Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase.

Treatment Order and Risk of Surgical Site Infection in Patients Undergoing Concurrent Operative Fixation of Closed and Open Fractures.

This study aimed to characterize postoperative infection rate among patients undergoing definitive fixation of both open and closed fractures during the same surgery. Outcomes between patients with open fractures (OF) treated first were compared to those with closed fractures (CF) treated first. We identified 303 adult patients with multiple (≥ 2) pelvis and extremity fractures who presented to our Level 1 Trauma hospital in 2017. Forty patients with at least one open and one closed fracture treated with operative fixation during the same surgery were included in analysis. Eight surgical site infections (SSI) developed in seven patients. There was no significant difference between treatment order groups (OF = 4 patients (5 fractures), CF = 3 patients (3 fractures); p > 0.99). This is the first study comparing different chronologies of operative fixation in coexisting open and closed fractures. Our study shows that the choice of treatment order does not influence SSI risk. (Journal of Surgical Orthopaedic Advances 31(3):181-186, 2022).

A role of flavonoids in cytochrome c-cardiolipin interactions.

The processes preceding the detachment of cytochrome c (cyt c) from the inner mitochondrial membrane in intrinsic apoptosis involve peroxidation of cardiolipin (CL) catalyzed by cyt c-CL complex. In the present work, we studied the effect of 17 dietary flavonoids on the peroxidase activity of cyt c bound to liposomes. Specifically, we explored the relationship between peroxidase activity and flavonoids' (1) potential to modulate cyt c unfolding, (2) effect on the oxidation state of heme iron, (3) membrane permeability, (4) membrane binding energy, and (5) structure. The measurements revealed that flavones, flavonols, and flavanols were the strongest, while isoflavones were the weakest inhibitors of the oxidation. Flavonoids' peroxidase inhibition activity correlated positively with their potential to suppress Trp-59 fluorescence in cyt c as well as the number of OH groups. Hydrophilic flavonoids, such as catechin, having the lowest membrane permeability and the strongest binding with phosphocholine (PC) based on the quantum chemical calculations exhibited the strongest inhibition of Amplex Red (AR) peroxidation, suggesting a membrane-protective function of flavonoids at the surface. The results of the present research specify basic principles for the design of molecules that will control the catalytic oxidation of lipids in mitochondrial membranes. These principles take into account the number of hydroxyl groups and hydrophilicity of flavonoids.

BurnCare tablet trainer to enhance burn injury care and treatment.

BACKGROUND: Applied Research Associates (ARA) and the United States Army Institute of Surgical Research (USAISR) have been developing a tablet-based simulation environment for burn wound assessment and burn shock resuscitation. This application aims to supplement the current gold standard in burn care education, the Advanced Burn Life Support (ABLS) curriculum. RESULTS: Subject matter experts validate total body surface area (TBSA) identification and analysis and show that the visual fidelity of the tablet virtual patients is consistent with real life thermal injuries. We show this by noting that the error between their burn mapping and the actual patient burns was sufficiently less than that of a random sample population. Statistical analysis is used to confirm this hypothesis. In addition a full body physiology model developed for this project is detailed. Physiological results, and responses to standard care treatment, are detailed and validated. Future updates will include training modules that leverage this model. CONCLUSION: We have created an accurate, whole-body model of burn TBSA training experience in Unreal 4 on a mobile platform, provided for free to the medical community. We hope to provide learners with more a realistic experience and with rapid feedback as they practice patient assessment, intervention, and reassessment.

Physiological response of 10 phytoplankton species exposed to macondo oil and the dispersant, Corexit.

