TISIGNER.com: web services for improving recombinant protein production.

Experiments that are planned using accurate prediction algorithms will mitigate failures in recombinant protein production. We have developed TISIGNER (https://tisigner.com) with the aim of addressing technical challenges to recombinant protein production. We offer three web services, TIsigner (Translation Initiation coding region designer), SoDoPE (Soluble Domain for Protein Expression) and Razor, which are specialised in synonymous optimisation of recombinant protein expression, solubility and signal peptide analysis, respectively. Importantly, TIsigner, SoDoPE and Razor are linked, which allows users to switch between the tools when optimising genes of interest.

Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites.

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann's ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.

Solubility-Weighted Index: fast and accurate prediction of protein solubility.

MOTIVATION: Recombinant protein production is a widely used technique in the biotechnology and biomedical industries, yet only a quarter of target proteins are soluble and can therefore be purified. RESULTS: We have discovered that global structural flexibility, which can be modeled by normalized B-factors, accurately predicts the solubility of 12 216 recombinant proteins expressed in Escherichia coli. We have optimized these B-factors, and derived a new set of values for solubility scoring that further improves prediction accuracy. We call this new predictor the 'Solubility-Weighted Index' (SWI). Importantly, SWI outperforms many existing protein solubility prediction tools. Furthermore, we have developed 'SoDoPE' (Soluble Domain for Protein Expression), a web interface that allows users to choose a protein region of interest for predicting and maximizing both protein expression and solubility. AVAILABILITY AND IMPLEMENTATION: The SoDoPE web server and source code are freely available at https://tisigner.com/sodope and https://github.com/Gardner-BinfLab/TISIGNER-ReactJS, respectively. The code and data for reproducing our analysis can be found at https://github.com/Gardner-BinfLab/SoDoPE_paper_2020. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Retrospective Review of Need for Delayed Naloxone or Oxygen in Emergency Department Patients Receiving Naloxone for Heroin Reversal.

BACKGROUND: Emergency departments (EDs) are experiencing an increasing number of heroin overdose visits. Currently, there is no generally agreed upon ED observation period for heroin overdose patients who receive naloxone. OBJECTIVES: We aimed to determine the safety of a 2-h observation period for heroin overdose patients who receive naloxone. METHODS: We performed a chart review of all patients who presented with any opioid-related complaint between 2009 and 2014 to our urban academic trauma center. Subset analysis of patients with isolated heroin overdose who received naloxone was performed, with the intent of excluding patients intoxicated with long-acting/enteral opioids. The primary outcome was the number of patients who required delayed intervention-specifically, additional naloxone or supplemental oxygen. RESULTS: Between 2009 and 2014, we recorded 806 visits to our ED for heroin use after receiving naloxone. Twenty-nine patients (3.6%) received a repeat dose of naloxone, and 17 patients (2%) received oxygen ≥2 h after initial naloxone administration. Our 2-h intervention rate was 4.6% (N = 37). This decreased to 1.9% (N = 15) after 3 h and 0.9% (N = 7) after 4 h. Patients with polysubstance use were more likely to receive repeat naloxone (p < 0.01), but not oxygen (p = 0.10). Preexisting cardiopulmonary conditions did not correlate with a need for supplemental oxygen (p = 0.24) or repeat naloxone (p = 0.30). CONCLUSIONS: A 2-h ED observation period for heroin overdose patients reversed with naloxone resulted in a delayed intervention rate of 5%. Clinicians may consider a 3-h observation period, with extra scrutiny in polysubstance abuse.

The Effect of Gastric Electrical Stimulation on Small Bowel Motility in Patients With Gastroparesis and Concomitant Pancreatic and Small Bowel Dysfunction: From Animal Model to Human Application.

