Identifying patients with prostate cancer at increased risk for haematuria during anticoagulation for venous thromboembolism.

BACKGROUND: Haematuria is a common complication in prostate cancer patients receiving anticoagulation for venous thromboembolism (VTE). Early identification of at-risk patients might help to reduce its incidence and severity. METHODS: We used data from the RIETE registry to develop a prognostic score for haematuria during the first year of anticoagulation for VTE. The prognostic score was built using regression coefficients. RESULTS: From March 2001 through March 2021, 1934 patients with prostate cancer and acute VTE were enrolled. Of these, 1034 (53 %) initially presented as pulmonary embolism and 900 (47 %) as isolated deep vein thrombosis (DVT). During anticoagulation (median 181 days; inter-quartile range: 97-354), 99 patients (5.1 %) developed haematuria (fatal 1, major 27, non-major 72). The incidence rate was: 8 events per 100 patient-years (95%CI 6.5-9.7). Median time to haematuria was 53 days (IQR 4-134). On multivariable analysis, recent haematuria, initial presentation as DVT, comorbidity, metastases, haemoglobin levels <11 g/dL, creatinine >1.2 mg/dL, and radiotherapy independently predicted the risk for haematuria. C-statistics was 0.71 (95%CI: 0.65-0.77). A cut-off of ≥1.5 points classified 312 patients (20 %) at high-risk and had the highest sensitivity (51 %; 95%CI: 39-62) and specificity (82 %; 95%CI: 79-83). Our score improved the performance and non-event net reclassification index (NRI) of the RIETE score (c-statistics: 0.61; 95%CI: 0.54-0.68; NRI: 0.09) or VTE-BLEED score (c-statistics: 0.64; 95%CI: 0.58-0.71; NRI: 0.76). CONCLUSIONS: A prognostic score for haematuria during anticoagulation for VTE performed well in patients with prostate cancer, and improved identification compared to other validated scores.

Risk factors for extended-spectrum beta-lactamase (ESBL)-producing E. coli carriage among children in a food animal-producing region of Ecuador: A repeated measures observational study.

BACKGROUND: The spread of antibiotic-resistant bacteria may be driven by human-animal-environment interactions, especially in regions with limited restrictions on antibiotic use, widespread food animal production, and free-roaming domestic animals. In this study, we aimed to identify risk factors related to commercial food animal production, small-scale or "backyard" food animal production, domestic animal ownership, and practices related to animal handling, waste disposal, and antibiotic use in Ecuadorian communities. METHODS AND FINDINGS: We conducted a repeated measures study from 2018 to 2021 in 7 semirural parishes of Quito, Ecuador to identify determinants of third-generation cephalosporin-resistant E. coli (3GCR-EC) and extended-spectrum beta-lactamase E. coli (ESBL-EC) in children. We collected 1,699 fecal samples from 600 children and 1,871 domestic animal fecal samples from 376 of the same households at up to 5 time points per household over the 3-year study period. We used multivariable log-binomial regression models to estimate relative risks (RR) of 3GCR-EC and ESBL-EC carriage, adjusting for child sex and age, caregiver education, household wealth, and recent child antibiotic use. Risk factors for 3GCR-EC included living within 5 km of more than 5 commercial food animal operations (RR: 1.26; 95% confidence interval (CI): 1.10, 1.45; p-value: 0.001), household pig ownership (RR: 1.23; 95% CI: 1.02, 1.48; p-value: 0.030) and child pet contact (RR: 1.23; 95% CI: 1.09, 1.39; p-value: 0.001). Risk factors for ESBL-EC were dog ownership (RR: 1.35; 95% CI: 1.00, 1.83; p-value: 0.053), child pet contact (RR: 1.54; 95% CI: 1.10, 2.16; p-value: 0.012), and placing animal feces on household land/crops (RR: 1.63; 95% CI: 1.09, 2.46; p-value: 0.019). The primary limitations of this study are the use of proxy and self-reported exposure measures and the use of a single beta-lactamase drug (ceftazidime with clavulanic acid) in combination disk diffusion tests for ESBL confirmation, potentially underestimating phenotypic ESBL production among cephalosporin-resistant E. coli isolates. To improve ESBL determination, it is recommended to use 2 combination disk diffusion tests (ceftazidime with clavulanic acid and cefotaxime with clavulanic acid) for ESBL confirmatory testing. Future studies should also characterize transmission pathways by assessing antibiotic resistance in commercial food animals and environmental reservoirs. CONCLUSIONS: In this study, we observed an increase in enteric colonization of antibiotic-resistant bacteria among children with exposures to domestic animals and their waste in the household environment and children living in areas with a higher density of commercial food animal production operations.