Culture experiments were conducted on ten phytoplankton species to examine their biological and physiological responses during exposure to oil and a combination of oil and dispersant. The species tested included a range of taxa typically found in the Gulf of Mexico such as cyanobacteria, chlorophytes, and diatoms. Cultures were exposed to Macondo surrogate oil using the water accommodated fraction (WAF), and dispersed oil using a chemically enhanced WAF (CEWAF) and diluted CEWAF, to replicate conditions following the Deepwater Horizon spill in the Gulf of Mexico. A range of responses were observed, that could broadly class the algae as either "robust" or "sensitive" to oil and/or dispersant exposure. Robust algae were identified as Synechococcus elongatus, Dunaliella tertiolecta, two pennate diatoms Phaeodactylum tricornutum and Navicula sp., and Skeletonema grethae CCMP775, and were largely unaffected by any of the treatments (no changes to growth rate or time spent in lag phase relative to controls). The rest of the phytoplankton, all centric diatoms, exhibited at least some combination of reduced growth rates or increased lag time in response to oil and/or dispersant exposure. Photophysiology did not have a strong treatment effect, with significant inhibition of photosynthetic efficiency (Fv /Fm ) only observed in the CEWAF, if at all. We found that the effects of oil and dispersants on phytoplankton physiology were species-dependent, and not always detrimental. This has significant implications on how oil spills might impact phytoplankton community structure and bloom dynamics in the Gulf of Mexico, which in turn impacts higher trophic levels.

Topographical gradients of semantics and phonology revealed by temporal lobe stimulation.

Word retrieval is a fundamental component of oral communication, and it is well established that this function is supported by left temporal cortex. Nevertheless, the specific temporal areas mediating word retrieval and the particular linguistic processes these regions support have not been well delineated. Toward this end, we analyzed over 1000 naming errors induced by left temporal cortical stimulation in epilepsy surgery patients. Errors were primarily semantic (lemon → "pear"), phonological (horn → "corn"), non-responses, and delayed responses (correct responses after a delay), and each error type appeared predominantly in a specific region: semantic errors in mid-middle temporal gyrus (TG), phonological errors and delayed responses in middle and posterior superior TG, and non-responses in anterior inferior TG. To the extent that semantic errors, phonological errors and delayed responses reflect disruptions in different processes, our results imply topographical specialization of semantic and phonological processing. Specifically, results revealed an inferior-to-superior gradient, with more superior regions associated with phonological processing. Further, errors were increasingly semantically related to targets toward posterior temporal cortex. We speculate that detailed semantic input is needed to support phonological retrieval, and thus, the specificity of semantic input increases progressively toward posterior temporal regions implicated in phonological processing. Hum Brain Mapp 38:688-703, 2017. © 2016 Wiley Periodicals, Inc.

Functional differences among stimulation-identified cortical naming sites in the temporal region.

To preserve postoperative language, electrical stimulation mapping is often conducted prior to surgery involving the language-dominant hemisphere. Object naming is the task most widely used to identify language cortex, and sites where stimulation elicits naming difficulty are typically spared from resection. In clinical practice, sites classified as positive undergo no further testing regarding the underlying cause of naming failure. Word production is a complex function involving multiple mechanisms that culminate in the identification of the target word. Two main mechanisms, i.e., semantic and phonological, underlie the retrieval of stored information regarding word meaning and word sounds, and naming can be hampered by disrupting either of these. These two mechanisms are likely mediated by different brain areas, and therefore, stimulation-identified naming sites might not be functionally equivalent. We investigated whether further testing at stimulation-identified naming sites would reveal an anatomical dissociation between these two mechanisms. In 16 patients with refractory temporal lobe epilepsy (TLE) with implanted subdural electrodes, we tested whether, despite inability to produce an item name, patients could reliably access semantic or phonological information regarding objects during cortical stimulation. We found that stimulation at naming sites in superior temporal cortex tended to impair phonological processing yet spared access to semantic information. By contrast, stimulation of inferior temporal naming sites revealed a greater proportion of sites where semantic access was impaired and a dissociation between sites where stimulation spared or disrupted semantic or phonological processing. These functional-anatomical dissociations reveal the more specific contribution to naming provided by these cortical areas and shed light on the often profound, interictal word-finding deficit observed in temporal lobe epilepsy. Additionally, these techniques potentially lay the groundwork for future studies to determine whether particular naming sites that fall within the margins of the desired clinical resection might be resected without significant risk of decline.

Shared space, separate processes: Neural activation patterns for auditory description and visual object naming in healthy adults.