BACKGROUND/AIMS: Patients with gastroparesis often have biliary/pancreatic and small bowel symptoms but the effects of gastric electrical stimulation on small bowel electrical activity of the mid-gut have not been studied. Animal model aim: Establish gastric and upper small bowel/biliary slow wave activity relationships with electrical stimulation. Human study aim: Demonstrate improvement in symptoms associated with proximal small bowel dysmotility in gastric stimulated patients. MATERIALS AND METHODS: Animal model: In vivo evoked responses of duodenal and Sphincter of Oddi measures recorded during gastric electrical stimulation in a nonsurvival swine model (N = 3). High-resolution electrical slow wave mapping of frequency, amplitude, and their ratio, for duodenal and Sphincter of Oddi electrical activity were recorded. Human study: Patients (N = 8) underwent temporary gastric stimulation with small bowel electrodes. Subjective and objective data was collected before and after temporary gastric stimulation. Symptom scores, gastric emptying times, and mucosal electrograms via low-resolution mapping were recorded. RESULTS: Animal gastric stimulation resulted in some changes in electrical activity parameters, especially with the highest energies delivered but the changes were not statistically significant. Human study revealed improvement in symptom and illness severity scores, and changes in small bowel mucosal slow wave activity. CONCLUSIONS: Gastric electrical stimulation in an animal model seems to show nonsignificant effects small bowel slow wave activity and myoelectric signaling, suggesting the existence of intrinsic neural connections. Human data shows more significance, with possible potential for therapeutic use of electrical stimulation in patients with gastroparesis and pancreato-biliary and small bowel symptoms of the mid-gut. This study was limited by the nonsurvival pig model, small sample size, and open label human study.

Prevalence of Emergency Department Patients Presenting with Heroin or Prescription Opioid Abuse Residing in Urban, Suburban, and Rural Jefferson County.

BACKGROUND: Heroin and prescription opioid abuse in the United States is exhibiting a growing geographic ubiquity. OBJECTIVES: This study characterizes the changing geographic distribution of patients presenting with heroin and prescription opioid abuse to one urban emergency department (ED). METHODS: A retrospective review of patients with heroin and prescription opioid abuse from 2009-2014 was conducted in one adult urban ED. The primary outcome was the prevalence of heroin and prescription opioid patients residing in urban, suburban, and rural ZIP codes over time. RESULTS: From 2009 to 2014, 2695 patients presented for either heroin (N = 1436; 53%) or prescription opioid (N = 1259; 47%) abuse from 32 Jefferson County ZIP codes. Of the 32 ZIP codes, 15 were urban (47%), 13 suburban (41%), and 4 rural (12%). The prevalence of heroin patients (per 10,000 population) increased in each ZIP code from 2009-2014. After 2011, prescription opioid prevalence decreased in urban and suburban ZIP codes but increased in rural ones. Using segmented regression analysis, the increase in patients who used heroin residing in all ZIP codes and the decrease in patients abusing prescription opioids residing in urban areas was statistically significant after 2011. CONCLUSION: From 2009 to 2014, there was an increasing trend in ED patients using heroin who resided in urban, suburban, and rural ZIP codes. There was an increasing trend in prescription opioid prevalence in all regions from 2009 to 2011. After 2011, prescription opioid prevalence decreased in urban and suburban ZIP codes but not rural ZIP codes.

Accuracy and reproducibility of virtual edentulous casts created by laboratory impression scan protocols.