Leveraging the COVID-19 pandemic as a natural experiment to assess changes in antibiotic use and antibiotic-resistant E. coli carriage in semi-rural Ecuador.

The coronavirus 2019 (COVID-19) pandemic has had significant impacts on health systems, population dynamics, public health awareness, and antibiotic stewardship, which could affect antibiotic resistant bacteria (ARB) emergence and transmission. In this study, we aimed to compare knowledge, attitudes, and practices (KAP) of antibiotic use and ARB carriage in Ecuadorian communities before versus after the COVID-19 pandemic began. We leveraged data collected for a repeated measures observational study of third-generation cephalosporin-resistant E. coli (3GCR-EC) carriage among children in semi-rural communities in Quito, Ecuador between July 2018 and September 2021. We included 241 households that participated in surveys and child stool sample collection in 2019, before the pandemic, and in 2021, after the pandemic began. We estimated adjusted Prevalence Ratios (aPR) and 95% Confidence Intervals (CI) using logistic and Poisson regression models. Child antibiotic use in the last 3 months declined from 17% pre-pandemic to 5% in 2021 (aPR: 0.30; 95% CI 0.15, 0.61) and 3GCR-EC carriage among children declined from 40 to 23% (aPR: 0.48; 95% CI 0.32, 0.73). Multi-drug resistance declined from 86 to 70% (aPR: 0.32; 95% CI 0.13; 0.79), the average number of antibiotic resistance genes (ARGs) per 3GCR-EC isolate declined from 9.9 to 7.8 (aPR of 0.79; 95% CI 0.65, 0.96), and the diversity of ARGs was lower in 2021. In the context of Ecuador, where COVID-19 prevention and control measures were strictly enforced after its major cities experienced some of the world's the highest mortality rates from SARS-CoV-2 infections, antibiotic use and ARB carriage declined in semi-rural communities of Quito from 2019 to 2021.

Risk factors for third-generation cephalosporin-resistant and extended-spectrum ß-lactamase-producing Escherichia coli carriage in domestic animals of semirural parishes east of Quito, Ecuador.

Extended-spectrum ß-lactamase (ESBL)-producing and other antimicrobial resistant (AR) Escherichia coli threaten human and animal health worldwide. This study examined risk factors for domestic animal colonization with ceftriaxone-resistant (CR) and ESBL-producing E. coli in semirural parishes east of Quito, Ecuador, where small-scale food animal production is common. Survey data regarding household characteristics, animal care, and antimicrobial use were collected from 304 households over three sampling cycles, and 1195 environmental animal fecal samples were assessed for E. coli presence and antimicrobial susceptibility. Multivariable regression analyses were used to assess potential risk factors for CR and ESBL-producing E. coli carriage. Overall, CR and ESBL-producing E. coli were detected in 56% and 10% of all fecal samples, respectively. The odds of CR E. coli carriage were greater among dogs at households that lived within a 5 km radius of more than 5 commercial food animal facilities (OR 1.72, 95% CI 1.15-2.58) and lower among dogs living at households that used antimicrobials for their animal(s) based on veterinary/pharmacy recommendation (OR 0.18, 95% CI 0.04-0.96). Increased odds of canine ESBL-producing E. coli carriage were associated with recent antimicrobial use in any household animal (OR 2.69, 95% CI 1.02-7.10) and purchase of antimicrobials from pet food stores (OR 6.83, 95% CI 1.32-35.35). Food animals at households that owned more than 3 species (OR 0.64, 95% CI 0.42-0.97), that used antimicrobials for growth promotion (OR 0.41, 95% CI 0.19-0.89), and that obtained antimicrobials from pet food stores (OR 0.47, 95% CI 0.25-0.89) had decreased odds of CR E. coli carriage, while food animals at households with more than 5 people (OR 2.22, 95% CI 1.23-3.99) and located within 1 km of a commercial food animal facility (OR 2.57, 95% CI 1.08-6.12) had increased odds of ESBL-producing E. coli carriage. Together, these results highlight the complexity of antimicrobial resistance among domestic animals in this setting.