Historically, both clinicians and cognitive scientists have used visual object naming measures to study naming, and lesion-type studies have implicated the left posterior, temporo-parietal region as a critical component of naming circuitry. However, recent results from behavioral and cortical stimulation studies using auditory description naming as well as visual object naming in left temporal lobe epilepsy patients suggest that discrete sites in anterior temporal cortex are critical for description naming, whereas posterior temporal regions mediate both visual object naming and description naming. To determine whether this task specificity reflects normal cerebral organization and processing, 13 healthy adults performed description naming and visual naming during functional neuroimaging. In addition to standard univariate analysis, multivariate, ordinal trend analysis examined the network character of the regions involved in task-specific naming. Univariate analysis indicated posterior temporal activation for both visual naming and description naming, whereas multivariate analysis revealed broader networks for both tasks, with both overlapping and task-specific regions, as well as task-related differences in the way the tasks utilized common regions. Additionally, multivariate analysis revealed unique, task-specific, regionally covarying activation patterns that were strikingly consistent in all 13 subjects for visual naming and 12/13 subjects for description naming. Results suggest a common neural substrate, yet differentiable neural processes underlying visual naming and description naming in neurologically intact individuals. These findings support the use of both types of tasks for clinical assessment and may have application in the treatment of neurologically based naming deficits. Inc.

Extraoperative neurostimulation mapping: results from an international survey of epilepsy surgery programs.

OBJECTIVE: Extraoperative electrical stimulation mapping (ESM) to identify functional cortex is performed prior to neurosurgical resection at epilepsy surgery programs worldwide. However, the procedure remains unstandardized, with no established clinical guidelines. We sought to determine the current range in ESM practice parameters across established epilepsy surgery centers. METHODS: We developed and distributed a 31-question survey to 220 epilepsy centers worldwide regarding current practice parameters of ESM. Questions addressed preoperative assessment, technical stimulation parameters, language testing protocols, criteria for identification of positive or negative functional sites, management of mapping complications, and postoperative functional outcome. RESULTS: Survey responses were obtained from 56 centers. These revealed marked practice variability in virtually all aspects of the ESM procedure. These aspects included critical procedure components such as electrical stimulation settings, the types of language functions tested, the operational definition of a language error, size of surgical resection margin, cortical locations mapped for language, testing in the presence of afterdischarges, and medical management of mapping complications. Forty-one percent of centers reported at least one persistent adverse language outcome despite preserving all eloquent sites defined by their stimulation mapping procedure. SIGNIFICANCE: The striking variations in practice across centers are likely to influence mapping results, which directly affect the boundaries of cortical resection and, consequently, might worsen either seizure or functional outcomes. Clearly, adverse functional outcomes occur despite mapping procedures that were perceived to be adequate. Investigation of critical technical and procedural aspects of stimulation mapping is warranted, with the ultimate goal of establishing empirically based practice guidelines to improve the safety and efficacy of ESM and resective epilepsy surgery. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Suspected gluteal compartment syndrome: Etiology predicts clinical course, outcomes and resource utilization.

Background: Gluteal Compartment Syndrome (GCS) is a rare subtype of acute compartment syndrome (ACS), complex to diagnose and potentially fatal if left untreated. The incidence of ACS is estimated to be 7.3 per 100,000 in males and 0.7 per 100,000 in females [1-3]. Given its rare occurrence, the incidence of GCS is not well reported. In the case of GCS, the most common etiologies are surgical positioning, prolonged immobilization secondary to substance use or loss of consciousness, and traumatic injury. Clinical findings are pulselessness, pallor, parasthesia, paralysis, and most notably pain out of proportion. Swift diagnosis and treatment are imperative to reduce morbidity and mortality, however the ideal management of GCS is difficult to ascertain given the rare occurrence and variable presentation. Methods: Orthopaedic trauma database at a level 1 trauma center was reviewed to identify patients for whom the orthopaedic service was consulted due to suspicion of gluteal compartment syndrome. This yielded 11 patients between 2011 and 2019. Patients with a measured ΔP greater than 30 upon initial consultation and with a concerning exam requiring monitoring were included. Patient demographics, comorbidities, GCS etiology, laboratory values, physical exam findings, pain scores (0-10) and patient outcomes were collected via chart review. Patient demographic and injury characteristics were summarized using descriptive statistics. Results: Prolonged immobilization patients had worse outcomes including longer hospital stays (40.5 days) compared to trauma patients (4.5 days). All adverse medical outcomes recorded including acute renal failure, prolonged neuropathic pain, cardiopulmonary dysfunction were exclusively experienced by prolonged immobilization patients. Conclusions: Our descriptive study demonstrates the bimodal distribution of GCS patients based on etiology. Prolonged immobilization patients have a longer hospital course and more complications. Our study confirms prior reports and provides information that can be used to counsel patients and families appropriately about treatment and recovery following GCS. Level of evidence: IV. Study type: Epidemiological.