STATEMENT OF PROBLEM: Although computer-aided design and computer-aided manufacturing (CAD-CAM) complete removable dental prostheses (CRDPs) have gained popularity, conventional impressions are still common for CAD-CAM CRDP treatment. These need to be digitized and converted into virtual edentulous casts with a laboratory impression scan protocol during prosthesis fabrication. How this can best be accomplished is unclear. PURPOSE: The purpose of this in vitro study was to compare the accuracy and reproducibility of virtual edentulous casts created by a dental laboratory laser scanner and a cone beam computed tomography (CBCT) scanner with a digitized master cast. MATERIAL AND METHODS: A master cast was digitized as the virtual reference cast. Ten polyvinyl siloxane impressions were made on the master cast and scanned with the dental laboratory laser scanner and CBCT scanner. The impressions were sprayed with antiglare spray and rescanned. Four groups of virtual study casts (N=40) were created from the impression scans. All virtual study casts and the reference cast were registered with surface-matching software, and the root mean square (RMS) values (representation of overall accuracy) and percentage of measurement data points within 1 standard deviation (SD) of mean RMS values (%, representation of overall reproducibility) among the 4 study groups were measured. Additionally, 95 numeric distance differences (representation of accuracy at each region) were measured in 5 distinct regions: the apex of the denture border, 6 mm from denture border, crest of the ridge, palate, and posterior palatal seal. The repeated-measures ANOVA and post hoc test (t grouping) were used to determine statistical differences (α=.05). RESULTS: The laboratory scanner group had a significantly larger RMS value (4.0 ±0.3 µm, P<.001) and smaller percentage of measurement data points within 1 SD of mean RMS value (77.5 ±1.0%, P<.001). The RMS values between the CBCT scanner (1.2 ±0.3 µm) and CBCT scanner-spray (1.1 ±0.2 µm) groups were not significantly different (P=.968), and the percentage of measurement data points within 1 SD of mean RMS values (90.1 ±1.1% versus 89.5 ±0.8%) were also not significantly different (P=.662). The numeric distance differences across 5 regions were affected by the scanning protocols (P<.001). The laboratory scanner and laboratory scanner-spray groups had significantly higher numeric distance differences at the apex of the denture border and crest of the ridge regions (P<.001). CONCLUSIONS: The CBCT scanner created more accurate and reproducible virtual edentulous casts, and the antiglare spray only significantly improved the accuracy and reproducibility of virtual edentulous casts created by the dental laboratory laser scanner. The accuracy of the virtual edentulous casts was different across 5 regions and was affected by the scanning protocols.

A generalized protein identification method for novel and diverse sequencing technologies.

Protein sequencing is a rapidly evolving field with much progress towards the realization of a new generation of protein sequencers. The early devices, however, may not be able to reliably discriminate all 20 amino acids, resulting in a partial, noisy and possibly error-prone signature of a protein. Rather than achieving de novo sequencing, these devices may aim to identify target proteins by comparing such signatures to databases of known proteins. However, there are no broadly applicable methods for this identification problem. Here, we devise a hidden Markov model method to study the generalized problem of protein identification from noisy signature data. Based on a hypothetical sequencing device that can simulate several novel technologies, we show that on the human protein database (N = 20 181) our method has a good performance under many different operating conditions such as various levels of signal resolvability, different numbers of discriminated amino acids, sequence fragments, and insertion and deletion error rates. Our results demonstrate the possibility of protein identification with high accuracy on many early experimental devices. We anticipate our method to be applicable for a wide range of protein sequencing devices in the future.

Anemia among Adolescent Girls Attending the Pediatric Outpatient Department of a Tertiary Care Hospital: A Descriptive Cross-sectional Study.

INTRODUCTION: Adolescents are children aged 10-19 years. Nutrition influences the growth and development during infancy, childhood and adolescence. Adolescent girls are at higher risk of anemia and undernutrition. This research was aimed to find the prevalence of anemia among adolescent girls in a tertiary care hospital. METHODS: A descriptive cross-sectional study was done in the adolescent girls attending the pediatric outpatient department of a tertiary care centre from October 2020 to May 2021. After the ethical clearance from the institutional review committee, 380 adolescent girls were taken using a convenient sampling technique. Anthropometric measurements, social demography and blood for hemoglobin estimation were taken and documented in predesigned proforma. Data were analyzed with Statistical Package for the Social Sciences version 20. Point estimate at 95% Confidence Interval was done, and frequency and proportion were calculated. RESULTS: Out of 380 adolescent girls, 230 (60.5%) at 95% Confidence Interval (55.56-65.41) were anemic with mean hemoglobin of 11.138±1.954 gm/dl. The mean age was 14.57±2.107 years. CONCLUSIONS: This study showed a higher prevalence of anemia than the national data. Proper education regarding personal and menstrual hygiene, weekly supplementation of iron in school, dietary habits and uplifting of economic status can prevent anemia in this population.