Influenza A-Associated In-Hospital Mortality in Very Older People: Does Inflammation Also Play a Role?

BACKGROUND: The aim of the study was to analyze the clinical manifestations and outcome of the oldest old (people aged ≥85 years) who were admitted to the hospital with a confirmed influenza A virus infection in comparison with younger patients and to assess the role of inflammation in the outcome of influenza infection in this population. METHODS: This is an observational prospective study including all adult patients with influenza A virus infection hospitalized in a tertiary teaching hospital in Madrid, in 2 consecutive influenza seasons (2016-17 and 2017-18). RESULTS: Five hundred nine hospitalized patients with influenza A infection were included, of whom 117 (23%) were older than 85 years (median age: 89.3 ± 3.2). We compared the clinical characteristics and outcome with those of the rest of the population (median age: 72.8 ± 15.7). Overall, mortality was higher in older patients (10% vs. 4%; p = 0.03) with no differences in clinical presentation. Patients older than 85 years who ultimately died (12 out of 117) showed increased systemic inflammation expressed by higher levels of C-reactive protein (CRP) and ferritin compared to survivors who were discharged (odds ratio [OR] of CRP >20 mg/dL: 5.16, 95% confidence interval [CI]: 1.29-20.57, and OR of ferritin >500 mg: 4.3, 95% CI: 1.04-17.35). CONCLUSIONS: Patients aged 85 and older with influenza A virus infection presented a higher in-hospital mortality than younger subjects. CRP and ferritin levels were higher in the oldest old who died, suggesting that inflammation could play a key role in the outcome of this subset of patients.

SARS-CoV-2 infection in cancer patients undergoing active treatment: analysis of clinical features and predictive factors for severe respiratory failure and death.

AIM: Previous studies have suggested a more frequent and severe course of novel coronavirus SARS-CoV-2 infection in cancer patients undergoing active oncologic treatment. Our aim was to describe the characteristics of the disease in this population and to determine predictive factors for poor outcome in terms of severe respiratory distress (acute respiratory distress syndrome [ARDS]) or death. PATIENTS AND METHODS: Patients consecutively admitted for SARS-CoV-2 infection were prospectively collected, and retrospective statistical analysis was performed. Univariate and multivariate analyses were performed to assess potential factors for poor outcomes defined as ARDS or death. RESULTS: Sixty-three patients were analysed, and 34 of them developed respiratory failure (70% as ARDS). Lymphocytes/mm3 (412 versus 686; p = 0.001), serum albumin (2.84 versus 3.1); lactate dehydrogenase (LDH) (670 versus 359; p < 0.001) and C-reactive protein (CRP) levels (25.8 versus 9.9; p < 0.001) discriminate those that developed respiratory failure. Mortality rate was 25%, significantly higher among ARDS, neutropenic patients (p = 0.01) and in those with bilateral infiltrates (44% versus 0%; p < 0.001). Multivariate logistic analyses model confirmed the predictive value of severe neutropenia (odds ratio [OR] 16.54; 95% confidence interval [CI] 1.43-190.9, p 0.025), bilateral infiltrates (OR 32.83, CI 95% 3.51-307, p 0.002) and tumour lung involvement (OR 4.34, CI 95% 1.2-14.95, p 0.02). CONCLUSION: Cancer patients under active treatment admitted for SARS-CoV-2 infection have worse outcomes in terms of mortality and respiratory failure rates compared with COVID-19 global population. Lymphopenia, LDH, CRP and albumin discriminate illness severity, whereas neutropenia, bilateral infiltrates and tumour pulmonary involvement are predictive of higher mortality.

Bioarchaeological evidence of decapitation from Pacopampa in the northern Peruvian highlands.