Efficacy and safety of implantable cardioverter-defibrillator implantation in the elderly-The I-70 Study: A randomized clinical trial.

Background: There is conflicting evidence on the efficacy of primary prevention implantable cardioverter-defibrillator (ICD) implantation in the elderly. Objective: The purpose of this study was to determine the efficacy and safety of ICD implantation in patients 70 years and older. Methods: Patients (n = 167) aged 70 years or older and eligible for ICD implantation were randomly assigned (1:1) to receive either optimal medical therapy (OMT) (n = 85) or OMT plus ICD (n = 82). Results: Of the 167 participants (mean age 76.4 years; 165 men), 144 completed the study protocol according to their assigned treatment. Average participant follow-up was 31.5 months. Mortality was similar between the 2 groups: 27 deaths in OMT vs 26 death in ICD (unadjusted hazard ratio 0.92; 95% confidence interval 0.53-1.57), but there was a trend favoring the ICD over the first 36 months of follow-up. Rates of sudden death (7 vs 5; P = .81) and all-cause hospitalization (2.65 events per participant in OMT vs 3.09 in ICD; P = .31) were not statistically significantly different. Eleven participants randomized to ICD received appropriate therapy. Five participants received an inappropriate therapy that included at least 1 ICD shock. Conclusion: The study did not recruit to target sample size, and accumulated data did not show benefit of ICD therapy in patients 70 years or older. Future studies similar in design might be feasible but will need to contend with patient treatment preference given the large number of patients who do not want an ICD implanted. Further research is needed to determine whether the ICD is effective in prolonging life among elderly device candidates.

Adjusting body weight for edema in severely burned patients.

Weight loss is difficult to quantify in critically ill burn patients, as the presence of edema can mask changes in dry body weight. We sought to estimate dry body weight using measured weights adjusted for reported extremity edema. We evaluated patients with at least 20% total body surface area (TBSA) burns admitted to our intensive care unit over a 3½-year period. Body weights were collected for this analysis from admission to the time of a recorded dry weight after wound healing. Extremity edema was collected at the time of each weight measurement and was categorized into three groups: (1) no edema, (2) 1 + pitting edema, (3) 2 + or 3 + pitting edema. Logistic regression yielded the following formula for estimating dry weight (in kg): 0.66 x measured body weight + 25 - (3 for 1 + pitting edema or 4 for 2 + or 3 + pitting edema of either upper extremity) - (4 for any pitting edema to either lower extremity) (p < 0.01, R2 = 0.81). These results may allow us to better estimate dry body weight changes in our edematous patients with severe burns. Nutrition goals can be adjusted earlier, when appropriate, based on these estimated dry body weight changes.

Determination of the drug-DNA binding modes using fluorescence-based assays.

