharide adhesion mechanism. Our study constitutes the first multiscale computational study to explore the plasminogen receptor function of GiENO for its further consideration as a potential therapeutic target for giardiasis treatment.