Occipital Encephalocele with Multiple Birth Defects: A Case Report.

A full-term female baby presented at 24 hours of life at the emergency department with occipital encephalocele, bilateral cleft lip, and cleft palate. She was born to a second gravida mother with no consanguinity between the parents. On examination, encephalocele was 10centimeters x 7centimeters in size with bilateral cleft lip and palate. It presents the opportunity for healthcare professionals to learn about a group of congenital neurological disorders in the content of a rare case presentation and highlights the importance of ultrasonography in the antenatal period for the detection of neural tube defects in the early stage for proper counselling and management. A compulsory prenatal diagnosis of the suspected family should be done by the intervention of the public sector of any country so that we can prevent and avoid abnormal birth.

Autonomic and enteric function profiling can predict disordered gastric emptying in diabetic gastropathy.

Gastric emptying tests (GET) are the gold standard for diagnosing gastroparesis, but many patients do not have delayed emptying. We aimed to examine the combination of autonomic nervous system testing (ANS) and the enteric measure (ENS) of electrogastrography (EGG) to predict disordered GET. Seventy-six patients (47 F, 29 M mean age 40 years) with diabetes mellitus underwent evaluation for end-organ failure including gastroparesis. ANS testing assessed autonomic function by finger capillary pulse to positional changes (PAR), vasoconstriction to cold (VC), and EKG R-R interval change (RRI) with deep breathing; the ENS measures of cutaneous EGG assessed gastric myoelectrical activity. Solid (S) GET subgroups were based on 50% emptying (TS50). Via linear regression analysis: VC, PAR, and EGG had a significant inverse correlation with GET TS50 and decreased in response to a delay in gastric emptying (p < 0.05). Via ordinal logistic regression RRI and EGG-predicted gastric emptying (p < 0.01). Patients with a higher RRI and EGG value were 0.93 and 0.14 times more likely to be diagnosed with rapid gastric emptying. The areas under the curve for receiver operator characteristics (AUROC) for all measures were 0.72 in comparison to 0.59 with EGG. Based on the results, four components (RRI, VC, PAR, and EGG) quantitatively describe gastric emptying in patients with signs of diabetic gastropathy better than EGG alone.

Calcineurin Inhibitor-Based Maintenance Immunosuppression in Lung Transplant Recipients: Optimal Serum Levels for Managing Acute Rejection and Renal Function.

BACKGROUND: Although effective for curtailing alloimmune responses, calcineurin inhibitors (CNIs) have an adverse-effect profile that includes nephrotoxicity. In lung transplant (LTx) recipients, the optimal serum levels of the CNI tacrolimus necessary to control alloimmune responses and minimize nephrotoxicity are unknown. METHODS: This retrospective, single-center study reviewed tacrolimus whole blood trough levels (BTLs), grades of acute cellular rejection (ACR), acute rejection scores, and creatinine clearance (CrCl) obtained in LTx recipients within the first year after their transplant procedure. Comparisons were made between the first 90 days post LTx (when tacrolimus BTLs were maintained >10 µg/L) and the remainder of the post-LTX year (when BTLs were <10 µg/L). RESULTS: Despite tacrolimus mean BTLs being higher during the first 90 days post LTx compared with the remainder of the first post-LTx year (10.4 ± 0.3 µg/L vs 9.5 ± 0.3 µg/L, P < .0001) there was no association with lower grades of ACR (P = .24). The intensity of ACR (as determined by acute rejection scores) did not correlate with tacrolimus mean BTLs at any time during the first posttransplant year (P = .79). During the first 90 days post LTx there was a significant decline in CrCl and a correlation between increasing tacrolimus mean BTLs and declining CrCl (r = -0.26, P = .03); a correlation that was not observed during the remainder of the year (r = -0.09, P = .52). CONCLUSIONS: In LTx recipients, maintaining BTLs of the CNI tacrolimus >10µg/L did not result in superior control of acute rejection responses but was associated with declining renal function.