Therapeutic drugs and environmental pollutants may exhibit high reactivity toward DNA bases and backbone. Understanding the mechanisms of drug-DNA binding is crucial for predicting their potential genotoxicity. We developed a fluorescence analytical method for the determination of the preferential binding mode for drug-DNA interactions. Two nucleic acid dyes were employed in the method: TO-PRO-3 iodide (TP3) and 4',6-diamidino-2-phenylindole (DAPI). TP3 binds DNA by intercalation, whereas DAPI exhibits minor groove binding. Both dyes exhibit significant fluorescence magnification on binding to DNA. We evaluated the DNA binding constant, K(b), for each dye. We also performed fluorescence quenching experiments with 11 molecules of various structures and measured a C(50) value for each compound. We determined preferential binding modes for the aforementioned molecules and found that they bound to DNA consistently, as indicated by other studies. The values of the likelihood of DNA intercalation were correlated with the partition coefficients of the molecules. In addition, we performed nuclear magnetic resonance (NMR) studies of the interactions with calf thymus DNA for the three molecules. The results were consistent with the fluorescence method described above. Thus, we conclude that the fluorescence method we developed provides a reliable determination of the likelihoods of the two different DNA binding modes.

In vitro studies of DNA damage caused by tricyclic antidepressants: a role of peroxidase in the side effects of the drugs.

Studies show that tricyclic antidepressants prescribed for migraines, anxiety, and child enuresis have numerous adverse effects in living cells. One of the undesired outcomes observed under treatment with these drugs is DNA damage. However, the mechanisms underlying damage have yet to be elucidated. We performed in vitro studies of the DNA damage caused by four tricyclic antidepressants: imipramine, amitriptyline, opipramol, and protriptyline. We focused particularly on the DNA damage aided by peroxidases. As a model of a peroxidase, we used horseradish peroxidase (HRP). At pH 7, reactions of HRP with excess hydrogen peroxide and imipramine yielded an intense purple color and a broad absorption spectrum with the maximum intensity at 522 nm. Reactions performed between DNA and imipramine in the presence of H(2)O(2) and HRP resulted in the disappearance of the DNA band. In the case of the other three drugs, this effect was not observed. Extraction of the DNA from the reaction mixture indicated that DNA is degraded in the reaction between imipramine and H(2)O(2) catalyzed by HRP. The final product of imipramine oxidation was identified as iminodibenzyl. We hypothesize that the damage to DNA was caused by an imipramine reactive intermediate.

A versatile chiral selector for determination of enantiomeric composition of fluorescent and nonfluorescent chiral molecules using steady-state fluorescence spectroscopy.

A fluorescent chiral molecular micelle (FCMM), poly (sodium N-undecanoyl-L-phenylalaninate) (poly-L-SUF), was developed as a chiral selector for enantiomeric recognition and determination of enantiomeric composition of four fluorescent and four nonfluorescent chiral molecules by use of steady-state fluorescence spectroscopy. The influence of FCMM concentration, buffer pH and complexation medium on FCMM-analyte host-guest complexation, and the emission spectral properties of the resulting complexes were investigated. The chiral interactions of the analytes,1,1'-binaphthyl-2,2'-diamine, 1-(9-anthryl)-2,2,2-trifluoroethanol, propranolol, naproxen, chloromethyl menthyl ether (CME), citramalic acid, tartaric acid, and limonene (LIM), in the presence of poly-L-SUF were based on diastereomeric complex formation. The figures of merit obtained from the partial-least-squares regression modeling of the calibration samples suggested good prediction ability for the validation of six of the eight chiral analytes. Better host-guest complexation of the more hydrophobic molecules, CME and LIM, were obtained in methanol/water mixtures, resulting in better predictability of the regression models. Prediction ability of the models was evaluated by use of the root-mean-square percent relative error (RMS%RE) and was found to range from 1.77 to 15.80% (buffer), 1.26 to 7.95% (25:75 methanol/water), and 1.21 to 4.28% (75:25 methanol/water).

Evidence for cortical reorganization of language in patients with hippocampal sclerosis.