Little is known about the precise date of the emergence of decapitation in a ritual context and the presence of systematic postmortem modification patterns in the ancient Central Andes. The ceremonial complex at Pacopampa in the northern Peruvian highlands provides early osteological evidence of decapitation in six individuals dating to the latter half of the Late-Final Formative Periods (500-50 BC) and to the Early Cajamarca Period (AD 200-450). Based on osteological evidence, and when taken together with archaeological settings and settlement patterns, researchers can be certain that those whose heads were disembodied were not likely to have been involved in organized battles. In addition, the similarities in the cut-mark distribution, direction, and cross-sectional morphology of each individual's remains, as well as the characteristics of selected individuals, imply that the decapitated individuals were carefully prepared using a standardized method and that those who modified the heads may have been professional decapitators. This study offers indisputable bioarchaeological evidence of ritualistic offerings of human skulls and systematic postmortem modification patterns, which is consistent with a contemporaneous iconographic motif of decapitation and extends the chronology of this practice back to the Formative Period in the northern Peruvian highlands.

High prevalence of self-reported tuberculosis and associated factors in a nation-wide census among prison inmates in Peru.

OBJECTIVE: To estimate the prevalence of self-reported tuberculosis TB diagnosed at Peruvian correctional facilities (CFs), and their associated factors. METHODS: Cross-sectional study based on secondary analysis of the National Census held in all Peruvian CFs in 2016. Outcome was defined as self-reported TB diagnosed by a healthcare professional intra-penitentiary. A descriptive bivariate analysis was carried out, followed by multivariate analysis using Poisson regression in order to calculate the adjusted prevalence ratios (PRa). Additionally, a mixed effects multilevel model adjusted by CFs as clusters was performed. RESULTS: Of 77 086 prison inmates in 66 CFs participated in the original census, of which 69 890 were included. Of these, 1754 self-reported TB diagnosed intra-penitentiary, yielding a prevalence of 2510/100 000 PDL. In the final model, self-reported TB was associated with younger age, male gender, lower educational level, not having a stable partner, having prison readmissions and having relatives in prison. There was also strong association with HIV/AIDS (PRa 2.77; 1.84-4.18), STIs (PRa 2.13; 1.46-3.10), DM (PRa 1.99; 1.59-2.50) and recreational drugs use (PRa 1.41; 1.23-1.61). The mixed model showed significant variance for belonging to different CFs (2.13; 1.02-4.44) and CF overcrowding (3.25; 1.37-7.71). CONCLUSIONS: Self-reported TB prevalence found was higher than reported by other lower/lower-middle income countries. Demographic factors, individual clinical features and overcrowding increases the likelihood of self-reported TB.

OBJECTIF: Estimer la prévalence de la tuberculose (TB) auto-déclarée, diagnostiquée dans des établissements pénitentiaires (EP) péruviens et les facteurs associés. MÉTHODES: Etude transversale basée sur une analyse secondaire du recensement national réalisée dans tous les EP péruviens en 2016. Le résultat était défini comme une TB auto-déclarée diagnostiquée par un professionnel de la santé intra-pénitentiaire. Une analyse descriptive à deux variables a été réalisée, suivie d'une analyse à plusieurs variables en utilisant une régression de Poisson afin de calculer les ratios de prévalence ajustés (PRa). En outre, un modèle multiniveau à effets mixtes ajusté selon les EP comme grappes a été réalisé. RÉSULTATS: 77.086 détenus de 66 EP ont participé au recensement initial, dont 69.890 ont été inclus. Parmi ceux-ci, 1.754 cas de TB auto-déclarée ont été diagnostiqués au sein des pénitenciers, soit une prévalence de 2.510/100.000 PDL. Dans le dernier modèle, la TB auto-déclarée était associée à un âge plus jeune, au sexe masculin, à un niveau d'éducation plus faible, à l'absence de partenaire stable, à la réadmission en prison et avoir des parents en prison. Il existait également une forte association avec le VIH/SIDA (PRa: 2,77; 1,84 - 4,18), les IST (PRa: 2,13; 1,46 - 3,10), le diabète (PRa: 1,99; 1,59 - 2,50) et la consommation de drogues à des fins récréatives (PRa: 1,41; 1,23 - 1,61). Le modèle mixte a montré une variance significative pour l'appartenance à différents EP (2,13; 1,02 - 4,44) et au surpeuplement des EP (3,25; 1,37 à 7,71). CONCLUSIONS: La prévalence auto-déclarée de la TB était plus élevée que celle rapportée par d'autres pays à revenu inférieur/moyen-inférieur. Les facteurs démographiques, les caractéristiques cliniques individuelles et le surpeuplement augmentent le risque de TB auto-déclarée.