Seroprevalence of Helicobacter pylori/CagA Antibodies in Guatemalan Gastric Cancer Patients: Association of Seropositivity with Increased Plasma Levels of Pepsinogens but not Soluble Urokinase Plasminogen Activator Receptor.

Infection by Helicobacter pylori is a major risk factor for gastric cancer (GC), the second leading cause of cancer-related death worldwide. Although biomarkers such as pepsinogens (PGs) and soluble urokinase plasminogen activator receptor (suPAR) may have diagnostic and/or prognostic value in patients with GC, their levels may be affected by H. pylori infection. The aim of this study was to investigate the association of the presence of antibodies to H. pylori and cytotoxin-associated gene A (CagA) with plasma levels of PGs and suPAR in a cohort of Guatemalan GC patients and controls. To this end, levels of suPAR, Pepsinogens I and II (PGI and PGII), and antibodies to H. pylori and CagA toxin were determined by ELISA in plasma samples from 67 GC patients and 136 matched healthy controls. Seropositivity for CagA was significantly higher in patients with GC than in controls. Pepsinogens II and suPAR levels were higher and PGI/PGII ratios were lower in GC patients than in controls. There was a significant association of H. pylori seropositivity status with increased levels of PGII and lower PGI/PGII ratios, particularly in the control (non-GC) population. The levels of suPAR were not significantly affected by H. pylori or CagA seropositivity status. These results suggest that the seropositivity status for H. pylori and CagA need to be taken into account during the GC diagnostic process.

Trending gabapentin exposures in Kentucky after legislation requiring use of the state prescription drug monitoring program for all opioid prescriptions.

OBJECTIVE: Gabapentin is a gamma-aminobutyric acid (GABA) analog approved by the Food and Drug Administration (FDA) for partial seizures and post-herpetic neuralgia. Due to its wide therapeutic window and minimal adverse effects, it is frequently prescribed for additional off-label uses. The purpose of this study was to characterize the number, exposure reason, medical outcomes, and disposition of gabapentin exposures reported to one regional poison control center (PCC). METHODS: A retrospective cross-sectional review of exposures reported to one regional PCC was performed from January 1, 2012 to December 31, 2015. The primary outcomes were the number of gabapentin-only exposures and multi-agent exposures including gabapentin reported. Exposure reason, medical outcome, and disposition were identified for each exposure. RESULTS: There were 424 gabapentin-only exposures during the study period. The number of exposures increased each year, from 39 in 2012 to 160 in 2015. There were 1321 multi-agent exposures that included gabapentin. These exposures increased from 165 in 2012 to 440 in 2015. Comparatively, total human exposures reported to the regional PCC decreased during the study period. The majority of gabapentin-only and multi-agent exposures was intentional versus unintentional. Nine patients (2%) had a major medical outcome and three patients (1%) died in the gabapentin-only group. Comparatively, 76 patients (6%) had a major medical outcome and 12 patients (1%) died in the multi-agent group. Almost half of the multi-agent exposures required admission to the intensive care unit (ICU). CONCLUSIONS: Both gabapentin-only and multi-agent exposures increased significantly from 2012 to 2015, with the majority of cases intentional ingestion, specifically suspected suicide. The increased number of gabapentin exposures coincided with Kentucky's implementation of prescription opioid reform legislation. Providers are encouraged to call their local PCC, regardless of exposure type, to effectively monitor and evaluate exposure trends.