Naming is mediated by perisylvian cortex in the left (language-dominant) hemisphere, and thus, left anterior temporal lobe resection for pharmacologically intractable temporal lobe epilepsy (TLE) carries risk for post-operative naming decline. Interestingly, this risk is lower in patients with hippocampal sclerosis (HS) relative to those without HS (non-HS). Although the hippocampus has traditionally been considered a critical structure for memory, without contribution to naming, this pattern might implicate direct hippocampal naming involvement. On the other hand, critical naming sites have been found in anterior, lateral temporal (i.e. extra-hippocampal) neocortex, the region typically removed with 'standard' TLE resection. We, therefore, speculated that the relative preservation of naming in post-operative HS patients might reflect cortical reorganization of language to areas outside this region. Using pre-resection electrical stimulation mapping, we compared the topography of auditory and visual naming sites in 12 patients with HS and 12 patients without structural brain pathology. Consistent with previous work, non-HS patients exhibited post-operative naming decline, whereas HS patients did not. As hypothesized, HS patients had proportionally fewer overall naming sites in anterior temporal cortex, the region typically removed with standard anterior temporal resection, whereas non-HS patients exhibited a more even distribution of naming sites in anterior and posterior temporal regions (P = 0.03). Although both groups exhibited the previously reported pattern of auditory naming sites anterior to visual naming sites, auditory naming sites had a significantly more posterior distribution in HS patients (P = 0.02). Additionally, non-HS patients exhibited a greater proportion of visual naming sites above the superior temporal sulcus, whereas visual naming sites in HS patients were scattered across superior and inferior temporal cortex. Results suggest that preserved naming ability in HS patients following anterior temporal resection might be attributable, at least in part, to intrahemispheric reorganization of language in response to the likely, early development of sclerosis in the medial temporal region. Furthermore, their more posterior distribution of naming sites is consistent with the more anterior propagation of EEG discharges in TLE. These results hold theoretical implications regarding the role of the dominant hippocampus in determining the cortical representation of semantic and lexical information, and raise questions regarding the specific roles of medial and lateral temporal cortex in targeted word retrieval. The different patterns of naming areas identified in patients with and without HS may also carry clinical implications, potentially improving efficiency during the time-constrained process of stimulation mapping.

Diagnostic tool to ascertain marine phytoplankton exposure to chemically enhanced water accommodated fraction of oil using Fourier Transform Infrared spectroscopy.

Phytoplankton alter their macromolecule composition in response to changing environmental conditions. Often these changes are consistent and can be used as indicators to predict their exposure to a given condition. FTIR-spectroscopy is a powerful tool that provides rapid snapshot of microbial samples. We used FTIR to develop signature macromolecular composition profiles of three cultures: Skeletonema costatum, Emiliania huxleyi, and Navicula sp., exposed to chemically enhanced water accommodated oil fraction (CEWAF) in artificial seawater and control. Using a multivariate model created with a Partial Least Square Discriminant Analysis of the FTIR-spectra, classification of CEWAF exposed versus control samples was possible. This model was validated using aggregate samples from a mesocosm study. Analysis of spectra and PCA-loadings plot showed changes to carbohydrates and proteins in response to CEWAF. Overall we developed a robust multivariate model that can be used to identify if a phytoplankton sample has been exposed to oil with dispersant.

The impact of dissolved inorganic nitrogen and phosphorous on responses of microbial plankton to the Texas City "Y" oil spill in Galveston Bay, Texas (USA).

Ongoing bioremediation research seeks to promote naturally occurring microbial polycyclic aromatic hydrocarbon (PAH) degradation during and after oil spill events. However, complex relationships among functionally different microbial groups, nutrients and PAHs remain unconstrained. We conducted a surface water survey and corresponding nutrient amendment bioassays following the Texas City "Y" oil spill in Galveston Bay, Texas. Resident microbial groups, defined as either heterotrophic or autotrophic were enumerated by flow cytometry. Heterotrophic abundance was increased by oil regardless of nutrient concentrations. Contrastingly, autotrophic abundance was inhibited by oil, but this reaction was less severe when nutrient concentrations were higher. Several PAH compounds were reduced in nutrient amended treatments relative to controls suggesting nutrient enhanced microbial PAH processing. These findings provide a first-look at nutrient limitation during microbial oil processing in Galveston Bay, an important step in understanding if nutrient additions would be a useful bioremediation strategy in this and other estuarine systems.