Apendicitis aguda en pacientes pediátricos oncológicos en el Instituto del Cáncer SOLCA-Cuenca. 2017 / Acute appendicitis in oncological pediatric patients at the Cancer Institute SOLCA-Cuenca. 2017

Antecedentes: La apendicitis aguda es una urgencia abdominal quirúrgica, en donde la presentación clínica y el diagnóstico generalmente son diferentes cuando se presenta en pacientes oncológicos a consecuencia de la alteración inmunológica propia del cáncer y secundarios al tratamiento con quimioterapia. Método: Se realizó un estudio descriptivo, retrospectivo desde enero del 2011 hasta junio del 2017. Se revisaron las historias clínicas de 433 pacientes con cáncer en menores de 18 años, que fueron atendidos en SOLCA-Cuenca, y se incluyó a 12 pacientes que presentaron apendicitis aguda. Se realizó una descripción de sus características clínicas, patológicas y terapéuticas. La información se incluyó en una base de datos y luego fueron analizados en el programa EXCEL y SPSSv20 portable. Resultados: El promedio de edad de los pacientes fue de 9.58 años en igual proporción para hombres y mujeres. La media de glóbulos blancos fue de 10.500 células por microlitro y su rango estuvo entre 400 y 21.600 células por microlitro. Los neutrófilos, plaquetas y hemoglobina tuvieron una media de 66%, 275.416 células por microlitro y 11 g/dl respectivamente, con un promedio de hospitalización de 18.5 días. El 66% de pacientes presentaron un valor mayor de 7 sobre 10 en la escala de Alvarado. La apendicitis aguda fue más frecuencia (4 casos) en la fase de mantenimiento del tratamiento oncológico. Los resultados patológicos más comunes fueron: apéndice congestivo, flemonoso, perforativo, con 3 casos cada uno. La ceftriaxona fue el antibiótico pre-quirúrgico más utilizado (5 pacientes) y la combinación de meropenem con metronizadol durante el pos-operatorio (4 pacientes). Una paciente (8,3%) falleció 9 días después del procedimiento quirúrgico debido a shock séptico secundario a peritonitis aguda. Conclusiones: La apendicitis aguda se presentó en el 2.7% de los pacientes oncológicos. La historia natural en lo que a manifestaciones clínicas se refiere, se presentaron en los pacientes oncológicos que en los no oncológicos. La escala de Alvarado fue una prueba diagnóstica válida en los dos grupos.

Background: Acute appendicitis is a surgical abdominal emergency, in which the clinical presentation and diagnosis are generally different when it occurs in oncological patients as a consequence of the immunological alteration characteristic of cancer and secondary to treatment with chemotherapy. Method: A descriptive, retrospective study was conducted from January 2011 to June 2017. The medical records of 433 patients with cancer in children under 18 years of age were reviewed; they were treated in SOLCA-Cuenca, and 12 patients who presented acute appendicitis were included. A description of its clinical, pathological and therapeutic characteristics was made. The information was included in a database and then analyzed in the portable program EXCEL and SPSSv20 Results: The average age of the patients was 9.58 years in the same proportion for men and women. The average of white blood cells was 10,500 cells per microliter and its range was between 400 and 21,600 cells per microliter. The neutrophils, platelets and hemoglobin had an average of 66%, 275,416 cells per microliter and 11g / dl respectively, with an average hospitalization of 18.5 days. A 66% of patients presented a value higher than 7 on to 10 in the Alvarado scale (method used to diagnose acute appendicitis). Acute appendicitis was more frequent (4 cases) in the maintenance phase of oncological treatment. The most common pathological results were: congestive appendix, phlegmonous, perforative, with 3 cases each. The ceftriaxone was the most used pre-surgical antibiotic (5 patients) and the combination of meropenem with metronizadol during the post-surgery period (4 patients). One patient (8.3%) died 9 days after the surgical procedure due to septic shock secondary to acute peritonitis. Conclusions: Acute appendicitis occurred in 2.7% of cancer patients. The natural history as far as clinical manifestations was presented in the oncological patients as in the non-oncological patients. The Alvarado scale was a valid diagnostic test in both groups. It is necessary to be careful with the interpretation of laboratory and image data.

Daptomycin Plus Fosfomycin as Salvage Therapy in a Difficult-to-Treat Total Femoral Replacement Infection.

The highly active anti-biofilm combination of daptomycin plus fosfomycin was successfully used in a difficult-to-treat infection of a total femoral replacement caused by multi-drug resistant Staphylococcus epidermidis in a 79-year-old woman. There was no need to remove the orthopedic hardware, and the patient is currently pain free and able to walk.

Impact of functional status on 6-month mortality in elderly patients with acute venous thromboembolism: results from a prospective cohort.

Functional status linked to a poor outcome in a broad spectrum of medical disorders. Barthel Activities of Daily Life Index (BADLI) is one of the most extended tools to quantify functional dependence. Whether BADLI can help to predict outcomes in elderly patients with acute venous thromboembolism (VTE) is unknown. The current study aimed to ascertain the influence of BADLI on 6-month all-cause mortality in aged patients with VTE. This is a prospective observational study. We included consecutive patients older than 75-year-old with an acute VTE between April 2015 and April 2017. We analyzed several variables as mortality predictors, including BADLI-measured functional status. Afterward, we performed a multivariate analysis, using logistic regression, to identify all-cause mortality independent predictive factors. Two hundred and two subjects were included. Thirty-five (17%) patients died in the first 6 months. The leading cause of death was cancer (59%). After multivariable logistic regression, we identified BADLI and Charlson index as independent predictors for 6-months mortality [BADLI (every decrease of 10 points) OR 1.21 95% CI (1.03-1.42) and Charlson index OR 1.71 95% CI (1.21-2.43)]. Body mass index (BMI) values were inversely related to mortality [OR 0.85 95% CI (0.75-0.95)]. In conclusion, BADLI, BMI, and Charlson index scores are independent predictive factors for 6-month all-cause mortality in old patients with VTE.

A case study of a high-status human skeleton from Pacopampa in Formative Period Peru.

The Pacopampa site is located in the northern highlands of Peru and is an archaeological site belonging to the Formative Period (2500-1 BC). The excavation of the Pacopampa site yielded unusual human skeletons from the main platform of a ceremonial center of the site during the 2009 field season. The skeletal remains were associated with a pair of gold earplugs, a pair of gold earrings, and shell objects. This specimen is possibly a female aged 20-39 years. Detailed examination of the neurocranium revealed the presence of artificial cranial deformation with decreased cranial length, increased cranial breadth, and lateral bulging of the parietal bones. The estimated stature of this individual was 162 cm, which is about 15 cm higher than that of contemporary females of Pacopampa and about 20-25 cm higher than that of other Formative Period sites in northern Peru. The peculiarity of this individual, detected not only in the cultural artifacts but also in the physical features, is possible evidence for social stratification in the Formative Period.

Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility.

BACKGROUND: Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes. RESULTS: We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes. CONCLUSIONS: These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: spatial propagation of differential flexibility in rubredoxin hybrids.

The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14-33(Pf)) Cp, that exhibits a 12 degrees increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14-33(Pf)) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14-33(Pf)) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs.

Contribution of the multi-turn segment in the reversible thermal stability of hyperthermophile rubredoxin: NMR thermal chemical exchange analysis of sequence hybrids.

Pyrococcus furiosus (Pf) rubredoxin is the most thermostable protein characterized to date. Reflecting the complications arising from irreversible denaturation of this protein, predictions of which structural regions confer differential thermal stability have utilized kinetic stability measurements, hydrogen exchange protection factors, long range hydrogen bond NMR spin couplings, and molecular dynamics simulations, and have primarily implicated the three-stranded beta-sheet and the adjacent metal binding site. Herein, NMR chemical exchange experiments demonstrate reversible two-state unfolding at the thermal transition temperature (T(m)) for hybrids of Pf and the mesophile Clostridium pasteurianum (Cp) rubredoxins which interchange residues 14-33, the so-called multi-turn segment. This complementary pair of hybrid rubredoxins exhibits largely additive incremental thermal stabilizations vs. the parental proteins. Both stabilization free energy measurements as well as incremental T(m) values indicate that a minimum of 37% of the total differential thermal stability resides in this multi-turn segment. Such a proportionality between DeltaDeltaG and incremental T(m) values is predicted for hybrid pairs exhibiting thermodynamic additivity in which the differential stability is predominantly enthalpic.